Albert Fl. Mann Library Cornell University Dr. Roger a. Morse 538 G3 = oi col Oi tvoi Oil COi CDi Cornell University Library The original of tinis bool< is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924090242698 SUREAU OF ENTOMOWay. L. O. HowABD, Entomologist and Ohief of Bureau. C. L. Mablatt, Entomologist and Acting Chief in absence Of Chief. R. S. Ci.ift6n, Chief Clerk. F. H. Chittenden, in charge of breeding experiments.^ A. D. Hopkins, in charge of forest, insect investigation^. W. D. IIuNTEB, in charge of cotton holl weevil investigations. F. M. Webster, in charge of cereal and forage-plant ijisect investigations. A. L. QuAiNfANCE, in charge of deciduous-fruit insect investigations. J)., M. UooERS, in charge of gipsy and hrown-tail moth work. A. W. MoERiix, engaged in white fly investigations. E. S. G. Titus, in charg^ of gipsy moth laboratory. C. J. GiLTJss, engaged in silk Investigations. R. P. CuKEiE , assistant in charge of, editorial^ work. Mabel Colcoed, librarian. Apictjltueal Investigations. . Feank Benton, in charge (absent). B. F. Phillips, acting in charge. ' J. M. Rankin, in charge of apicultural station, Chico, Cfit. Jessie E. ^abks, apicultural clerk. Technical Series, No. 14. U. S. DEPARTMENT OF AGRICULTURE, L. 0. HOWARD, Entomologist and Chief of Bureau. THE BACTERIA OF THE APIARY, WITH SPECIAL REFEREI^CE TO BEE DISEASES. GERSHOM FRANKLIN WHITE, Ph. D., Expert in Animal Bacteriology, Biochemic Division, Bureau of Animal Industry. Issued November 6, 1906. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1906. LEHER OF transmittal; U. S. Department or Agricultttee, Bureau of Entomology, Washington, D. G., September 2Ji., 1906. Sir: I have the honor to transmit the manuscript of a paper on the bacteria of the apiary, with special reference to bee diseases, by Dr. G. F. ^Vhite, expert in animal bacteriology in the Biochemic Division of the Bureau of Animal Industry. This paper was pre- pared by Doctor "White as a thesis in part fulfilment of the require- ments for the degree of doctor of philosophy, at Cornell University, in June, 1905. The Bureau of Entomolo^ considers itself fortu- nate in obtaining it for publication, since in this way a wider distri- bution can be made than would be possible were it published in a journal devoted exclusively to bacteriological investigations. It is hoped that the publication of these facts may help to clear up the confusion which now exists concerning the causes of the two most common diseases of the brood of bees. I recommend that the manu- script be published as Technical Series, No. 14, of this Bureau. Doctor White wishes to acknowledge his indebtedness to Dr. Veranus A. Moore, professor of comparative pathology and bac- teriology of Cornell University, under whose direction this work was done; to Dr. E. F. Phillips, acting in charge of apiculture, Bureau of Entomology, United States Department of Agriculture, for encouragement and assistance in the preparation of this manu- script; and to Messrs. Mortimer Stevens, Charles Stewart, N. D. West, and W. D. Wright, bee inspectors of the State of New York, for their interest in the work. EespectfuUy, L. O. Howard, Entomologist and Chief of Bureau. Hon. James Wilson, Secretary of Agriculture. PREFACE The spread of diseases of the brood of bees is to-day a great menace to the bee-keeping industry of the United States. It is therefore of great importance that all phases of these diseases should be investi- gated as thoroly as possible, and this paper, it is believed, will help in clearing up some disputed points in regard to the cause of the two most serious brood diseases. Dr. G. F. White has offered this paper for publication as a bulletin in the Bureau of Entomology because in that way the statements herein contained may become more widely known than would be the case were it published in some journal devoted exclusively to bacteri- ological investigations. Obviously there are many points still un- settled, and it is hoped that some of these may be taken up for in- vestigation in the near future, but the results so far obtained should by all means be made known to the persons practically engaged in bee keeping. The necessity for the study of nonpathogenic bacteria found in the apiary may not be at first evident to the ordinary reader. When it is seen, however, that some of the investigators of bee diseases have apparently mistaken Bacillus A or some closely allied species for Bacillus alvei it will be evident that a study of nonpathogenic germs is necessary to a thoro investigation of the cause of these diseases and a full understanding of the confusion which has existed. The names which should be used for the diseased conditions of brood was a matter which arose after this paper was offered for pub- lication. It was desired that out of the chaos of names in use cer- tain ones be chosen which would be distinctive and still clear to the bee keepers who are interested in work of this nature. Unfortu- nately, after a short investigation. Dr. W. K. Howard, of Fort Worth, Tex., gave the name " New York bee disease," or " black brood," to a disease which Cheshire and Cheyne described in 1885 as " foul brood." Since this is the disease in which Bacillus alvei is present, we can not drop the name " foul brood," and the word " European " is used to distinguish it from the other disease. The bee keepers of the United States have been taught that the type of brood disease characterized by ropiness of the dead brood is true foul brood, 3 4 PREFACE. but since Bacillus alvei is not found in this disease it obviously is not the same disease as that described by Cheyne. It would be well-nigh impossible, however, to change the name of this disease, and any effort in that direction would merely result in complicating laws now in force which control the infectious diseases of bees and would serve no good purpose. This disease is here designated "American foul brood." These names have been chosen only after consultation with some of the leading bee keepers of the United States, and these distinguishing terms were chosen by the majority of those consulted as indicating the place in which the diseases were first investigated in a thoroly scientific manner. Both diseases are found in Europe, as well as in America, so that the names indicate nothing concerning the geo- graphical distribution of the maladies. Strangely enough, certain writers for our American apicultural papers have seen fit to take exception to some of the statements made in this paper without having first found out the reasons for the de- cisions herein published. Apiculture will not be advanced to any appreciable extent by such eagerness to rush into print, especially when there is not a semblance of scientific investigation back of the criticism. E. F. Phillips, Acting in Charge of Apiculture. CONTENTS. Page. Introduction 7 Technique 7 Obtaining material for study 7 Obtnining^ cultures 7 Differentiation and identification of bacteria 9 Tbe cultures which are described 9 Morphology, staining properties, and oxygen requirements, with sug- gestions on variations 9 Media employed and suggestions as to the description of cultures 10 PART I. BACTERIA OF THE NORMAL APIARY. Bacteria from the combs 13 Bacteria from pollen 15 Bacteria in honey and normal larvae 16 Bacteria upon the adult bees 16 Bacteria of the intestine of the healthy honey bee 18 Saccharomyces and fungi 25 Tabulation of micro-organisms normally present in the apiary 28 Summary to Part I 29 Bibliography to Part I 29 PART II. THE DISEASES OF BEES. Brief history ^ 30 The term "foul brood" as hitherto applied 31 European foul brood (foul brood of Cheyne) 32 Symptoms 32 Confusion regarding foul brood in America 33 The present investigation 34 ' Bacillus alvei 36 Inoculation experiments 37 Distribution of Bacillus alvei in infected hives 38 Experiments with formaldehyde gas . 39 American foul brood 40 Symptoms 40 The present investigation 41 Bacillus larval 42 The so-called " picljle brood" 43 The so-called " blacic brood" 43 Palsy or paralysis 44 Summary to Part II 44 Conclusions 45 Bibliography to Part. II 46 Index 47 6 THE BACTERIA OF THE APIARY WITH SPECIAL REFERENCE TO BEE DISEASES. INTBODTJCTION. Since bacteriology is one of the youngest of the sciences, it is only natural that there should be many problems concerning which there is much confusion, and many others concerning which nothing is known. In a study of the saprophytic bacteria this is especially true; the exploration of this jungle of micro-organisms is scarcely begun. Comparatively few species have been studied and named, and a much less number can be identified. From studies that have been made one is led to believe that the species which might be classed under bacteria outnumber by far all the macroscopic plants known. Comparatively little is as yet known concerning the dis- tribution of these minute organisms in nature, their needs for multi- plication and growth, their power of endurance, their relations the one to the other, their relations to man and industries, and their relation to pathogenic species. Both from the standpoint of scien- tific interest and from the standpoint of practical economy these problems call for further investigation. By far the greatest amount of work which has been done in the science of bacteriology has been prompted by the direct or indirect economic importance of the question. This is largely true of the present investigation, since honey bees suffer from a number of diseases, some of which are considered in Part II. TECHNIQUE. Obtaining Material for Study. If necessary, bees may be conveniently shipped alive by mail in cages constructed for that purpose. Combs also may be sent by mail in small boxes. If combs, honey, pollen, or larvae are desired, the hive must be entered. In case older adult bees are wanted it is not difficult to supply the needs from the entrance to the hive. To capture them one may stand at the entrance and catch the unwary toiler as she 7 9583— No. 14—06 m 2 8 THE BACTEEIA OF THE APIAEY. comes in loaded with pollen and honey. After the victim alights on the entrance board, by the aid of a pair of forceps, before she disap- pears within, one can easily lodge her safely in a petri dish. It is, however, an advantage to study the young adult bees as well as the older ones, and if young ones are desired they may be taken from the combs or from the front of the hive, near the entrance. Obtaining Cultures. (a) From combs. — With sterile forceps small pieces of the comb are put directly into gelatin or agar for plates or incubated in bouil- lon for 24 hours and then plated. Growing in bouillon and plat- ing on gelatin is usually preferable. {h) From pollen. — The same technique is used as for combs, but the direct inoculation of gelatin tubes for plates is generally pre- ferable. (c) From honey. — With sterile loops honey is taken from uncapped and capped cells. The caps are removed with sterile forceps and the honey is plated directly on gelatin or agar. Bouillon tubes are in- oculated also with varying quantities of the honey. {d) From larvm. — The larva is carefully removed to a sterile dish, and with sterile scissors the body is opened and the contents plated directly, or bouillon cultures are first made and later plated, if a growth appears. (e) From parts of the adult hee. — In studying the adult bee, a small piece of blotting paper wet with chloroform is slipt under the cover of the petri dish in which the insects have been placed, and in a short time the bees are under the influence of the anesthetic. Then with sterile scissors a leg, a wing, the head, the thorax, or the abdomen, the intestine being removed, is placed in bouillon and, after 24 hours incubation, plated, preferably on gelatin. When it is desired to make a study of the bacteria of the intestine, the intestinal tract is removed and studied as follows: The bee is flamed and held in sterile forceps. With another sterile pair of for- ceps the tip. of the abdomen is seized and, by pulling gently, the tip and the entire intestine are easily removed. This can then be plated directly. If gelatin, which is preferable, is used, the intestine itself must not be left in the gelatin or the medium will become liquefied by the presence of the tissue. If one desires to obtain cultures of the anaerobe, which is quite common in the intestine, it is most easily obtained in pure culture by the use of the deep glucose agar (Liborius's method). Cover glass preparations made direct from the walls of the intestine or its contents give one some idea of the great number of bacteria frequently present. MORPHOLOGY, STAINING PKOPEETIES, ETC. 9 Differentiation and rdentification of Bacteria. These very low forms of plant life show a marked susceptibility to environmental conditions and those desirous of speculating on prob- lems in evolution may find here food for thought and experimenta- tion. On account of this susceptibility, various cultures which belong to the same species may possess slight variations in some one or more specific characters. Consequently one can not say that a species must possess certain definite characters and no others. It is convenient, then, to think of a species as more or less of a group of individuals whose characters approximate each other very closely. In this paper are described a number of species each of which, in fact, represents a group, the individual cultures of which approxi- mate each other so closely in character that the differences may be easily attributed to environmental conditions which are more or less recent. Concerning the identification of species, the conditions have been well summed up by Chester. He says: Probably nine-teuths of tbe forms of bacteria already described might as well be forgotten or be given a respectful burial. This will then leave comparatively few well-defined species to form the nuclei of groups In one or another of which we shall be able to place all new sufficiently described forms. The variations which occur and the very incomplete descriptions which can be found make it impossible to identify many species even to a more or less restricted group. For these reasons some of the cultures are not identified or named, but letters are used for conven- ience in this paper to represent the specific part. Migula's classifica- tion has been used. The Cultures Which are Described. Plate cultures were observed for some weeks, the different kinds of colonies which appeared being especially noted. Subcultures were then made in bouillon, and after 24 hours the subculture was re- plated. Subculturing and replating were then repeated. From this last plate the pure culture was made on agar for study. These were not studied culturally, as a rule, for some weeks, thus allowing time for the organism to eliminate any character due to recent environ- mental conditions (1)." Morphology, Staining Properties, and Oxygen Bequirements, with Sug- gestions on Variations. (a) Size.— The length and thickness of a micro-organism often varies so much with its environmental conditions that certain re- o Numbers in parentheses refer to papers in the bibliography at the end of Part I or that at the end of Part II. 10 THE BACTEEIA OF THE APIAEY. corded dimensions should always be accompanied by facts concerning the medium, age, and temperature of incubation. The measure- ments recorded in this paper were all taken of organisms in prepara- tions made from a 24-hour agar culture stained with carbol-fuchsin. The involution forms are not reckoned in the results. (5) Spores. — The presence of spores was determined in each case by staining the various cultures at different ages. A check was made on their presence by means of the thermal death point. (c) Flagella. — Loeffler's method, as modified by Johnson and Mack, was used for staining the flagella (2). {d) Motility. — Motility may be present in cultures when first iso- lated, but after artificial cultivation appear to be entirely lost. The reverse of this also may be noted. No cultures should be recorded as nonmotile until cultures on various media at different temperatures and of different ages shall have been studied. Hanging-drop prepar- tions were made from cultures on agar and bouillon, both incubated and not incubated, and on gelatin. (e) Staining froperties. — Basic carbol-fuchsin was the stain used almost exclusively. In the use of Gram's staining method, carbolic gentian violet (5 per cent carbolic acid 20 parts, saturated alcoholic solution gential violet 2 parts) was applied to a cover-glass prepara- tion from a 24-hour culture on agar for 5 minutes, placed in Lugol's solution 2 minutes, and placed, without rinsing, in 95 per cent alcohol for 15 minutes, removed, washt in water, and allowed to dry. (/) Oxygen requirements. — Determinations were made by ob- serving whether a growth took place in the closed or open arm or both, of the fermentation tube containing glucose bouillon. Media Employed and Suggestions as to tlie Description of Cultures. {a) Bouillon. — All bouillon used was made from beef (meat 1 part, water 2 parts) , to which infusion 1 per cent Witte's peptonum siccum and one-half per cent sodium chlorid were added. The re- action of the solution was then determined by titrating, and made -j-1.5 to phenolphthalein. In describing a culture growing in bouillon as a medium, there is usually a more extended description given than in the case of sugar and sugar-free bouillons, since cultures in these media do not differ materially in gross appearance from those observed in the plain bouillon. (6) Sugar-free houillon. — This bouillon is made free from sugar by the use of B. coli communis, after which peptone and sodium chlorid (NaCl) were added as in bouillon. (c) Sugar bouillons. — Five different sugars — glucose, lactose, sac- charose, levulose, and maltose, as well as mannite — were used in the study. If a 1-per-cent solution of glucose in plain bouillon Avas fer- MEDIA EMPLOYED, ETC. 11 merited with the production of gas, fermentation tubes were used for all the sugars and mannite. If no gas was formed in the glucose, the straight tubes were inoculated. The sugars and mannite were used in a 1-per-cent solution in sugar-free bouillon. {d) Rcaetion of media. — The reaction of cultures is determined as it appears on the fifth day in the different media, unless otherwise stated. The medium in the open arm is used to determine the re- action in the fermentation tube. Beginning with a reaction of -|-1.5 to phenolphthalein, or slightly alkaline to litmus, the detection of an increase in acidity is not difficult. But inasmuch as the production of an alkali is very frequently small in degree, cultures are often in this paper recorded alkaline in reaction when probably the reaction has not changed. (e) Fermentation with the production of gas. — Gas may be formed in such small quantities as not to be observed as such, but to be en- tirely absorbed by the medium. Whenever gas formation is men- tioned as a character, visible gas is meant. The analysis of the gas was made in the usual manner by absorbing a portion with potassium hydrate (KOH) and testing the remainder with the flame. The amount absorbed by potassium hydrate (KOH) is referred to as carbon dioxid (CO,) and the remainder, if an explosion is obtained, as hydrogen (H). This is, naturally, only approximately correct. Since the gas formula may vary from day to day, too much value must not be given to the exact proportion. It is well to observe whether the proportion of hydrogen to carbon dioxid is greater or less than 1. (/) Agar. — One per cent agar is used. The description of the growth on this medium is made from the appearance as seen on the surface of an agar slant. The description is usually very brief, since it has, as a rule, little differential value. {g) Acid agar. — This medium is made acid by titrating to +3 to phenolphthalein. The absence or presence, as well as the degree of growth, is noted. (A) Serum. — The serum used is taken from the horse, sterilized at 55° C. and congealed at 80° C. Deep inoculations are made, and the surface of slanted serum is also inoculated. The degree of growth is usually noted. Cultures are observed for 6 weeks to 2 months. The presence or absence of liquefaction is the chief character sought for. Since room temperature varies so greatly, the time at which liquefac- tion begins varies, and little differential value, therefore, can be given to the exact time of this phenomenon. («') Potato. — The composition of potato varies so markedly that a description of a culture on this medium may differ materially from that which is observed on another tube of the same medium. It is the aim to omit for the most part the observed variations due to the composition of the different potatoes. 12 THE BACTEKIA OF THE APIAEY. (j) Potato water.— To potatoes sliced very thin is added an equal amount of water by weight and the mixture is then boiled. This is btrained and distributed in straight and fermentation tubes. The reaction of the solution was made +1.5 to phenolphthalein. If any of the micro-organisms ferment glucose with the production of gas, fermentation tubes are inoculated to test the fermentation of starch ; if not, straight tubes are inoculated. (k) Milk.— If a micro-organism breaks up glucose with the forma- tion of gas, a fermentation tube of milk is inoculated with the culture; if not, straight tubes are used. Separator milk is used. The coagulation of the casein with or without liquefaction is the chief character noted. Very little stress is laid upon the time ele- ment in the coagulation of the casein and the other phenomena which are to be observed in milk. Different samples of milk and the different environmental conditions are factors which vary the length of time at which the different phenomena appear. (1) Litmus milk. — The reaction as shown by the litmus and the dis- charging of the color are the chief points observed. (m) Gelatin. — The color, degree of growth, the presence or absence of liquefaction, and the form of liquefaction are the chief points observed. The cultures are kept under observation 2 months or longer and, as in serum, the time given at which liquefaction takes place is only approximate. (w) Indol. — The cultures are allowed to grow in sugar-free pep- tonized bouillon for 3 to 5 days, and are tested with potassium nitrite (KNOj) and sulfuric acid (H,S04) after the ring method. Too much stress may be placed upon the ability of an organism to form indol. This character has been shown to be a somewhat transient one (3). {o) Reduction of nitrates to nitrites. — Cultures are cultivated 7 days in a solution of 1 gram of Witte's peptonum siccum and one- fifth gram of sodium nitrate in 1,000 c. c. of tap water. To such a culture and to a control tube are added a mixture of naphthylamine and sulfanilic acid (napthylamine, 1 part; distilled water, 1,000 parts: sulfanilic acid, one-half gram, dissolved in dilute acetic acid in the proportion of 1 part of acid to 16 parts of water) . If nitrate is reduced to nitrite, a pink color develops. The control tube should remain clear, or slightly pink — owing to the absorption of a trace of nitrite from the atmosphere. PART I. BACTERIA OF THE NORMAL APIARY. Before studying the cause of a disease it is necessary that we know what bacteria are normally present, so that later, in studying diseased conditions, a consideration of these nonpathogenic species may be eliminated. In view of this necessity a bacteriological study BACTEEIA PROM THE COMBS, 13 of the hives, combs, honey, pollen, larvae, and adult bees was begun, to determine the bacteria normally preseftt. It was not hoped that all the species isolated could be easily identified, or that all would merit a careful description, but it was hoped that those species which seemed to be localized in any part of the apiary, or upon or within the bees, might be studied and described with sufficient care to guarantee their identification upon being isolated again. The chance of varia- tion in morphology, pathogenesis, and cultural characters due to environmental conditions to which these micro-organisms were being subjected at the time, or to which they had been subjected before isolation or study, has been carefully borne in mind. BACTERIA PBOM THE COMBS. One might naturally suppose that very many species of bacteria would be present on combs, since these are exposed more or less to the contaminating influence of the air. The reverse, however, seems to be true. The number of different species isolated is comparatively small. Those which appear most often are described below. Some other species mentioned in this paper are found on combs, but inas- much as they appear most frequently from other sources they are described there. One species of Saccharomyces from the comb, also, is described under the heading " Saccharomyces and fungi." Bacillus A. {B. mesentericus?) Occurrence. — Found very frequently on combs, on scrapings from hives, and on the bodies of bees, both diseased and healthy. Oelatin colonies. — Very young colonies show irregular edges, but very soon liquefaction takes place and the colony gives rise to a circular liquefied area, covered with a gray membrane, which later turns brown. Agar colonies. — Superficial colonies present a very irregular margin consist- ing of outgrowths taking place in curves. Deep colonies show a filamentous growth having a moss-like appearance. Morphology. — In the living condition the bacilli appear clear and often grauu lar, arranged singly, in pairs, and in chains. The flagella are distributed over the body. The rods measure from Sn to 4/i in length, and from 0.9/4 to L2|U in thickness. Motility. — The bacUli are only moderately motile. Spores. — Spores are formed in the middle of the rod. Gram's stain. — The bacilli take Gram's stain. Oxygen requirements. — Aerobic and facultatively anaerobic. Bouillon. — Luxuriant growth in 24 hours, with cloudiness of medium ; a gray flocculent membrane is present. Later, the membrane sinks and the medium clears, leaving a heavy, white, flocculent sediment, with a growth of the organ- isms adhering to the glass at the surface of the medium. Reaction alkaline. Glucose. — Luxuriant growth takes place in the bulb, with a moderate, floccu- lent growth in closed arm. The gradual settling of the organisms causes a 14 THE BACTEEIA OF THE APIARY. heavy white sediment to form in the bend of the tube. The reaction is at first slightly acid, but subsequently becomes alkaline. No gas is formed. Lactose. — Reaction alkaline. Saccharose. — Reaction alkaline. Levulose. — Reaction acid. Maltose. — Reaction acid. Mannite. — Reaction alkaline. Potato water. — Reaction alkaline. Agar slant. — A luxuriant growth takes place on this medium. The growth gradually increases to a moist, glistening one, being then friable and of a grayish brown color. Serum. — A luxuriant, brownish, glistening, friable growth spreads over the entire surface. No liquefaction is observed. Potato. — An abundant fleshy growth of a brown color spreads over the entire surface. The water supports a heavy growth. The potato is slightly discolored. Milk. — Precipitation takes place rapidly, followed by a gradual digestion of the casein, the medium changing from the top downward to a translucent liquid, becoming at last semi-transparent and viscid. Litmus milk. — Precipitation of the casein takes place usually within 24 hours, followed by a gradual peptonization. Reduction of the litmus occurs rapidly, leaving the medium slightly brown ; later the blue color will return on exposing the milk to the air by shaking. Reaction alkaline. Gelatin. — An abundant growth takes place with rapid, infundibuliform lique- faction. A heavy, white, friable membrane is formed on the surface of the liquefied medium. A flocculent sediment lies at the bottom of the clear lique- fied portion. Acid agar. — Growth takes place. Indol. — None has been observed. Nitrate. — Reduction to nitrite is positive. Bacterium acidiformans. (Sternberg, 1892.) Occurrence. — Isolated from the scraping of propolis and wax from the hives and frames of healthy colonies. Gelatin colonies. — The superficial colonies are friable, convex, opaque, and white with even border ; when magnified they are finely granular, sometimes radiately marked. They are from 1 to 4 millimeters in diameter. The deep colonies are spherical or oblong and entire. Morphology. — When taken from an agar slant 24 hours old, the rods are short, with rounded ends, singly and in pairs. Length about 1.6|ti, thickness O.Sfi. They stain uniformly with carbol-fuchsin. Flagella are apparently ab- sent. Motility. — No motility has been observed in any medium. Spores. — Spores are apparently absent. Gram's stain. — The bacteria are decolorized by Gram's method. Oxygen requirements. — Facultatively anaerobic. Bouillon. — The medium becomes slightly clouded with a feeble ring of growth on the glass at the surface of the liquid. A moderate amount of white friable sediment is formed. Reaction alkaline. Glucose. — Uniformly and slightly clouded. No gas is formed. Reaction acid. Lactose. — Reaction acid. Saccharose. — Reaction alkaline. Levulose. — Reaction acid. BAOTEEIA FROM POLLEN. ' 15 Maltose. — Reaction acid. Mannite. — Reaction acid. Potato water. — Reaction acid. Agar slant. — A moderate, gray, glistening growth, confined to tlie area Inocu- lated with the loop, is formed on the inclined surface. Serum. — A feeble gray growth Is formed only on the inoculated surface. No liquefaction taljes place. Potato. — A gray growth covers the inoculated surface. Milk. — Heat causes a ready coagulation of the casein. Reaction acid. I/itmus milk. — Coagulation of casein occurs promptly on boiling a culture 2 weeks old. Reaction acid. Gelatin. — Growth of spherical colonies appears along the line of inocula- tion, the surface growth being grayish and spreading slowly. No liquefaction takes place. Acid agar. — Growth takes place. Indol. — A trace was observed. Nitrate. — No reduction to nitrite could be observed. BACTERIA PROM POLLEN. As in the case of the examination of the combs, the number of spe- cies of bacteria found in pollen is comparatively small. The follow- ing are often found to be present. Other species have been isolated, but their distribution in the pollen is not at all constant. Bacillus B. Occurrence. — Found frequently in pollen and in the intestine of healthy honey bees. dclatin colonies. — The colonies are egg-yellow with even border. Liquefac- tion takes place slowly. Surface colonies are about 1.5 millimeters in diameter, have coarsely granular center, finely granular margin, and clear and sharply defined border. A peculiar toruloid growth is often observed. Morphology. — The organisms are short rods with rounded ends, which stain uniformly with carbol-fuchsln, and are 1/i to 2|H in length. Few short involu- tion forms occur. Motility. — The bacilli are actively motile in young cultures. Spores. — No spores have been observed. Oram's stam.— .The bacilli are decolorized by Gram's stain. Oxygen requirements. — Facultatively anaerobic. Bouillon. — This medium becomes uniformly clouded, frequently with a scanty, friable membrane. Sometimes the organisms settle, clearing the medium and forming a viscid sediment. A growth of the culture adheres to the glass at the surface of the liquid. This, together with the membrane, is of a light egg-yellow color, which deepens somewhat with age. Reaction alkaline. Glucose. — At first both arms of the fermentation tube are clouded slightly, and the cloudiness later Increases. Sometimes a stronger growth occurs in the closed arm than in the open one. Reaction Is at first acid, but slowly changes to alkaline. Lactose. — Reaction alkaline. Saccharose. — Reaction alkaline. Levulose. — Reaction alkaline. Maltose. — Reaction slightly acid. 9583— No. 14—06 m 3 16 THE BACTERIA OF THE APIAKY. Mannite. — Reaction slightly acid, later alkaline. Agar slant. — A moderate, slightly yellow, nonviscid glistening gi:pwth appears along the inoculated surface. This growth gradually spreads and deepens in color to an egg-yellow. Potato. — A moderate, egg-yellow, nonviscid, glistening growth spreads over the entire surface. The potato Is slightly discolored. Milk. — The milk is covered by a yellow growth of the culture, resembling cream. Coagulation takes place on boiling. Litmus milk. — Reaction alkaline. Gelatin. — Growth takes place along the line of inoculation. Deep in the medium the colonies are white and spherical ; the surface growth is yellow. After a few days liquefaction begins, and at the end of 2 weeks one-half the tube is liquefied. The liquefaction is infundibuliform. Liquefied gelatin is sur- mounted by a friable, egg-yellow pellicle. The growth in the liquefied portion is flocculent, which, on settling, forms a yellow sediment at the apex. Indol. — None could be observed. Nitrates. — No reduction to nitrites occurs. BACTEEIA IN HONEY AND NORMAL IiAIlV.ai. Comb honey from a large number of sources has been examined and found to be quite uniformly sterile. The healthy larvae likewise are usually sterile. BACTERIA UPON THE ADULT BEES. On the external part of the bee we again find only a few different species. Bacillus A, described as found upon the combs, is fre- quently isolated from the bee. Other species which are found fre- quently are described below. Bacterium cyaneus (Micrococcus cyaneus). Occurrence. — Isolated from the body of a healthy honey bee and from pollen. Gelatin colonies. — The colonies are lemon-yellow, with entire border, growth taking place readily on this medium. The superficial colonies, having well- defined border, are finely granular, and liquefy the medium within 3 to 6 days. Morphology. — Short oval rods 0.8/n to l.T/j, in length, O.Y/i to 0.8|U in thickness. Short involution forms are present. The rods occur singly, paired, and in clumps. No flagella have been demonstrated. Motility. — No motion has been demonstrated. Spores. — No spores have been demonstrated. ' Gram's stain. — The bacterium takes Gram's stain. Oxygen requirements. — Aerobic. Bouillon. — At first a slight cloudiness appears, the medium becoming turbid in old cultures. A heavy yellowish-white, slightly viscid ring forms on the tube at the surface of the medium. The sediment, and sometimes the medium, show marked viscidity. Reaction alkaline. Glucose. — ^The growth of the culture is confined entirely to the open bulb, in which the medium becomes turbid. No gas is formed. Reaction alkaline. Lactose. — Reaction alkaline. Saccharose. — Reaction alkaline. Levulose. — Reaction alkaline. BACTEEIA UPON THE ADULT BEES. 17 Maltose. — Reaction allialino. Mannitc. — Reaction allialine. Potato water. — Reaction alkaline. Agar slant. — On the surface of the agar there takes place an abundant growth, which is confined to the surface inoculated with the loop. The culture is tleshy, nonviscid, and lemon-yellow. It produces a soluble pigment that dif- fuses thru the agar, giving it a dark-pink color. Scniiii. — Luxuriant growth takes place, lurompanled by liquefaction. Potato. — A lemon-yellow, fieshy, glistening growth spreads over the inclined surface of the potato. Milk. — Precipitation followed by slow liquefaction of the casein occurs ; later the medium becomes alkaline and very viscid. Litmus iiiillc. — The litmus is discharged and the casein is liquefied. Reaction alkaline. Gelatin. — Infundibuliform liquefaction soon begins, which is followed by stratiform liquefaction. The liquefied gelatin is turbid and viscid. Acid agar. — On this mediimi a moderate lemon-yellow growth is observed. Indol. — None could be observed. Xitrates. — No reduction of nitrates could be observed. Micrococcus C. Occurrence. — Isolated from the body of a healthy honey bee. Gelatin colonics. — The surface colonies are round and slightly yellow. Liquefaction, begins in from 2 to 4 days. The magnified colonies are finely granular, with sharply defined, entire border. Morphology. — Cocci, about 0.8|U in diameter, occur in pairs and in small clusters. Motility. — Nonmotile. Spores. — Spores are apparently absent. Grants stain. — The coccus takes the Gram's stain. Oxygen requirements. — Aerobic. Bouillon. — ^This medium becomes uniformly clouded in 24 hours after in- oculation, growth increases, and friable sediment forms. The liquid clears somewThat on standing. Reaction at first slightly acid ; later returns to neutral. Glucose. — The medium in the bulb becomes cloudy, while that in the closed arm remains clear. White friable sediment forms in bend of tube. Reaction acid. No gas is formed. Lactose. — Reaction slowly becomes acid. Saccharose. — Reaction acid. Levulose. — Reaction acid. Maltose. — Reaction acid. Mannite. — Reaction acid. Potato water. — Reaction acid. Agar slant. — A grayish white, fleshy, nonviscid, glistening growth takes place along the inoculated surface. It does not spread, and retains a dis- tinct boundary. Serum. — A spreading growth takes place, accompanied by liquefaction. Potato. — A gray, fleshy, glistening, nonviscid growth forms over the entire cut surface of the potato. The potato is slightly discolored. Milk. — This medium becomes firmly coagulated and later the casein liquifies with the formation of a milky serum. 18 THE BACTEEIA OF THE APIARY. Litmus milJc. — In this medium coagulation takes place, accompanied bj reduction of the litmus. Reaction slightly acid. Gelatin. — After a day or two infundibuliform liquefaction occurs, being followed by stratiform liquefaction; the liquefied. gelatin is turbid. Growth below this portion Is in the form of small spherical colonies. Acid agar. — A white, fleshy, nonviscid growth is observed. Indol. — A trace was observed. y Urates. — Reduced to nitrites. BACTERIA OF THE INTESTINE OF THE HEALTHY HONEY BEE. A great many investigations have been made in recent years on the bacteria found present in the intestines of vertebrates (4, 5, 6, Y, 8, 9), and striking similarities are noticed in the species found in many of them. In this investigation the intestinal contents of about 150 bees, mostly from one apiary, have been studied more or less thoroly. Several species which are found to be constant in many of the verte- brates are found in the intestine of the honey bee. Since the tem- perature of the bee approximates much of the time, especially when in the hive, that of the warm-blooded animals, many of the same species of bacteria inhabit the intestine of this insect as are found thriving in the same locality in man and other^ animals. A stained cover-glass preparation made directly from a healthy adult field bee reveals, almost without exception, a multitude of bacteria. In a study of the bacterial flora stress has been placed upon the different species which were found to be more or less constant, rather than upon the actual number of bacteria oi- species in any quantity of material from a single bee. From the observations which have been made, it appears that the number of species in any individual is comparatively small, but the number of bacteria is in many cases very large. Sometimes, however, the plates show very few colonies, while cover-glass preparations show a very large number of bacteria. These organisms are probably the anaerobe, which is quite constant, as shown by cultures made direct from the intestine into glucose agar (Liborius's method). When a loopful of the material from the intestine was used for the inoculation, the following data give the approximate findings : Bee No. 1, 300 to 400 yellow colonies, probably alilie. Bee No. 2, a few colonies of fungi only. Bee No. 3, 500 colonies, mostly yeast. Bee No. 4, 100 or more colon-like colonies. Bee No. 5, 2,000 or more, mostly yellow. Bee No. 6, 20 or more colonies, mostly yeasts. Bee No. 8, 400 or more yellow colonies. Bee No. 9, 30 yeasts with a few fungi. Bee No. 10, 50 yeast colonies with a few fungi. Bee No. 11, no growth. Bee No. 12, 300 colonies, slightly yellow. BACTERIA OP THE INTESTINE. 19 Bee No. 13, 2,000 or more gray colonies. Bee No. 14, yeast colonies and a few colonies of bacteria showing ground- glass appearance. Bee No. 15, 2,000 or more colon-like colonies {B. cloaca;). The following are the species which have been found to be most constant. The reader is referred also to the description of the yeast plant found very frequently in the intestine of the normal honey bee, described under " Saccharomyces and fungi." Bacterium S. Occurrence. — Frequent in the intestine of the healthy honey bee. Agar colony. — Deep colonies when magnified are coarsely granular, showing a dark brown center, with a thin and ill-deflned border. Morphology. — A preparation made from a young culture taken from a glu- cose fermentation tube shows rods with rounded ends, occurring singly and in pairs', staining easily and uniformly with carbol-fuchsin, and measuring 0.7^ to 1.5/» in length and 0'.5/i to 0.7/t in thickness. Motility. — No motility could be observed. Spores. — No spores could be demonstrated in young cultures. In old cultures their presence is questionable. Oxygen requirements. — Strictly anaerobic. Bouillon. — In straight tubes no growth occurs. Olucose. — A moderate cloudiness can be, seen in the closed arm, while the open bulb remains clear. No gas is produced. Reaction about neutral. Glucose agar (Liborius's method). — Growth is rather slow. After 3 days a moderate growth may be observed; later, if cultures have recently been iso- lated from the bee's intestine, the growth imparts to the medium a diffused haziness or cloudiness. After many generations the culture loses this property. Glucose gelatin (Liborius's method). — Very slow growtji occurs in the depth of the mediiuu. No liquefaction takes place. ' Bacillus cloacae. Occurrence. — Found in the intestine of a large number of healthy honey bees. Oelatin colonies. — Superficial colonies are thin and blue to gray In color ; deep colonies, brown, regular, granular, and spherical to lenticular. Agar colonies. — Superficial colonies are partially opaque, brown, finely granu- lar, with well-defined margin ; deep colonies are regular, spherical, or lenticular, with well-defined margin. Morphology. — The rods from 24-hour agar cultures have rounded ends, vary- ing in length from V to 2 it and in width from 0.7/i to 0.9 /i». They are usually found singly or in pairs. Involution forms are not uncommon. With carbol- fuchsin they stain uniformly. This species possesses a few peritrichic flagella. Motility. — Active motility is observed In young cultures. Spores. — No spores are formed. Gram's stain. — ^The bacillus does not take Gram's stain. Oxygen requirements. — Facultatively anaerobic. Bouillon. — A uniform cloudiness appears in 24 hours. Growth continues until the medium becomes heavily clouded, followed by a gradual settling of many of the organisms, forming a viscid grayish-white sediment. A gray friable mem- brane, which adheres to the sides of the tube at the surface of the medium, is sometimes produced. Upon agitation this membrane breaks up and sinks to the 20 THE BACTERIA OF THE APIAEY. bottom, leaving a gray ring of the growtli adhering to the glass. Reaction alkaline. Glucose.— The medium in the bulb becomes turbid, while that in the closed arm is uniformly cloudy. A heavy grayish-white sediment is formed. The reaction is at first slightly acid, but in a few days becomes alkaline. Abundant and rapid gas formation takes place, filling usually from one-half to nine-tenths of the closed arm. The ratio of hydrogen to carbon dioxid is approximately 1 to 2 ; that is, the ratio of hydrogen to carbon dioxid is less than 1. Lactose. — In this medium gas formation takes place more slowly than in glucose. At the end of 8 days one-fourth of the closed arm is filled with gas. The ratio of hydrogen to carbon dioxid is greater than 1. Reaction acid. Saccharose. — Gas is formed abundantly and rapidly; more than one-half of the tube is usually filled with gas. The ratio of hydrogen to carbon dioxid is less than 1. Reaction alkaline. Levulose. — A rapid fermentation takes place ; more than one-half of the closed arm is filled with gas. The ratio of hydrogen to carbon dioxid is approximately 1 to 5 ; that is, less than 1. A slight formation of acid takes place at first, but the reaction rapidly becomes alkaline. Maltose. — Formation of gas takes place with the result that at the end of 5 days approximately one-half of the tube is filled. The ratio of hydrogen to carbon dioxid will approximate that of 1 to 1. Reaction acid. Mamnite. — Gas is formed rapidly and abundantly ; at the end of 5 days the closed arm is usually much more than half filled with the gas. The reaction is at first slightly acid, but soon becomes alkaline. The ratio of hydrogen to car- bon dioxid is approximately 1 to 2 ; that is, less than 1. Potato loater. — Gas forms rapidly and fills half the closed arm. The ratio of hydrogen to carbon dioxid is as 1 to 2 ; that is, less than 1. Agar slant. — A moderate, grayish-white, glistening, friable growth appears along the line of inoculation, which usually spreads to the sides of the tube. Serum. — Moderate gray growth appears, which is confined quite closely to the line of inoculation. Liquefaction takes place slowly after .3 weeks. Potato. — A moderate amount of gray fleshy growth covers the slope. The potato is slightly discolored. MilJc. — Coagulation takes place after 4 days' growth. Gas is formed. Litmus, milk. — A marked production of acid takes place, followed by firm coagulation. Gelatin. — A heavy white growth takes place along the line of inoculation ; the surface growth is flat, bluish-white, and spreads with an uneven margin. Slow infundibuliform liquefaction takes place after 2 weeks. Acid agar. — A growth takes place. Indol. — A trace is sometimes produced. Nitrates. — Reduction to nitrites is positive. B. coli communis. Occurrence. — Pound in the intestine of healthy honey bees. Gelatin colonies. — The superficial colonies are blue, lobate-lobulate, and slightly spreading; when magnified they are brownish yellow in the center and more transparent toward the margin; the deep colonies are spherical to lenticular and brownish yellow, with well-defined borders. Morphology.— The short rods with rounded ends measure 1.5^ to 2^1 in length and 0.7/t to O.S/j. in thickness. They occur singly or in pairs, stain uniformly, and are motile by means of a few peritriehie flagella. BACTERIA OP THE INTESTINE. 21 ■ Motility. — ^The bacilli are actively motile from some cultures. Spores. — No spores are formed. Oi-am's stain. — The bacillus is decolorized by Gram's method. Oxygen requirements. — It is a facultative anaerobe. Bouillon. — The medium becomes uniformly clouded in 24 hours, with a slight acid reaction ; the medium later becomes allialine, ^yitll a gray and friable sediment. A feeble pellicle is formed and u growth of the, organism often adheres to the glass at the surface of the liquid. Glucose. — Both branches of the fermentation tube become clouded. The sugar splits by fermentation into gas and acid, one-half or more of the closed arm being filled. The ratio of hydrogen to carbon dioxid is 2 to 1. Lactose. — Gas fills one-fourth of the closed tube. Reaction acid. Saccharose. — Gas fills one-sixth of the closed tube. Reaction acid. Levulose. — Gas fills one-half of the closed tube. The value of hydrogen to carbon dJoxid is 2 to 1. Reaction acid. ^ Maltose. — One-sixth of the closed arm is filled with gas. Reaction acid. Mannite. — One-half of the closed tube is filled with gas. Reaction acid. Potato tratei: — Reaction acid. Agar slant. — A moderate, gray, nonviscid, spreading growth takes place on the surface of the inclined agar. Serum. — A gray, glistening, nonspreading growth is observed on the inclined serum. No liquefaction takes place. Potato. — A moderate, fleshy, glistening growth spreads over the inoculated surface. Potato slightly discolored. Milk. — Coagulation of the casein takes place in about 4 days. A small quan- tity of gas is produced. Litmus milk. — Coagulation occurs. Reaction strongly acid. Gelatin. — ^A moderate growth occurs along the line of inoculation ; the growth is spreading with an irregular margin on the surface. No liquefication occurs. Acid agar. — A moderate grayish growth occurs on surface. Indol. — A trace was obtained in some cultures. Nitrates. — Reduced to nitrites. B. cliolerse suis. Occurrence. — Isolated from the intestine of healthy honey bees. Gelatin colonies. — Colonies are translucent by transmitted light; bluish to gray by reflected, the border being uneven and well defined. When the colonies are magnified they appear brownish and finely granular. Morphology. — ^The rods are short, with rounded ends, occurring singly and in pairs, and staining uniformly with carbol-fuchsin, 1 to 2.8^ in length, and 0.6/1 to 0.8/1 in thickness. A few peritrichic flagella are present. Motility. — ^Usually only jl few are motile at a time in the field, and these present a rapid whirling motion. Spores. — ^No spores are formed. Gram's stain. — ^The bacteria are decolorized by Gram's stain. Oxygen requirements. — Facultatively anaerobic. Bouillon. — A uniform, moderate cloudiness arises in this medium in 24 hours; later a grayish-white membrane is formed which, upon shaidng the tube, sinks to the bottom, forming a gray sediment. The reaction is at first slightly acid, but later becomes alkaline. Glucose. — The medium becomes clouded in both arms of the fermentation tube, with the production of a small amount of gas. Reaction acid. 22 THE BACTERIA OE THE APIARY. Lactose.— Growth takes place in both arms of the tube, but the sugar Is not split into either acid or gas. Saccharose.— Giowth occurs in both arms of the tube, neither acid nor gas being formed. f Levulose.— Growth takes place in both arms with the production of gas and acid; one-third of the closed arm is filled. The ratio of hydrogen to carbon dioxid is about 3 to 1 — that is, greater than 1. Maltose. — The medium in both arms of the tube becomes clouded. Fermenta- tion results in the production of gas sufficient to fill about one-fifth of the tube. Only a small portion of the gas is absorbed by sodium hydroxid, leaving behind an explosive gas. Mannite. — The medium in both branches of the tube becomes clouded; gas is not formed. Reaction alkaline. Potato water. — ^About one-fifth of the closed arm is filled with gas. Reaction acid. Agar slant. — A moderate, grayish-white, glistening, nonspreading growth is formed along the surface inoculated with the loop. Serum. — A moderate, gray, glistening, nonspreading growth takes place on the inclined surface. No liquefaction occurs. Potato. — A feeble, grayish growth is observed. The potato becomes slightly discolored. Millc. — No coagulation occurs, and no gas is produced. Reaction alkaline. Litmus milk. — The medium slowly becomes more and more alkaline. Gelatin. — A moderate, white growth takes place along the line of inocula- tion. On the surface it spreads with irregular margin. No liquefaction occurs. Acid agar. — ^A moderate growth appears. Indol. — Indol is produced. Nitrates. — Reduction to nitrites (?). Bacillus E. Occurrence. — Isolated from the intestine of healthy honey bees. Oelatin colonies. — The colonies are lemon-yellow. Surface colonies are con- vex, smooth, with entire margin ; when magnified ' they are finely granular. Deep colonies, when magnified, are lenticular, finely granular, and may appear dark green. Liquefaction takes place slowly. Morphology. — The rods are short, with rounded ends, and usually occur singly. The bacilli are l.5/i to 2/< in length and 0.7ju. in thickness. This species pos- sesses a few peritrichic flagella. Motility. — ^The bacteria are actively motile. Spores. — No spores are present. Gram.'s stain. — They stain with Gram's stain. Oxygen requirements. — Aerobic. Bouillon. — The medium becomes uniformly clouded in 24 hours. Later a tough, yellowish-white membrane is formed, which sinks upon shaking. The medium is very viscid in old cultures. Reaction alkaline. Glucose. — Growth is confined to the open bulb. No gas formation occurs. Reaction slightly acid. Lactose.— There is a marked mucous-like appearance in the medium. Reac- tion alkaline. Saccharose. — Reaction acid. Levulose. — Reaction alkaline. Maltose. — Reaction alkaline. Mannite. — Reaction slightly acid. BACTERIA OF THE INTESTINE. 28 Potato iratcr.— Reaction iilkaline. Agar slant.— A moderiite, yellowish-gi-iiy, iioiivisoid growth takes place on the surface. Serum.— A strong growth takes place and the inediuiii is liquefiea. Potato. — A yellowish-gray, noiuMscid growth is observed over the entire inclined surface. ilfiifc.— Precipitation of casein takes place with very slight digestion (V). Litvius milh: — Precipitation of the casein occurs. Kenctioii alkaline. Oelatin.—A white growth forms along the line of inoculation, which becomes slowly liquefied from above. Acid agar. — A moderate, slightly yellow growth is observed. Indol. — None demonstrated. Nitrates. — No reduction to nitrites occurs. Bacillus subgastricus. Occurrence. — Isolated from the Intestine of a healthy honey bee. Qelatin colony. — The colon-like, superficial colonies are thin, blue, spreading, and lobate-lobulate. When magnified they are finely granular, with brown center. Deep colonies are spherical and yellow. Mwphology. — Short rods, singly and in pairs, are from 1.5/i to 2.5/n long and from 0.6/1 to 0.8^ thick. They stain uniformly with carbol-fuchsin. Motility. — Marked whirling motion from gelatin cultures. Spores. — No spores could be demonstrated. Oram's stain. — The bacilli are decolorized with Gram's stain. Oxygen requirements. — Facultatively anaerobic. Bouillon. — This medium becomes clouded in 24 hours. A slight band of growth is formed on the glass at the surface of the liquid. Later a feeble pellicle Is sometimes formed. Reaction at first slightly acid, later becomes alkaline. Glucose. — The medium in both branches of the tube becomes clouded. Gas is readily formed until about one-fourth of the closed branch is filled. The ratio of hydrogen to carbon dioxid is 2 to l — that is, greater than 1. Reaction strongly acid. Lactose. — Gas formation occurs. About one-sixth of the tube is filled with gas, part of which is absorbed by sodium hydroxid and another part is explo- sive. Reaction acid. Saccharose. — This sugar is fermented to the point of formation of acid, but no gas is formed. Levulose. — This sugar splits in the process of fermentation to form acid and gas, the gas filling about one-sixth of the tube. A pgrtion of the gas is absorbed by sodium hydroxid, the remainder being explosive. I Maltose. — Fermentation takes place with the formation of acid. No gas is produced. Mannitc. — One-fifth of the closed arm is filled with gas. A portion of the gas is absorbed by sodium hydroxid and a portion is explosive. Reaction acid. Potato water. — Reaction alkaline. Agar slant. — A moderate, translucent, gray, nonviscid and glistening growth spreads slowly from the surface inoculated with the loop. Serum. — A moderate, glistening growth appears along the surface inoculated. No liquefaction occurs. Potato. — A grayish growth takes place on the sloped surface. Milk. — Firm coagulation of the milk takes place with the formation of a small amount of clear serum. A small amount of gas is produced, 9583— No. 14—06 m i 24 THE BACTERIA OF THE APIAEY. Litmus milk. — Reaction .strongly acid. Coagulation occurs in about six days. Gelatin. — Wtiite, spherical colonies appear along the line of inoculation. The surface growth is grayish blue and spreading, with irregular margin. Slow liquefaction takes place, beginning usually in 2 weeks. Acid agar. — A growth takes place. Indol. — None could be demonstrated. Nitrates. — No reduction to nitrites occurs. Bacterium mycoides. Occiirrrnec. — Isolated from the intestine of a healthy honey bee. Gelatin coJouiea. — A rapid growth of root-like colonies appears in 24 hours. In macroscopic appearance it somewhat resembles cotton fibers ; when magni- fied these appear thick and somewhat felted in the center, while toward the margin they are beautifully filamentous. After a day or two the gelatin begins to liquefy. ilorpliologii. — The rods are large, scarcely rounded at the ends, and frequently in chains. They measure from 2.;V to 5.5/n long and 1.5/(. thick. No flagella have bpen demonstrated. Motility. — No motility could be demonstrated. Spores. — Spores are present. Gram's stain. — The bacteria are not decolorized by Gram's stain. Oxygen requirements. — Facultatively anaerobic. Bouillon. — A decided fieecy gi'owth with heavy, cotton-like sediment occurs. Glucose. — No gas is formed. Reaction acid. Lactose. — Reaction acid. Saccharose. — Reaction acid. Levulose. — Reaction acid. Maltose. — Reaction acid. Mannitc. — Reaction acid. Potato tcatcr. — Reaction alkaline. Agar slant. — A luxuriant growth that appears root-like takes place on this medium. This growth tends to extend into the agar, which causes it to adhere to the medium. Serum. — A luxuriant growth is formed, accompanied by liquefaction. Potato.— A thick, gray, moist growth is found, the potato not being discolored. 3IUJc. — Coagulation occurs promptly, with formation of a clear serum. Litmus milk. — The color is discharged in 48 hours. Ge/aM».— Hair-like outgi-owths occur along the line of inoculation. Lique- faction begins at the surface and proceeds along the needle tract. In a few days the entire medium is liquefied. Indol. — No indol isproduced. ^"itrates. — Reduction to nitrites is positive. Pseudomonas fluorescens liquefacierLs. Occurrence. — Isolated from the Intestine of the healthy honey bee. Gelatin colonies.— Betore liquefaction, the superficial colonies, when magni- fied, are finely granular, Avith regular margin; deep colonies are spherical, brown, with regular margin. Liquefaction takes place rapidly. The surface of liquefied gelatin is covered by a friable membrane. Later the liquefied gela- tin takes on a green fluorescence. Morphology.— The bacteria are short rods, varying from l/i to 2^^ in length and from 0.5/t to 0.7/1 in thickness. They stain uniformly with carbol-fuchsin and are motile by means of one or more polar fiagella. SAOCHAROMYCES AND FUNGI. 25 Spores. — No spores could be demonstrated. Oram's stain.— The bacteria do not take Gram's stain. Oxygen rc(iitirciiients. — Aerobic Teiiiiii'ratiire require luents. — Culture must be grown at room temperature. Bouillon. — Tbe medium becomes clouded in 48 hours, forming a moderately tough pellicle. A greenish-yellow fluorescence begins at the surface, which gradually increases until the entire medium talcos on that appearance. Rfcac- tion alkaline. Gliico!se. — A cloudiness is formed in the open arm, but the closed arm is clear. Reaction ailjaline. Lactose. — Iteaction allialine. Saccharose. — Reaction allcaiine. Levulose. — Reaction alkaline. Maltose. — Reaction alkaline. ilannitc. — Reaction alkaline. Agar slant. — At first a gray friable growth is formed confined to the surface inoculated, which later takes on a brown hue. Greenish-yellow fluorescence is observable in the medium. * Serum. — A slow liquefaction occurs. Potato. — Very scanty growth occurs with slight discoloration. MiVc. — Rapid liquefaction of the casein takes place. Litmus milk. — Rapid liquefaction of the casein takes place. Reaction alkaline. Gelatin. — Infundibuliform liquefaction takes place rapidly. Acid agar. — No growth occurs. Indol. — No indol observed. Nitrates. — No reduction to nitrites occurs. SACCHAROMYCES AND rtTNGI. The first yeast plant described below is of very frequent occurrence in the intestine of the normal bee. Saccharomyces roseus can^be iso- lated from the comb. A large number of common fungi wei-e found in the flora of the intestines and in cultures from the pollen and combs. In addition to the above the third Saccharomyces here described was found in two samples of brood apparently diseased, which could not be diagnosed as any disease commonly known. Saccharomyces P. Occurrence. — Very common in the intestine of healthy honey bees. Oelatin colonies. — Colonies form slowly; the superficial colonies are white, glistening, convex, capitate, and about 1 to 2 millimeters In diameter. When magnified they are finely granular, brownish yellow, with entire margin. Deep colonies are finely granular, with uniform margin, spherical to lenticular, and brownish green. Morphology. — The cells are oval and on agar in 24 hours .attain their full size of 4.5/tt in length and 3.5(1. in thickness. They stain uniformly with carbol fuchsin. Motility. — The yeast is not motile. Oram's stain. — The cells take the Gram's stain. Oxygen requirements. — Aerobic 26 THE BACTEEIA OF THE APIAEY. Bouillon.— This medium remains flour, with tbe formation of a friable white sediment. Reaction neutral. Glucose.— The closed arm remains clear. No gas is formed. Reaction acid. Lactose. — Reaction neutral. Saccharose. — Reaction neutral. Levulose. — Reaction neutral. Maltose. — Reaction neutral. Mannite. — Reaction neutral. Agar. — A white, nonspreading growth occurs. Serum.— ^yhite, moderate, nonviscid, nonspreading growth occurs along the surface inoculated. No liquefaction takes place. Potato water. — Reaction neutral. Potato. — Gray, luxuriant, fleshy growth occurs. Alilk. — No change occurs. Litmus milk. — No change occurs. Oelatim. — A moderate growth is formed, accompanied by no liquefaction. Acid agar. — Moderate growth takes place. Indol. — Negative. Nitrates. — Reduced to nitrites. Saccharomyces roseus. Occurrence. — Isolated from comb of healthy hive. Gelatin colonies. — Superficial colonies' are pink, convex, capitate, with lobate- lobulate margin ; when magnified, the deep colonies are irregular, brownish- yellow, and finely granular. Morpliology. — This cell is oval, attaining about 6.5,14 in length and 3.5/i in thickness. The cells stain uniformly. Motility. — No motility occurs. Grain's stain. — The cells are not decolorized by Gram's stain. Oxygen requirements. — Aerobic. Bouillon. — This medium remains clear, forming a pink, friable sediment. A pink band forms at the surface of the medium and adheres to the glass. Glucose.— ^The closed arm remains clear. No gas is formed. Reaction acid. Lactose. — Reaction neutral. Saccharose. — Reaction neutral. , Levulose. — Reaction slightly acid. Maltose. — Reaction slightly acid. Mannite. — Reaction neutral. Potato water. — Reaction acid. Glucose agar. — Luxuriant, red growth forms on the surface. Serum. — A pink, fleshy, nonspreading growth is formed, accompanied by no liquefaction. Potato. — ^A thick, nonspreading, red growth occurs. Millc. — No apparent change takes place. The milk coagulates on boiling. ■Litmus milk. — Reaction alkaline. Gelatin. — Moderate pink growth is formed, accompanied by no liquefaction. Aoid agar. — Slow growth occurs. Indol. — Negative. Nitrates. — Reduction to nitrites is positive. Saccharomyces G. Occurrence. — Found in the dead larvse of diseased adult bees. Morphology.— Thej appear in hanging-drop preparation in large clusters SACCHABOMYCES AND PUNGT. 27 stain uniformly witli carbol-fuclisin and are oval, nearly spherical, attaining (he length of i.ofi and thickness of 3.5/1. Gram's stain. — The cells are not decolorized by Gram's stain. Oxygen reqii iromeii ts. — Aerobic. BoHiUon. — A slight, friable, white sediment is formed, with a clear medium above. Reaction slightly acid. Glucose. — The medium in the closed ai-m remains practically clear and about one-fifth of the closed arm Is filled with gas. Reaction acid. Lactose. — Reaction neutral. Saccharose. — Reaction neutral. Levulqse. — Reaction slightly acid. Maltose. — Reaction slightly acid. ilannitc. — Reaction neutral. Potato water. — Reaction acid. Agar. — A moderate, white growth is formed. Serum. — Very feeble growth occurs, accompanied by no liquefaction. Potato. — A luxuriant, moist, white growth occurs. Milk. — No appreciable change taiies place. Litmus milk. — No appreciable change takes place. Gelatin. — A moderate, white growth occurs along needle tract and on the surface. No liquefaction results. Acid agar. — A feeble white growth occurs. Indol. — None could be demonstrated. Nitrates. — No reduction to nitrites occurs. Glucose agar. — A thick, white, fleshy growth occurs. 28 THE BACTEEIA OF THE APIABY. o c/j o a ■73 a; ^ C1 -tJ fcXI c t» n o bn C3 H M c o be 'lupui ■3:iu^TU o ; o;^ntM •3n![B3[IP nitH •^iinw •ojinod •asott^M •3S0inA8T ■asoj'BqDOiJg •aso:^OTiT ■Qsoonto •m\n •uoijo^pnbiT -3SO^[13IM • 3s'0tnA9T asoiBqao^g •asoonio •uias^O •ni:^'Bi30 •0ouaos9ion^j[ ■ivSy ■sisa -naSonioiiio ■jbSv •Qjmoj •^niK •pagjBqosrp '^t^ •uOTli3in3i300 •jvS^ piov UQ qiMOJQ •Tii^'G[ao •paj0[0osiq •aajgaq •jvSy •noiimoa; •sam -0[00 xipBpo ■jtpnoio 'aai3jqniapi ■papno ■ainns •a^iii-noiOQ •K^uamajmbaj uaSA-KQ •Q oL2 113 mAtoiQ •ajniBjadmai m ooj j-b q^Avoio ■sniBjg Aq niBig •saiodg •uoijisatuiiao •poi ni nopjsoj •jtliliJOM I + I I ■ ! ++ I I I I I I + :++^- I I + I ++ I + I+H I i ++ I :+ I + I + I I + :+H I + ; I I I + : + + III : ! ++ ; I + I +++ I I I I ; I ++ I I ++ + + I I ■ + + I ++ I I +H I I I I + : I + I +++ I I I I I + I I + :++ I I ++ I I I I I + I I + : I +++++ I +++ :++ I :++H ;++ I :+ I +++ I + I I I I ;++ + 1 I ++ : I + I +++ I + I I I II M ++ I ++ COMOKNC* + + + + + + I + I I :+++ + MM' I + I I I I + I M ++ 1^+^ I i +++++: I, ++ + f+++++-i-+.++ I i WMrH0100Q0r-00»QI^ldOiO J. J," •X) l-OOO i'V 'T"^ '^ '^ <^ (^ ^ ^ "^ v> -^ CO r* li lOioii H i-H i-H 1-1 C-l 1-H =^i2«§S o o mm o3 3 CJ O • ^ . o CJ o c a; m .; ai o d I) S a) a o o aa S £ d 03 J3Si O C3 BIBLIOGRAPHY TO PART I. 29 SUMMARY TO PART I. The results of the study of the bacteria found normally in the apiary may be briefly summarized as follows : (1) The temperature of the hive approximates that of warm- blooded animals. (2) Upon adult bees and upon the comb there occurs quite con- stantly a species of bacteria which we refer to in this paper as BaciUiis A, and which, it is believed, is the organism that some workers have confused with Bacillus alvei, the cause of European foul brood (p. 33). (3) There occurs very constantly in the pollen and intestine of adult bees a species here referred to as Bacillus B. (4) From the combs Bacterium cyaneus, Saccharomyces roseus, and a Micrococcus referred to here as Micrococcus C, have been iso- lated and studied. (5) Honey from a healthy hive is, as a rule, sterile. (6) The normal larvae are, as a rule, sterile. (7) There is an anaerobe found quite constantly in the intestine of the healthy honey bee. It is referred to in this paper as Bacterium D. (8) From the intestine there have been isolated and studied the following micro-organisms: Bacillus cloacw, Bacillus coli communis. Bacillus cholercp suis, Bacillus subgastricus. Bacterium mycoides, Pseudomonas fluorescens liquefaciens, and two referred to as Bacillus E, and Saccha/romyces F. Others less frequently present have been isolated, but not studied. (9) In two samples of brood with unknown disease there was found a species of yeast plant here referred to as Saccharomyces G. BIBLIOGRAPHY TO PART I. 1. Fuller, Geo. W., and Johnson, Geo. A. On the Differentiation and Distribu- tion of Water Bacteria. i\io of inoculation. June 30, 1916. Sept. 2, 1916.. Aug. 15, 1916. Sept. 7, 1915.. Julys, 1916.. Sept. 10, 1915, Julys, 1916... Aug. 23, 1915. Aug. 30, 1915. Period of putre- faction. Eosults of inoculation. Bays. 9 7 8 13 15 16 18 19 28 ISuropoan toulbrood produced. No disease produced. Do. Do Do Do. Do Do. Do Table XII. — Bac-illioi pluton in titc presence of putrefactive processes at room temperature Date of inoculation. Period of putrefac- tion. Results of inoculation. Aug. 4, 1914. July 17, 1915. July 5, 1916.. July 23, 1915. Aug. 14, 1914. Sept. 17, 1915 Aug. 25, 1916. Sept. 2, 1916. Aug. 3, 1915.. Aug. 28, 1916. Sept. 1,1914. Sept. 16, 1914 Aug. 12, 1916. Days. European foulbrood produced. Do. Do. Do. Do. Do. Do. Do. Do. No disease produced. Do. Do. Do. As shown by Tables XI and XII Bacillus pluton is destroyed in the presence of putrefactive processes. At incubator temperature it resisted the* effects of these processes for from 7 to 13 days and at room temperature for from 21 to 35 days. During August and September, 1916, preliminary experiments were made testing the resistance of Bacillus flvion to putrefaction at outdoor temperature. The parasite was alive and virulent after 40 days. The maximum period during which it will remain so has not been determined. VIABILITY OF BACILLUS PLUTON IN HONEY Honey suspensions of Bacillus pluton from the stomach contents of larvse sick or recently dead of European foulbrood were made and distributed in flasks each containing about 300 c. c. These were allowed to stand at room temperature shielded from the light. At intervals thereafter colonies fre6 from the disease were inoculated 24 BULLETIN 810, U. S. DEPAETMENT OF AGEICtJLTURE. each, with the contents of a single flask. A summary of the exi^eri- ments is contained in Table XIII : Table XIIL — Resistance of Bacilhis phiton in lionen at room temperature Bate of inoculation. Period in honey. Results of inoculation. May 22, 1915.. June 12,1915.. July 23, 1915.. June 25, 1915.. Aug. 23, 1915.. Aug. 3, 1915... July 12, 1915... Aug. 23, 1915.. Sept. 10, 1915.. Aug. 16, 1916.. May 19,1916.. May 4, 1915... June 7, 1913... June 13, 1913.. May 13, 1915... May 14, 1915.. May 22, 1915.. May 24, 1915.. July 31, 1916. - May 15, 1916.. Months. Days. 4 25 6 11 15 17 25 7 25 17 25 26 5 S 11 European foulbrood produced. Do. Do. Do. Do. Do. Do. Do. Do. Do. No disease produced. Do. Do. Do. Do. Do. Do. Do. Do. Do. Experimental evidence recorded in Table XIII shows that the virus of European foulbrood when suspended in honey at room tem- perature ceased to be virulent in from 3 to 7 months. VIABILITY OF BACILLUS PLUTON IN POLLEN Preliminary experiments were made to determine the viability of Bacillus pluton in pollen. Pollen is removed from brood-comb, and an aqueous suspension of the organism obtained from the stomachs of larvae sick or recently dead of the disease is added to it until a moderately thick, pastelike mass is obtained. This is distributed in Petri dishes and allowed to stand at room and refrigerator tempera- tures, respectively. After different intervals of time Jhe contents of a single dish, after being suspended in water, are added to about 300 c. c. of sirup and the suspension is fed to a colony, using the indirect method. The results show that Bacillus pluton was viru- lent after 7 months at room temperature and for more than 10 months in the refrigerator. The maximum period during which the organism will remain alive in these two environments has not been determined. RESISTANCE OF BACILLUS PLUTON TO CARBOLIC ACID Preliminary experiments were made to determine the effect of carbolic acid on the virus of European foulbrood. An aqueous sus- pension of the contents of the stomachs of larvte sick or dead of the disease is" first made. A measured quantity of this suspension is added to an equal quantity of an aqueous suspension of carbolic acid EUROPEAN rOULBKOOD. 25 of a strength twice that dewired in the experiment. After shaking, it is allowed to stand at room temperature. At intervals brood free from the disease is fed a bit of this suspension, using the direct method. Table XIV summarizes the experiments performed : Table XIV. — Effect of carbolic acid on Bacillus pluton Bate of inoculation. Aug. 22, 1914 Aug. M, 1914 Julys, 1915.. Aug. 21, 1914 Sept. 4, 1914. June 29, 1915 Julys, 1915.. June 29, 1915 Aug. 22, 1914 Aug. 14, 1914 Aug. 17, 1914 Aug. 25, 1914 Aug. 22, 1914 June 29, 1915 Strength of solution. Per cent, i 1 Period of sus- pension. Days. i 1 4 8 18 15 4 '5 '18 1 4 4 9 '5i Results of inoculation. European foulbrood produced. Do! Do. No disease produced. European ioulbrood produced. No disease produced. Do. Do. Do. Do. Do. Do. Do. 1 Hours. The experiments outlined in Table XIV show that Bacillus pluton withstood a one-half per cent solution of carbolic acid for 8 days but not for 18 days; that it withstood 1 per cent for 5 hours but not for 4 days ; and that it was destroyed by 2 and 4 per cent solu- tions, respectively, in less than 6 hours. Probably it is destroyed by these latter strengths in considerably- less time than this. It is seen by these preliminary experiments that Bacillus pluton is destroyed easily by carbolic acid as a disinfectant. As a drug, however, less can be expected of it, inasmuch as a strength twice that which the bees will accept in honey (Table XV) requires days to destroy the germ. While the fact does not furnish conclusive proof of the value of carbolic acid as a drug, it indicates what might be expected of it in the treatment of the disease. In using the results recorded on the foregoing pages for the purpose of destroying the virus of European foulbrood and con- trolling the disease in practical apiculture, it must be borne in mind, as has been urged in the discussions on the other bee diseases, that due allowance must be made by the beekeeper for variations which always occur. These, however, are relatively slight and can lye met readily. In the destruction of the virus through heating, for example, the temperature can be raised a few degrees above that which is found to be the minimum required, or the time can be extended somewhat. Similarly for the other destructive agencies the effectiveness of the process can be increased. 26 BULLETIN 810, U. S. DEPARTMENT OF AGRICULTUEB. EFFECT OF DRUGS ON EUROPEAN FOULBROOD rreliminary experiments have been made to obtain data relative to the effect of drugs on Bacilhis pinion. In conducting the experi- ments a suspension of the stomach contents of larvae sick or recently dead of European f oulbrood is made in an aqueous solution of the drug. This is added to diluted honey and healthy brood is fed this sus- pension. In some instances the direct and in others the indirect method was followed. In Table XY are summarized the experiments which were performed : Table XV. — The effect of drugs on European foulbrood Date of experiment. July 11.. May 31.. June T.- July 11. . June 21. Do.. May 31.. July 11.. June 7-- July 11. . Do.. May 31. . June 7. . July 11.. May 31.. June 7. - July 11. . May 31.. June 7. . Drugs. Betanaphthol. -...do do Carbolic acid. do do Oil of eucalyptus. . do Formic acid do Salicylic acid .do. do. Salol... ..-.do. .do. Quinin. . do.. do-. Strength. 1:2000 1:1000 2:1000 1:2000 1:1000 2:1000 4:1000 4:1000 1:1000 3:1000 1:2000 1:1000 2:1000 1:2000 1:1000 2:1000 2:1000 4:1000 10:1000 Hesults of inoculation. European foulbrood produced. It will be observed from Table X^' that European foulbrood was produced in all cases in which larvae were fed a suspension of Bacillus pluton in sirup medicated with betanaphthol, carbolic acid, eucalyptus, formic acid, salicylic acid, salol, and quinin (bisulphate of quinin) , respectively, in the proportions noted. The strongest solutions of the drugs used in the experiments are in most instances approximately the maximum proportion of the chemical in honey that will be taken by the bees. These prelimi- nary results indicate that drugs should not be depended upon, for the present at least, in the treatment of European foulbrood, and emphasize the fact that beekeepers should make sure that the value of a drug has been demonstrated fully before it is used. TRANSMISSION OF EUROPEAN FOULBROOD ^Vhile there is yet much to be learned concerning the transmission of European foulbrood, the data at hand relative to this important phase in the study of the disease justify certain statements in regard to it. The disease can be produced experimentally by feeding a healthy colony the crushed larva sick or dead of the disease, sug- gesting that infection takes place by way of the alimentary tract. ^EnjRDPE7nr~F0ULBR00D. 27 Through the study of microtome sections of such larvae, it has been conclusively proved that infection takes place in this way. The fact is naturally one of special moment in the solution of the transmission of the disease. There is a tendency on the part of adult bees to remove sick and dead larvffi from the brood comb. This is done largely at least in a piecemeal manner. Were the fate of the frag- ments removed known definitely the solution of the problem natu- rally would be aided greatly. If infective material thus removed were fed to susceptible healthy larvae, disease would result. On the other hand should the fragments of diseased larvae be stored with the honey of the hive or with the pollen, or consumed by the adult bees, or by larvae later in the feeding stage, the chances that such material would ever reach susceptible larvae to cause infection are very much re- duced. Stored in honey the virus remains virulent only a few months (p. 24) ; in pollen, however, it remains virulent much longer (p. 24). Drying within the hive Bacillus pluton would probably remain alive more than a year (p. 19). The chances that any portion of the infectious material of any given fragment, if it is removed entirely from the hive by the bees of the colony, and released from them, will be taken up by other bees and carried to healthy brood and cause infection are compara- tively slight. If thus removed and exposed to the direct rays of the sun, the virus will be destroyed within a few hours (p. 19) ; or if subjected to fermentative or putrefactive processes it will be destroyed in a few weeks (p. 23). If BaeilJms pluton is present in honey extracted from diseased colonies it will be destroyed within a few months while in storage (p. 24). It is seen, therefore, that in nature there are many means that destroy the virus of European foulbrood and thus limit the spread of the disease. All of the colonies of the experimental apiary used in making the inoculations cited in the present paper had free access to the fields and there was no evidence at anj- time of the transmission of the disease from infected to healthy colonies. This fact supports the conclusion that the disease is not spread by way of flowers visited by bees from healthy colonies which had been visited previously by bees from diseased ones. The fact further indicates that if the dis- ease is transmitted at all by way of the water supply of the bees, it takes place to a limited extent only. The fact still further indicates that if drones or straying or drifting workers transmit European foulbrood they do so to a slight extent only. If these observations are at variance with the experience of the practical beekeepers, as the writer has been informed that they are, they will probably be of particular interest. 28 BULLETIN 810, p. S. DEPARTMENT OF AGKICULTURE. Observations made during the present studies indicate that queens from European foulbrood colonies are not likely to transmit the disease when introduced into healthy colonies. The experiences fur- ther show, and the facts in general regarding the disease support the conclusions, that the infection will not be transmitted by the hands or clothing of the beekeeper, or by visitors to the apiary when the manipulations ordinarily practiced are followed. Tools and equip- ment used about the apiary are not to be feared unless they supply a source for robbing. Hives which have housed infected colonies are not likely to be a medium for the spread of the disease. Eobbing of infected colonies is the most fruitful source of infec- tion. A colony weakened by disease (p. 5) becomes a prey for other bees. Infectious material is carried to other colonies, thereby trans- mitting the infection. Manipulations in the apiary, whereby brood combs from diseased colonies are placed in healthy ones, are another fruitful source for the transmission of the disease. Preliminary Avork^ indicates that stored brood combs from European foulbrood colonies may transmit the disease after a considerable period. The disease, it would seem, might be spread through the medium of honey from infected colonies. The danger from this source, how- ever, probably has been overestimated at times (p. 23). That pollen stored in the comb would serve as a protection to Bacillus fluton, if the parasite were lodged with it, has been determined (p. 24). DIAGNOSIS The diagnosis of European foulbrood offers more difficulty than does that of either American foulbrood or sacbrood. It can usually be made, however, from the symptoms alone. Inasmuch as these symptoms (p. 4) are rather varied, much care should be exercised in diagnosing the disease. The appearance of the adult bees does not aid in the diagnosis. A weak colony should arouse suspicion. Increased suspicion is jus- tified when no other readily discernible cause for the weakness is to be observed. The disease may be present, however, in a strong colony. Such a case may be one of recent infection or one which late in the recovery from the disease has gained in strength. It may be, how- ever, a colony which has suffered only a slight attack of the disease. The following outstanding gross characters are often sufficient for a diagnosis : The dying of the brood before the time for capping (Pis. 1 Brood combs were removed from European foulbrood colonies in October, 1914, and stored in the laboratory. In May, 1915, one frame of brood comb was placed In each of two colonies with the result that European foulbrood was produced In both, instances. When a frame of the comb was placed In the colony in May, 1916, no disease resulted. After 6 months tjie combs were still able to transmit the disease ; after 18 months they did not. These experiments are not sufficient to Justify definite conclusions but are suggestive. EUROPEAN rOULBROOD. 29 II, III, IV), the yellow hue of the larvae more recently dead, and the brown shade of those longer dead, the irregidarity of the brood (PL I), and the absence of a disagreeable odor. Not infrequently, however, the diagnosis is not so simple. During recovery from the disease scales (PI. V, F, I) of larvae dying in capped cells may be the only remains of diseased brood to be found, all of the younger larvaj having been removed by the bees. These scales^ are, as a rule, comparatively few in number and resemble somwhat those of American foulbrood, but would rarely be mistaken for those of sacbrood. In these cases a diagnosis can be made fre- quently by a microscopic examination alone. Cultures, however, are needed in some instances. Special attention is needed in cases of early infection and in other instances where only a small amount of diseased brood in uncapped cells is present (PI. I, A). The symptoms manifested by larva sick or only recently dead of the disease furnish often the readiest and most conclusive evidence of the presence of the disease. Larvae of the age at which they comfortably fill the bottom of the cell exhibit- ing increased peristalsis-like movements of the body suggest European foulbrood. Increased transparency of larvae of this age (PI. II, B) is also suggestive. The presence of a white or yellowish-white mass within the stomach (midgut) as seen through the dorsal median line of the body is strong evidence of the presence of the disease. If on puncturing the body of larvae nearly dead or only recently dead the contents of the stomach flows out as a fluid and more or less finely granular mass, the fact furnishes further evidence of European foul- brood. A symptom which is pathognomonic of the disease is to be seen in larvae that have been infected somewhat more than two days, but wherein the disease has not reached an advanced stage. The test (15) involves the removal of the stomach contents, which con- sist of a bacterial mass, together with a small amount of larval food and a clear envelope (PI. VIII, a, b, c). The slight tension necessary to remove the contents stretches the envelope and breaks the whitish bacterial mass into a number of fragments. 1 The number of larrse that die of European foulbrood In capped cells after assuming the endwise position represents a very small percentage of the brood that dies of the disease. These remains may be found in practically all colonies in which the disease has been present for a suflaclently long period and in which a considerable amount of dead brood has resulted. Before becoming dry they are somewhat viscid and are less easily removed than are those of larvae dying at an earlier age. These and the scales resulting from them are used In diagnosis principally (1) when the younger larvse sick or dead of the disease have been removed, (2) when a demonstration of the presence of Bacillus alvei Is desired, and (3) when both European foulbrood and American foulbrood infection is suspected. Such a double infection has been encountered in the writer's experience very rarely. In making diagnoses, therefore, after European foulbrood has been found in the sample American foulbrood Is seldom looked for. 30 BUIXETIN 810, U. S. DEPARTMENT OF AGRICULTURE. By one or more of these colony symptoms manifested by larvae sick or only recently dead of the disease the experienced can diagnose European foulbrood definitely without a microscopic examination. The methods not only give definite results, but are also easy of application. They have been indispensable in much of the writer's experimental work and it is believed that the beekeeper will find them to be valuable in practical apiculture where other gross meth- ods fail. BACTERIOLOGICAL EXAMINATION The findings from microscopic examinations and from cultures liave been set forth in an earlier publication (10). These are always adequate for a definite diagnosis when a suitable sample is at hand. Baoillus alvei (p. 11) (fig. 2; PI. VII, D, F) frequently overshadows all other species. In larvje sick of the disease Bacilkis pluion (PL Yll, A, B) overshadows all others. With experience one learns to recognize this species in stained preparations. The individuals are seen frequently in groups. They are more or less lancet shaped, and a variation in size is often sufficient to be noticeable (fig. 1).^ In larvae nearly dead and in those only recently dead Bacterium, eurydice (p. 13) (fig. 4; PI. VII, C) is frequently encoimtered. Streptococ- cus apis (p. 12) (fig. 3; PI. VII, E) occurs in a small number of cases. Bacillus orpheus (p. 14) (fig. 5; PI. VII, H), B. vulgatvis, and B. mesentencus are occasionally encountered. While B. pluton is present in all cases of European foulbrood, not infrequently in routine examinations it is so masked by the secondary invaders that the microscopic examination fails to reveal it. In many cases B. alvei and B. orpheus are recognized microscopically. Cultures are necessary for the differentiation of B. vulgatus and B. mesentericus. In many cases cultures are needed to differentiate Strep, apis and B. pluton. Strep, apis grows on the ordinary media, B. pluton does not. DIFFERENTIAL DIAGNOSIS AMEMOAN rOUIBEOOD American foulbrood is recognized by the death of larvae in capped cells and of pupse soon after transformation, the viscidity of the decay- ing remains of the brood, and the " foulbrood " odor which is fre- quently present. The presence of the spores of Bacillus larvae in large numbers and the absence of other species is conclusive proof of American foulbrood. 1 Smears made from laryse sick of European foulbrood and quite early In the course of tlie disease were selected in malsing a study of the morphology of B. pluten. These were stained with iron hematoxylin. In smears made from dead larrse and stained with carhol fuchsin, as is usually done, the pointed ends and the more or less rod-shaped forms are less prominent than illustrated in figure 1. EUROPEAN FOULBROOD. 31 S&CBBOOn Sacbrood is recognized by the death of larvae after capping, by the saclike appearance, the watery granular consistency of the larval remains, and the absence of viscidity. The absence of microorgan- isms characterizes the microscopic picture in sacbrood. OTHER CONDITIONS Conditions referred to as chilled brood, overheated brood, and starved brood must be differentiated from European foulbrood. This can usually be done with little diiRculty by a comparison of the symp- toms present with those of European foulbrood. The history of the case is of much value. Brood dying after being removed from the hive and before examination is made shows often an interest- ing similarity to European foulbrood. B. alvei and B. flmton are not found in these conditions. The absence of bacteria, or their presence in small numbers only, and a lack of uniformity of the species when present, characterize the bacteriological findings in these cases. PROGNOSIS There is no uniformity in the prognosis in European foulbrood. The diseased colony may recover completely from the infection, suf- fering only a slight loss in strength as a result of it ; the colony may recover but sustain considerable loss ; or it may die out entirely, as a result of the disease. The infection may spread only slightly to other colonies of the apiary or the entire apiary may become infected. The losses sustained vary from slight to total. The tendency for Euro- pean foulbrood to disappear is greater after midsummer than before. Whether a larva once infected ever recovers from this disease is not known, but the evidence at hand indicates that it may. This seems to be especially probable when the infection takes place during the latter part of the feeding period of the larva. Queen larvse are susceptible to infection, but sufficient data are wanting from which to estimate the extent to which queenlessness may result from the disease. In experimental colonies queens have been reared in the presence of a considerable amount of European foulbrood infection. The prognosis for the colony in the case of European foulbrood may be said, therefore, to vary from very good to very grave, many recovering entirely from the infection without treatment and without appreciable losses, while others rapidly decline and finally die out. SUMMARY AND CONCLUSIONS The following is a brief summary of facts regarding European foulbrood, together with some conclusions based upon them: 1. European foulbrood is an infectious brood disease of bees caused by Bacillus pluton. 32 BULLETIN 810, U. S. DEPARTMENT OF AGKICtTLTURE. 2. All larvae — worker, drone, and queen — are susceptible to the dis- ease; adult bees are not. 3. Man evidently is not susceptible to infection with Bacillus pluton nor are the experimental animals. 4. As far as is known insects other than bees are not susceptible. 6. Brood can be infected by feeding the colony a suspension of crushed larvae sick or dead of the disease. This is described in the present paper as the indirect method. 6. The virus contained in a single larva recently dead of European foulbrood will produce a considerable amount of disease when fed to a colony. 7. The larvae can be infected also by a more direct method. A fraction of a drop of a suspension of the stomach contents of a larva sick of the disease, added with a capillary pipette directly to the food surrounding the larva to be inoculated will result in infection. 8. BaciUus pluton gains entrance to the larva by way of the mouth. The growth and multiplication of the parasite take place within the stomach (mid-intestine) of the larva and do not, during the life of the larva, get beyond the peritrophic mem- brane. The tissues, therefore, are not invaded by it. 9. The secondary 'invaders in European foulbrood. Bacillus alvei, Streptococcus apis, Bacterium, eurydice, and Bacillus orpheus, rarely, if ever, invade the tissues until the larva is dead or nearly so. In a few instances in microtome sections rod fonns have been encountered in the act of invading the tissues of living larvae. The species, however, was not determined defi- nitely. 10. The period of incubation is slightly less than 3 days. 11. Brood is susceptible to infection at all seasons of the year. 12. More brood die of the disease during the first half of the brood- rearing season than during the second half. 13. The writer has examined samples of the disease from Canada and the United States. From written reports it seems quite certain that it occurs also at least in Denmark, England, Ger- many, France, and Switzerland. 14. Occurring as it does in this somewhat wide range of climatic conditions, the presence of the disease in any particular locality can not be attributed entirely to the prevailing climatic con- ditions. 15. The quality of food obtained by the bees does not affect greatly, if at all, the course of the disease in the colony, although the quantity may affect it to a variable extent. EUROPEAN FOULBEOOD. 33 16. Experimental colonies may be inoculated and kept in the apiary without transmitting the disease to others. This fact is of special importance, not only in connection with the technique of making studies on the disease, but also in the control of the malady. 17. The thermal death point of Baoillm pluton suspended in water is approximately 63° C. maintained for 10 minutes. 18. ^Vhen suspended in honey Baoillus pluton is destroyed in 10 minutes at approximately 79° C. 19. Drying at room or incubator temperature Bacillus phiton re- mains alive and virulent for approximately one year. 20. Wlien dry, Bacillus pluton resisted the direct rays of the sun for from 21 to 31 hours. 21. When suspended in water Bacillus pluton was destroyed by the direct rays of the sun in from 5 to 6 hours. 22. "When suspended in honey and exposed to the direct rays of the sun Bacillus pluton was destroyed in from 3 to 4 hours. 23. In the presence of fermentative processes in a 10 per cent sugar solution Bacillus plvion was destroyed in from 3 to 6 days at incubator temperature and in from 11 to 21 days at room temperature. 2-1. In a fermenting honey solution outdoors Bacillus pluton was still alive and virulent after one month. 25. In the presence of putrefactive processes at incubator tempera- ture Bacillus pluton was destroyed in from 7 to 13 days and at room temperature in from 21 to 35 days. 26. In a putrefying medium at outdoor temperature Bacillus pluton remained alive and virulent for more than 40 days. The maxi- mimi period has not been determined. 27. In honey at room temperature Bacillus pluton ceased to be viru- lent in from 3 to 7 months. 28. Mixed with pollen. Bacillus pluton remained alive and virulent for more than 7 months at room temperature and more than 10 months at refrigerator temperature, the maximum time not being determined. 29. In one-half per cent carbolic acid solution Bacillus pluton was destroyed in from 8 to 18 days ; in 1 per cent it was destroyed in from 5 hours to 4 days, and in 2 and 4 per cent ip less than 6 hours. The probability is that at these higher strengths of the solution minutes rather than hours are sufficient for the destruction of the virus. 30. Experimental evidence indicates that at the present time drugs should not be depended upon in the treatment of European foulbrood. 34 BULLETIN 810, U. S. DEPARTMENT OF AGEICXJLTUKE. 31. Robbing from diseased colonies of the apiary or from neigh- boring apiaries is the most likely manner in which European foulbrood is transmitted in nature. 32. Brood-combs containing diseased brood, if given to a healthy colony, serve as a medium for the transmission of the disease. 33. European foulbrood is not likely to be transmitted by queens or drones. Whether they ever do so has not been demonstrated. 34. As a rule a hive which has housed a European foulbrood colony should not be considered as a fruitful source of infection. The facts indicate that often such hives could be used with im- punity for housing colonies without treatment. Flaming them inside certainly removes all danger. 35. The transmission of European foulbrood by way of flowers, visited by bees from diseased colonies and subsequently by those from healthy ones, is not to be considered as a likely source of infection. Whether the water supply is ever a source of danger is not known. It is evidently not a fruitful source. 36. The disease is not likely to be transmitted through the medium of the clothing or hands of the apiarist. 37. Tools and bee supplies in general do not serve as means for the transmission of the disease in the absence of robbing from such sources. 38. It is usually possible to diagnose European foulbrood from the symptoms alone. A definite diagnosis can be made from suit- able samples by bacteriological methods. 39. The prognosis in European foulbrood varies from very good to exceedingly grave. The tendency for a colony to recover en- tirely from the disease is much greater than in American foulbrood. 40. Considered from the technical point of view, much is yet to be learned concerning European foulbrood. For practical pur- poses, however, it can be said that sufficient knowledge has been gained to make it possible for the beekeeper to devise a treatment which will be logical, efficient, and at the same time economical. LITERATURE CITED (1) Bahb, Louis. 1904. "Vore bisygdomme. Foredrag holdt ved DBF's Diskusslonsm0de i Grejsdalen den 11. Septbr. 1904. (Efter Foredragsholderens manuskript.) RoskUde [1904] 17 p. ( Saertryk af " Tldsskrif t for Biavl," Nr. 16 og 17, 1904. (2) 1915. Sygdomme hos Honningbien og dens Yngel. Meddelelser fra den Kgl. Veterinser-og Landboh^jskoles Serumlaboratorinm XXXVII. 109 p., 11 fig. EUROPEAN FOULBROOD. 35 (3) Btjkri, R. 1906. Bacteriologlsche Untersuchungen fiber die Faulbrut und Saw-r- brut der Blenen. 39 p., 1 pi. Vorwort von U. Kramer, January. (4) Cheshihe, F. R., and Cheyne, W. W. 1885. The pathogenic history and history under cultivation of a new bacillus (B. alvel), the cause of a disease of the hive bee hitherto known as foul brood. In Jour. Roy. Micros. Soe. Ser. II, vol. V, pt. 2, p. 581-601, pi. X-XI, August. (5) Ford, W. W., Laubach,.C. A., Lawrence, J. S., and Rice, J. L. 1916. Studies on aerobic spore-bearing non-pathogenic bacteria. Pt. II. In Jour, of Bact.. vol. I, No. .5, p. 493-533, 15 pis. September. (6) Howard, W. R. 1900. New York bee disease, or black brood. In Gleanings in Bee Culture, V. 28, no. 4, p. 121-127, February 15. (7) Maassen, Aisert. 1907. t'ber die sogenannte Faulbrut der Honigbienen. Mitteilungen aus der Kaiserlichen blologischen Anstalt fiir Land- und Forst- wirtschaft, Hft. 4, p. 51-53, 6 fig. February. (8) 1908. Vber die unter der Namen " Faulbrut " bekannten seuchenhaften Bruterkrankungen der Honigbiene. IMittellungen aus der Kaiserlichen konlgliehen Anstalt fiir Land- und Forstwirtschaft, Hft. 7, 24 p., 4 pi. September. (9) McCrat, a. H. 1917. Spore-forming bacteria of the apiary. In V. S. Dept. Agr. Jour. of Agr. Research, v. 8, no. 11, p. 399-^20, 6 fig., pi. 93-94, March 12. (10) McCrat, A. H., and White, G. F. 1918. The diagnosis of bee diseases by laboratory methods. U. S. Dept. Agr. Bui. 671. 15 p., 2 pi. June 21. (11) Moore, Veranus A., and White, G. F. 1903. A preliminary investigation into the cause of the infectious bee diseases prevailing in the State of New York. In N. Y. [State] Dept. Agr. 10th Ann. Rept. Com. Agr. for 1902, p. 255-260, 2 pi. January 15. (12) Muck, Oswald. 1915. Seuchen der Bienenbrut. In Wiener tierarztlichen Monatsschrift, Jahrg. 11, Hft. 3, p. 124-139, 6 fig., 2 pi. (13) White, G. F. 1906. The bacteria of the apiary, with special reference to bee dis- eases. XJ. S. Dept. Agr. Bur. Ent. Tech. Ser. no. 14. 50 p. (14) 1908. Miscellaneous papers on apiculture. The relation of the etiology (cause) of bee diseases to the treatment. XJ. S. Dept. Agr. Bur. Ent. Bui. 75, pt. 4, p. 33-42. December 26. (15) 1912. The cause of European foulbrood. U. S. Dept. Agr. Bur. Ent. Giro. 157. 15 p., 10 fig. May 10. (16) 1914. Destruction of germs of infectious bee diseases by heating. U. S. Dept. Agr. Bui. 92. 8 p. May 15. 36 EVLLETIN 810, U. S. DEPARTMENT OF AGRICULTURE. (17) White, G. F. — Continued. 1917. Sacbrood. U. S. Dept. Agr. Bui. 431. 55 p., 33 fig., 4 pi. Feb- ruary 9. (18) 1918. Nosema-dlsease. U. S. Dept. Agr. Bui. 780. 59 p., 7 fig., 4 pi. (Professional paper.) June 12. (19) 1920. American foulbrood. U. S. Dept. Agr. Bui. 809. 46 p., 9 fig., 8 pi. March 10, 1920. (20) Zandee, Enoch. 1910. Die Faulbrut und ihre Bekampfung. 32 p., 8 fig., 4 pi. Stutt- gart. (Handbueh der Bienenkunde I.) EXPLANATION OF PLATES Plate I Brood-combs containing larvse that are sick and others that are dead of Euro- pean foulbrood, showing the Irregular appearance of the brood. About one-half natural size. A. — The dead larv£e have all been removed. Some of the remaining larvse are sick, others are not infected. The disease was produced by experimental inoculation. B. — Many of the dead larvce have not been removed. The comb had been out of the colony for a considerable period. The larvae that are quite young showing abnormal position and appearance are not sick or dead of European foulbrood, but are so as a result of the comb being away from the colony. Disease was produced by experimental Inoculation. C. — ^The comb was taken from a colony in which the disease had appeared in nature and not as the result of artificial inoculation. Before being photo- graphed the brood-comb had been out of the hive for a few days. Aside from the larvse which are dead of European foulbrood, other larvse present are dead from lack of attention by adult bees — starvation, exposure, and other causes. Plate II A. — Live larva showing first symptoms of European foulbrood. The tur- gidity is slightly less than in a healthy larva (D). B. — ^Live larva showing early symptoms of European foulbrood. The body is more transparent than that of a healthy larva (D). Small opaque areas give it a punctate appearance. C. — ^Larva dead of European foulbrood contained within a chitinous envelope iilled with a watery-appearing fluid. D. — ^Healthy larva of the earliest age at which larvse die of European foul- brood. Turgidity marked. E. — European foulbrood larva which may or may not be dead. Surface less glistening than in healthy larvse. Marked turgidity lost. Prominence of tracheae not increased. P. — Scale formed by drying of larvae dead at early age. Prominence of tracheae marked. G. — View of healthy larva in normal position with roof of cell removed. Larva turgid. Surface glistening. H. — ^Larva sick with European foulbrood. Lack of turgidity and increased prominence of tracheae observed. I. — European foulbrood larva which may or may not be dead. Less turgidity, a relative dullness in the surface appearance, and punctate condition present. Similar to B. Plate III A. — Healthy lai;va immediately preceding the age at which the capping of the cell is done. Dorsal surface turned toward the observer. The narrow trans- parent area along the dorsal median line is prominent. B. — ^Larva dead of European foulbrood of the same age as A. The turgidity, glistening surface, and transparent area are less marked. 0. — ^Larva dead of European foulbrood partly coiled and partly endwise in cell. 37 38 BULLETIN 810, U. S. DEPAETMENT OF AGRICtrLTTJRE. D. — ^Healthy larva near the age at which capping takes place. E.— Dorsolateral view of a larva dead of European foulbrood. The ends are directed toward the bottom of the cell. F.— Larva dead of European foulbrood. The body occupies a spiral position in the cell. G.— Healthy larva approaching the age at which capping takes place. H. — Lateral view of larva dead of European foulbrood seen with the roof of the cell removed. The ends are directed toward the bottom and the dorsal surface toward the mouth of the cell. L — Dead larva similar to H but having been dead somewhat longer. Plate IV A. — ^Toung larva dead of European foulbrood. The chitinous capsule and tracheae are prominent. B. — Fragments of young larva dead of European foulbrood, a portion having l)een removed by adult bees after its death. C. — Lateral view of larva dead of European foulbrood, the roof of the cell having been removed. The ends in this instance are directed more or less to- ward the mouth of the cell. D. — Lateroventral view of larva dead of European foulbrood. The body lies with the dorsal portion against the floor of the cell. E. — Larva dead of European foulbrood lying on the floor of the cell in some- what lengthwise position. F. — Scale of European foulbrood larva which had occupied a somewhat spiral position in the cell. G. — Scale of a European foulbrood larva which had occupied a position some- what as shown in D. This scale and the one shown in F can be removed intact rather easily and without tearing the waU of the cell. Plate V Larvaa (prepupse) of bees dead of European foulbrood which had already assumed before death a lengthwise position in the cell. A. — Fragment of European foulbrood soon after death. A portion of the larva has been removed by the adult bee. B. — Entire cap of ceU containing larva dead of European foulbrood. C. — Punctured cap of cell containing the remains of a larva dead of Euro- pean foulbrood. D. — End view of larva dead of European foulbrood. E. — End view of larva dead of European foulbrood, lying with its dorsal surface against the floor of the cell. Considerable drying of the remains has taken place. F. — End view of scale of European foulbrood larva which had reached be- fore death the age at which the endwise position in the cell is assumed. G. — Ventral view of European foulbrood larva. Stage similar to D. Turgid- ity is lost to a large extent and the segmented markings are less distinct than in healthy larvae. H.— Larva which has been dead of European foulbrood for a longer period than illustrated in G. The ridge and furrows indicating the segments of the body are not marked. I.— Scale of European foulbrood similar to F. The larva before death had reached the endwise position in the cell. These scales resemble very much those of American foulbrood. They are more easily removed, however, do not adhere so closely to the floor of the cell, and are more rubberlike in 'con- sistency, breaking less readily than those of American foulbrood. EUROPEAN POULBROOD. 39 Plate VI A view of the exptMiiuental tiiiinry of 54 colonies iu which tlie inoculation ex- periments made during the sunnner of 1915 were conducted. Plate VII Photomicrosniphs illustrutinn the wore commonly encountered bacteria in European foulbrood. A. — Bacillus phuton: A smear from tlie siomacli of a larva sick with Euro- pean foulbrood. Note the paired forms and short chains. These forms are numerous In a recent infection, suggesting the organism in the process of mul- tiplication. The lancet-shaped form is by far the predominant one in all later stages of the disease. X 1000. B. — Bacillus phiton: A smear from a larva quite recently infected. The multiplying paired forms are at this stage present almost exclusively. X 1000. C. — Bacterium curydkc: Stained preparation from a pure culture on the surface of agar. X 1000. D. — Bacillus alvci: Stained preparation showing spores and spore forma- tion. X 800. E. — Streptococcus apis: Stained preparation from a pure culture. X 800. P. — Ba-cillus alrci: The peculiar arrangement of the spores as sometime.s seen. From a pure culture, the smear having been made by suspending the culture on the slide in normal salt solution. X 1000. G. — Bacillus orpheus: Stained preparation made from a pure culture only a few hours old. Grown on the surface of agar. X 1000. H. — Bacillus orpheus: Stained preparation showing spore formation. Note the stained portion along one side and about both ends of the spore. The stage is soon reached in a culture at incubator temperature. At room tempera- ture it remains in this stage for a considerable period. X 800. I. — Longisection of a young larva showing early infection in European foulbrood. The bacterial growth is seen as a narrow black area just within the peritrophic membrane on one side of the food mass. J. — ^Longisection of larva sick of European foulbrood, showing a later stage of infection than that present in I. The dark area in the food mass shows the bacterial growth. Note that the growth mass does not extend beyond the peritrophic membrane and that it does not extend uniformly along this membrane and throughout the food mass. K. — Transverse section of larva about the time of its death from European foulbrood infection. Note the bacterial mass along the peritrophic mem- brane and extending from the membrane into the food mass. As seen within the living larva this bacterial mass in the sick larva is practically white, but is more or less yellowish white when present with larval food material. The gelatinous-like envelope outside the peritrophic membrane and inside the stom- ach epithelium in healthy larvje thins out as the disease advances. Plate Vni The stomach contents of larviB sick of European foulbrood removed from the organ. The anterior end of the larva is shown. Fairly early stage of infection (a) showing the white bacterial mass broken into fragments as a result of the tension produced in removing the stomach contents from the organ. A somewhat later stage (b) in the course of the disease, show- ing the bacterial growth contained in the stomach fragmented, also the mucous or gelatinous envelope surrounding the petritrophic membrane. The stomach contents removed from a European foulbrood larva (c) about the time of its death. The bacterial growth at this time is surrounded by very little other than the peritrophic membrane. When this membrane is ruptured the contents flow out as a thin yellowish-white mass. • Plate I. fill ^ istfai«fyjigg&^ v't-v-.>"^V : -^ - cr-'^:-^- T:.-cr«r-' i^ i yiS^i ^Si^^^^rT^jE^^j^^HlHl^li^na^Si MiffliPi^EMi HkJflSU<^JMKr jB^H European Foulbrood. Bui. 810, U. S. Dept. of Agriculture. A B Plate II. D H European Foulbrood. Bui, 810, U. S. Dept. of Agriculture. PLATE III. B D European Foulbrood. Bui. 810, U. S. Dept. of Agriculture. PLATE IV. European Foulbrood. Bui. 810, U. S. Dept. of Agriculture. PLATE V. H European Foulbrood. Bui. 810, U. S. Dept. of Agriculture. Plate VI. Bui, 810, U. S. Dept. of Agriculture. PLATE VII. European Foulbrooo. Bui. 810, U. S. Dept. of Agriculture. PLATE VIII, European Foulbrood. UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 431 Oonlrtbntlon IVom the Bureau of Entomologr t. O. HOWARD. CUef Washington, D. C. PROFESSIONAL PAPER Febraary 9, 1917 SACBROGD G. F.WHITE Expert, Engaged in the Investigation of Bee Diseases CONTENTS Blitoiical Aceouit ..•....•. Nwne of the Disease AReennce of Healthy Breed at the Ace MlindchitDleBefSacbnMd . . . . ItoaiBof Sacbrood ....... e«rSacbroad . I Effect of Sacbrood Upon a i&luinnt of Tiros Bevdred to Produce ■ )ie Dbease. and the Bapidtty of its in Making Ezperimentai Page I 2 4 6 10 24 SO 31 _ _ 32 sDeMmefienof the Tims of _-^ to Destroy Sacbrood iSnapended In Water . . 34 IjMuired to Destroy Saclwoad "^"■Saqeaded in Glycerine . 35 ^ aired to Itestroy Sacbrood TlQuinien Suspended in Honey . . 36 Rcdstsnee of Sacbrood Tins to Oifiac atBoantltaBpantua. ... ._. . . 37 Bcststaoo) of Sacbrood Ttrha to Direct Snnllrin When Dry ........ BeslBtanee of Sacbrood Tims to Direct Sunlight When Suspended in Water . BeslBtanee of Sacbrood Tirni to Direct Sunlight When Sumended in Honey . length of Time that Sacbrood Tiroa Be> mains Tiralent in Honey Besistance of SaCbrood Tims to thePraS' eilceofPennentatiTePiocesaea . . . Resistance of Sacbrood Tints to Fer- mentation in Diluted Honey at Out. door Temperature . . . '. . . . . Besistance of Sacbrood Tims to the Frea. enceof Pnirefacttire ProcesseO . •„. Resistance of Sacbrood Tims to Carboue Acld^. . . . . . Modes of Transmission of Sacbrood . . Dhigno^ of Sacbrood Prognosis .......••.•. Belation ofTfaese Studies to the Treat* Page 38 otSacMtiod . . . Coachisfonji. Summary and . UtetatuM Cited 39 40 40 41 42 43 44 46 48 49 80 82 63 WASHINGTON GOVERNMENT PBtNTINa OFilO 1917 UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 431 Contribution from the Bureau of Entomology L. O. HOWARD, Chief Washington, D. C. PROFESSIONAL PAPER February 9, 1917 SACBROOD. By G. P. White, Expert, Engaged in the Investigation of Bee Diseases. CONTENTS. Introduction 1 Historical aoootmt 2 Name of the disease * Appearanceof healthy brood attheageat which it dies of sacbrood 6 Symptoms of sacbrood 10 Cause of sacbrood 24 Weakening effect of sacbrood ujMn a colony. . . 30 Amount of virus required to produce the dis- ease, and the rapidity of its increase 31 Methods used in maldng experimental inocula- tions 32 Means for the destruction of the virus of sac- brood 34 Heating required to destroy sacbrood virus when suspended in water 34 Heating required to destroy sacbrood virus when suspended in glycerine 35 Heating required to destroy sacbrood virus when suspended in honey 36 Kesistance of sacbrood virus to drying at robm temperature ■ ''' Resistance of sacbrood virus to direct sunlight when dry 38 Resistance of sacbrood virus to direct sunlight when suspended in water 39 Resistance of sacbrood virus to direct sunlight when suspended in honey 40 Length of time that sacbrood virus remains virulent in honey 40 Resistance of sacbrood virus to the presence of fermentative processes 41 Resistance of sacbrood virus to fermentation in diluted honey at outdoor temperature 42 Resistance of sacbrood virus to the presence of putrefactive processes 43 Resistance of sacbrood virus to carbolic acid . . . 44 Modes of transmission of sacbrood 46 Diagnosis of sacbrood 48 Prognosis *9 Relation of these studies to the treatment of sacbrood 50 'summary and conclusions 52 1 Literature cited 53 INTRODUCTION. Sacbrood is an infectiotis disease of the brood of bees. It is fre- quently encountered and has often been the cause of fear on the part of beekeepers through a suspicion that one of the more serious maladies— the foulbroods— was present. The disease is more benign than mahgnant. It is insidious m its nature and somewhat transient in its character. The number of colonies that die as a direct result of sacbrood is comparatively small; the loss of individual bees from it, however, in the aggregate is enormous. The loss tends naturally to weaken the colony m which the disease is present, a fact whicli makes the disease one of great economic importance. 5S574°-BuU. 431—17 1 2 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE. Until recently no laboratory study has been made of this disease. Circular No. 169, Bureau of Entomology, is a preliminary report on recent studies made by the writer. The present buUetm represents the results obtained from a contiauation of these studies. In it are included only such results as it is beheved can be applied by the beekeeper directly to his needs or as will be otherwise of particular interest to him. HISTORICAL ACCOUNT. There are a nmnber of references in beekeeping literature to a dis- order of the brood of bees which had been recognized by the presence of dead brood that was different from that dead of "foulbrood." It will be profitable to cite here a few of these articles: Langstroth (1857) writes as follows: There are two kinds of foul-brood, one of which the Germans call the dry and the other the moist or fcetU. The dry appears to be only partial in its effects and not contagious, the brood simply dying and drying up in certain parts of the combs. The moist differs from the dry in this that the brood dies and speedily rots and softens, diffusing a noisome stench through the hive. In this statement it will be seen that beekeepers had already recognized differences in the brood diseases which caused Langstroth to write that there were two kinds of ' ' foulbrood." The ktad referred to as "dry" foulbrood might easily have been sacbrood. Doolittle (1881), following a description of "foulbrood," writes: We have been thus particular in describing the disease [foulbrood] so none can mistake it; and also because there is another disease similar, called foul brood, which is not foul brood. With this last-named, the caps to the cells have very much the same appearance as in the genuine, but the dead larva is of a grayish color, and instead of being stretched out at full length in the cell, it is drawn up in a more compact shape. After a time it so dries up that the bees remove it, and no harm seems to arise .from it, only as there are a few larvae that die here and there through the combs at different periods; sometimes never to appear again, and sometimes appearing with the next season; * * *. Doohttle, therefore, as early as 1881, had also observed a brood disease which he says is similar to foulbrood and called foulbrood, but which is different from the genuine foulbrood. From his description one can readily beheve that the disease which he says was not foul- brood was sacbrood. Jones (1883), of Beeton, Ontario, Canada, writes the following: There is also another disease of the larvse which is sometimes found both in Europe and America, which is more like foul brood than any of the above [chilled, starved, or neglected brood] and which frequently deceives those who we might claim should be good judges, but which, however, is not the genuine article. It is a dying of the brood both before and after it has been capped over. The appearance of this and the genuine is much the same during the earlifer stages of their existence, but the former is usually removed by the bees and no further trouble ensues. SACBBOOD. 3 It will be noted that Jones also recognized that there was a disease that resembled somewhat the genuine foul brood, but was different from it, and that it was also different from chilled, starved, or neglected brood. Most likely the disorder referred to in his article was sacbrood. Sunmins (1887), writing from Kottingdean, England, points out the difference between "deadbrood' ' and foulbrood: That loul brood is ol'lon confused with eimplo dead brood I am well aware. * * * But that every beo keeper may decide for himself without the aid of a microscope, which is the genuine foulbrood and which is not, I will show how I have always been able to detect the difference. With simple deadbrood, while some may appear like the foul disease, much of the older brood dries up to a white cinder, in many cases retaining its original form, which I have never found to occur when genuine foul- brood is present. Chilled brood can be distinguished from the more serious malady in like manner. In addition to emphasizing the difference between "deadbrood" and "foulbrood," Simmias says that these two diseases are in turn to be differentiated from chiUed brood. He adds the additional £act also that Cheshire had examuied this "deadbrood" and failed to find any microscopic evidence of disease. Cook (1904), under the heading "New Bee Disease," writes as follows: In California and some other sections the brood dies without losing its form. We use the pin-head, and we draw forth a larva much discolored, often black, but not at all like the salvy mass that we see in foulbrood. . From his description, and from the fact that the disease is quite prevalent ia California, it is very probable that the disorder men- tioned by Cook is sacbrood. A study of this "dead brood" recognized by the beekeepers as being different from foulbrood was begun by the writer in New York State in 1902, under the direction of Dr. V. A. Moore. In a brief report on the work (1904) the following is found: The beekeepers are sustaining a loss from a diseased condition in their apiaries which they are diagnosing as "pickled brood." The larvse usually die late in the larval stage. The most of them are found on end in the cell, the head frequently blackened and the body of a watery granular consistency. * * * The results of the examinations showed that Aspergillus pollinis was not found. Further investigations must be made before any conclusion can be drawn as to the real cause of this trouble. It will be observed from, this quotation that the so-called pickled brood did not conform to the description of pickled brood and could not therefore be the condition which had called forth the- description of and the name, "pickled brood" (see p. 4). Burri (1906), of Switzerland, writes: Dead brood, said to have been black brood, I have occasionally met with in my investigations. It occiured in the older larvae, and showed a gray to blackish colora- 4 BULLETIN 431, XT. S. DEPAETMENT OF AGEICULTTJKE. tion, pai-tially drying the lai-vse until mummified. Tliese larvss of the black-brood type gave a negative result both in microscopic examination and in the usual bac- teriological culture experiments. Bacteria seem to take no part in this disease, and so far as I have come in contact with black brood, I have been able to reach no certain opinion as to its cause. [Translation.] It is very probable that the disorder encountered by Burri, which was free from bacteria, was sacbrood. Out of 25 samples examined between 1903 and 1905, he found four samples containing this dis- ease alone, while in a few of the samples the disorder was accom- panied by one of the other brood diseases. Kursteiner (1910), of Switzerland, gives a summary of all samples exammed by Burri and hunself from 1903 to 1909. Out of 360 samples of suspected disease examined, 94 were diagnosed as " dead brood free from bacteria." These were probably samples of sac- brood. As shown by his later reports, Km^teiner has continued to find this disease in the examination of suspected samples. The foregoing references to the literature show that beekeepers in different countries had been observing dead brood in their apiaries which was imhke brood dead of "foulbrood." On this point all of the observers practically agreed. No name had been given to the disorder. NAME OF THE DISEASE. Before 1912, very httle definite information concerning this somewhat mysterious disorder of the brood had been obtained. After discovering its cause and determining its true nature, the writer (1913) used the name "sacbrood" to designate it. The name was coined to suggest the saclike appearance of the dead larvae in this disease at the time they are most frequently seen by the bee- keeper. The fact should here be emphasized that sacbrood is not a new disease. It is only the knowledge concerning the disease and its name that is of recent origin. It is far better, and in aU probabiHty much more accurate, to think of sacbrood as a disease which has affected bees longer than history records the keeping of bees by man. The disease, therefore, has been collecting its toll of death for centuries, often \mawares to the beekeeper. Simply knowing that there is such a disease should not be the cause of any additional anxiety concern- ing its losses. On the other hand, less fear should be experienced, since by knowiag of it hope may be entertaiaed that the losses resulting from it may be reduced. PICKLED BROOD. The term "pickled brood" was introduced into beekeeping litera- ture 20 years ago (1896), by WUliam E. Howard of Texas. The condition which he described imder this term he declared was caused SACBEOOD. 5 by a fungus to which he yavd tho\ iiuine Aapergillus polUni. In a second article (1898) ho writes that pupaj and adult bees, as well as the larvae, are attacked by the disease, stating his behef that the disease in adult bees had been diagnosed as paralysis. Technically, therefore, the term "piclded brood" refers to an infectious disorder of bees affectmg both the brood and adult bees and caused by a specific fungus, Aspergillus pollini. It was particularly unfortunate that these articles on pickled brood should have appeared at the time they did, as through them some beekeepei-s have been led to the mistaken belief that the brood disease, which they had so long observed as being similar to "foul- brood," but differing from it, had been described in his articles as pickled brood. Whether such a disease (pickled brood) does exist, can not be defi- nitely stated. It may be said, however, that it probably does not. The writer has not encountered such a disorder during his study on the bee diseases. He believes that if the condition is present it cer- tainly has not attracted the attention of beekeepers to any great extent. It can safely be advised, therefore, that all fear of losses from such a possible condition should be dispelled, at least until the disease is met with again. It would seem that the name "pickled brood" is being used among beekeepers at present in a very general sense. Root (1913) writes: The name pickled brood has been applied to almost any form of dead brood that was not foul brood. In a rather general way, it seems to cover, then, any form of brood that is dead from some natural causes not related to disease of any sort. This quotation suggests that a number of conditions are most Hkely included under the term "pickled brood" as it is popularly used. Brood dead of starvation and that foimd dead before capping and not dead of an infectious disease seem to be referred to especially by the name. Beekeepers sending samples of disease to the laboratory have been asked the question : " What disease do you suspect V In the replies received more than one disease was sometimes suggested as being suspected. Out of 189 repHes received from beekeepers sending samples of sacbrood, European foulbrood was suggested in 55 repHes, pickled brood in 39, foulbrood in 19, blackbrood in 15, poisoned brood in 7, chilled brood in 5, starved brood in 6, American foulbrood in 13, dead brood in 3, neglected brood in 1, scalded brood in 1, suffocated brood in 1, and in 24 cases the reply was: "Don't know." These replies show that beekeepers generally had not learned to recognize the disorder which is now called sacbrood by any one name. It is natiiral to suppose that sacbrood would have been one of the conditions occasionally referred to under the term "pickled brood." 6 BULLETIN 431, TJ. S. DEPAETMENT OF AGKICULTtJBE. As sacbrood has been proved, ho\\-(:'ver, to be a disUiift disease and different from all other disorders, iiaturaUy it js incorrect to use the terms "sacbrood" and "pickled brood" synonymously, either m the popular or in the technical sense.^ APPEARANCE OF HEALTHY BROOD AT THE AGE AT WHICH IT DIES OF SACBROOD. By comparing the appearance of healthy brood with that of brood dead of a disease, both the description and the recognition of the symptoms of the disease are often materially aided. Before discuss- ing the symptoms of sacbrood, therefore, a description of the healthy brood at the age at which it dies of sac- brood wiU be given. In this description the same method will be used and simi- lar terms employed as will be found in the description of the symptoms of the disease. It will be recalled by those who are at all familiar with healthy comb in which brood is being reared that the brood is arranged in such a way that capped and uncapped areas occur alter- nately and in more or less semicircular fashion . Practically aU cells in the un- capped areas will be without caps while practically all in the capped areas wiU be capped. Since the brood that dies of sac- brood, with but few exceptions, does so in capped cells, a description of such brood involves the form, size, and position of these cells. A cell (figs. 1 and 2) may be described as having six side walls, a bottom or base, andacap. (The cap has been removed by the bees from the cells from which these figures were drawn.) In general the six side walls are rectangular and equal. These walls form six equal obtuse angles within the cell (fig. 1 ) . The angle which is uppermost in the cell (Ai) is formed by two sides which together may be termed the roof of the cell. The angle which is lowermost (figs. 1 and 2, A^) is formed by two sides which with equal propriety may together be termed the floor of the cell (fig. 2, F). When a cell is cut along its long axis 1 For the purpose of an explanation for those who may have learned to refer to sacbrood by the term "pickled brood," it might be felt advisable by some to continue for a while in some way a reference to the latter term. In such an event, the expression "so-called pickled brood" is suggested as being more nearly accurate than the term "pickled brood." Fig. 1. — Looking into an empty worker cell uncapped by bees. The uppermost angle (Ai),the lowermost angle (As), the lateral wall (L), and the wrinkling of the inner sur- face of the cell near the opening, indicating the presence of a mass of cocoons (C), are shown. Enlarged about 8 diameters. (Original.) SACBROOD. the cut surface of the older ones shows the presence of a varying num- ber of old cocoons (fig. 2, C). Near the mouth of the ceU on the side walls (figs. 1 and 2, C) will often be noted a wrinkling of the surface. This wrinkling is caused by the presence of old cocoons. The two remaining walls are parallel and wiU be referred to as the lateral walls (fig. 1, L). The bottom is concave on the inside. The cap Fig. 3.— End view oJ cell capped. Tlie cap is convex, being recently constructed. (Origi- nal.) is also concave on the inside, making it convex on the outside. When freshly constructed the sur- face of the cap (fig. 3) is smooth and and entire and shows considerable convexity. Later, not infrequently it is found to be less convex and somewhat irregular. The cap should remain normally for the most part entire (fig. 8) . While this is the rule, there are exceptions to it. The bee- keeper is familiar with the appear- ance which suggests that it had not been entirely completed (fig. 11 ; PI. n, I). X ,. „ The long axis of the cell is nearly horizontal, the bottom of the cell being normally only shghtly lower than the mouth. The long axis measures approximately one-half inch, while the perpendicular dis- tance between any two diametrically opposite side walls is approx- imately one-fifth of an inch. The side walls are each approximately one-tenth of an inch wide. It is in such a cell, then, that the brood of the age at which it rHes of sacbrood is found. Fig. 2. — Empty worker cell cut in half along the long axis ol the cell, showing cocoons (C) at the base and near the mouth of the cell, and the lowermost angle (As) formed by the two walls which constitute the floor (F) of the cell. Enlarged about 8 diameters. (Original.) 8 BULLETIN 431, TJ. S. DEPAETMENT OF AGEICULTUBE. APPEARANCE OF A HEALTHY LARVA AT THE AGE AT WHICH IT DIES OF SACBROOD. The symptoms which differentiate sacbrood from the other brood diseases are to be found primarily in the post-mortem appearances of the larvffi dead of the disease. As an aid in interpreting the description of these appearances a description of the healthy larvae is &st made. Larvse ' that die of sacbrood do so almost invariably after capping and at some time dm-ing the four days just preceding the change in form of the maturing bee to that of a true pupa. During the jBrst two days of this prepupal period the larva moves about more or less in the cell and spins a cocoon. It is then com- paratively quiet for about two days, lying on its dorsal side and ex- FiG. 4,— Lateral view of healthy worker larva showing the normal position within the cell. For conven- ience of description the length is divided into thirds— anterior third (AT), middle third (MT) and posterior third (PT). Enlarged about 8 diameters. (Original.) tended lengthwise in the cell. At the close of this two-day period of rest, as a result of the metamorphosis going on, the larva changes very rapidly to a true pupa, assuming the outward form of an adult bee. Although many larvae die of sacbrood during the first two days or active period, of the 4-day prepupal period, by far the greater number of deaths occur during the last two days, the period of rest. A healthy larva at this resting period of its development is chosen, therefore, for description. As dead worker larvae are the ones usually encountered in sacbrood and the ones almost invariably chosen in discussing the symptoms of the disease, the worker larva is here described. The normal larva lies extended in the cell (fig. 4) on its dorsal side, motionless, and with its head pointing toward the mouth of the cell. Its posterior or caudal end lies upon the bottom of the cell, i As beekeepers usually refer to the brood at this age as "larvae," the term is used here to designate the developing bee at this stage of its growth. SAOBKOOD. 9 while Its extreme anterior or cephalic end extends almost to the cap and roof. The length of the larva is approximately one-half inch, being nearly that of the cell. Its two lateral sides cover about one- half each of the two lateral walls. The width of the larva is approxi- mately one-fifth of an inch, being the distance between the two lateral walls of the cell. The dorsal portion of the larva lies against the floor of the cell, being more or less convex from side to side and also from end to end. Its ventral surface is convex from side to side, and is, generally speak- ing, concave from end to end. Considerable empty space is found between the larva and the roof of the cell. The spiracles are visible. The glistening appearance, characteristic of a larva before capping, very largely disappears after capping. Although larvae at this age might be thought of as white, they are in fact more or less bluish white in color. It is possible to remove a healthy larva at this age from the cell without rupturing the body wall, but care is required in doing so. For purposes of description it is con- venient to divide the length of the larva into three parts. These may be denom- inated the anterior (AT), middle (MT), and posterior thiMs (PT). Anterior third. — On removing the cap from a cell the anterior cone-shaped portion of the larva is seen (fig. 5; PI. II, g). The apex of this cone-shaped third is directed upward toward the angle in the roof of the cell, but is not in contact with the roof or the cap. Transverse segmental markings are to be seen. Along a por- tion of the median dorsal line there is frequently to be observed a narrow transparent area. A cross section of this third is circular in outline. The anterior third passes rather abruptly into the middle third. At their juncture on each lateral side, owing to a rapid increase in the width of the larva at this point, there is presented the appear- ance of a "shoulder." Middle third. — This third (figs. 6 and 4 ; PI. II, m) lies with its dorsal portion upon the floor of the cell, its axis being nearly horizontal. The ventral surface is convex from side to side, and is considerably below the roof of the cell. This upper surface is crossed from side to side by well-marked furrows and ridges representing ^segments of the larva. These furrows and ridges produce a deeply notched appearance at the lateral m argins. In some of the segments a trans- verse trachea may be seen appearing as a very fine, scarcely per- 58574°— Bull. 431—17 2 Fig. 5.— End view of healthy worker larva in normal position in the cell. Cap torn and turned aside with forceps. En- larged about 8 diameters. (Original.) 10 BULLETIN 431, XT. S. DEPABTMENT OF AGEICTJLTUKE. ceptible, white line. Sometimes there may be seen a narrow area along the median line of the ventral sm-f ace that is more nearly trans- parent than the remaining portion of the surface. This area may extend slightly into the anterior and posterior thirds. It is sunilar in appearance to the one on the dorsal side, but less distinct. A cross section of this third is slightly eUiptical in outline. The middle third passes more or less gradually into the posterior third. The ]uncture on the ventral surface is indicated by a wide angle formed by the ventral surfaces of these two thirds. Posterior third.— In form the pos- terior third (figs. 6 and 4) is an im- perfect cone, the axis of which is directed somewhat upward from the horizontal. This third occupies the bottom portion of the cavity of the cell. Its dorsal surf ace lies upon the bottom wall, with the extreme caudal end of the larva extending to the roof of the cell (fig. 4). The third is marked off into segments by ridges and furrows similar to, but less regular than, those of the middle third. TISSUKS OF A HEALTHY LABVA AT THE AGE AT WHICH IT DIES OP SACBROOD. Upon removing a larva in the late larval stage and pimcturing its body wall lightly, a clear fluid almost water-like m appearance flows out. This fluid consists chiefly of larval blood. By heating it, or by treat- ing it with any one of a number of different reagents, a coagulum is formed in it. Upon rupturing the body wall sufficiently, the tissues of the larva flow out as a semiliquid mass. The more nearly solid portion of the mass appears almost white. This portion is suspended in a thin liquid, chiefly blood of the larva. A microscopic examination shows that the cellular elements of the mass are chiefly fat cells. Many fat globules suspended in the liquid tend to give it a milky appearance. SYMPTOMS OF SACBROOD. The condition of a colony depends naturally upon the condition of the individual bees of which it is composed. In the matter of diseases in practical apiculture the beekeeper is interested piimarily in the Fig. 6.— Healthy larva and cell viewed from above and at an angle. (Original.) SACBKOOD. 11 colony as a whole, and not in individual bees. Therefore, in describ- ing the symptoms of a bee disease, the colony as a whole should be considered as the unit for description, and not the individual bee. \ symptom of tUsease manifested by an individual bee, broadly con- sidered, is, in fact, also a colony symptom. The symptoms of sacbrood as described in this paper are, therefore, those evidences of disease that are manifested by a colony affected by the disease. It has been found that sacbrood can be produced in a healthy colony by feeding it a suspension in sirup of crushed larvae dead of the disease. With sacbrood thus produced in ex- perimental colonies the symptoms of the disease have been studied, and the desci'iption of these symptoms given here is based chiefly upon observations made in these experimental studies. The facts thus obtained are in accord with those observed in numerous sam- ples of the disease sent by beekeepers from various localities in the United States for diagnosis. They are in ac- cord, furthermore, with the symptoms as they have been observed in colonies in which the disease has appeared, not through experimental inoculation but naturally. The symptoms of sacbrood which would ordinarily be observed through a more or less casual examination of the disease will first be considered. It must be remembered that the brood is susceptible to the disease, but that the adult bees are not. SYMPTOMS AS OBSEKVED FROM A CASUAL EXAMINATION. Fig. 7.— Larva dead of sacbrood lying in tlie cell as viewed from above and at an angle. It may have been dead a month. Cap of cell removed by bees. Enlarged about 8 diameters. (Original.) The presence of dead brood is usually the first symptom observed. An irreg- ularity in the appearance of the brood nest (PI. I, figs. 1 and 2; PI. IV) frequently attracts attention early in the examination. The strength of a colony in which the disease is present is often not noticeably diminished. Should a large amount of the brood become affected, however, the colony naturaUy becomes weakened thereby, the loss in strength soon becoming appreciable. Brood that dies of the disease does so almost invariably in capped cells, but before the pupal 12 BULLETIN 431, U. S. DEPAETMENT OF AGBICULTUEE. Fig. 8. — End view of capped cell wliich con- tains a larva dead of sacbrood, being simi- lar to the one shown in figure 9. The cap here is not difierent from a cap of the same age over a healthy larva. (Original.) stage is reached. It is rare to find a pupa dead of sacbrood (PL II, zz). The larv^ that die (fig. 7) are found lying extended lengthwise with the dorsal side on the floor of the cell. They may be found in capped (fig. 8) cells or iu cells which have been ^mcapped (fig. 9), as bees often remove the caps from cells containing dead larvae. Caps that are not removed are more often en- tire, yet not infrequently they are foimd to have been pxmctured by the bees. Usually only one ptmcture is found in a cap (PI. II, d), but there may be two (fig. 10) or even more (PI. H,/). The punctures vary in size, sometimes approximating that of a pinhead, although usually smaller, and are often irregular in outline. Sometimes a cap (fig. 11, PL II, h) has a hole through it which suggests by its position and uniform circumference that it has never been completed. Through such an opening (fig. 11; PL II, e) or through one of the larger punctiires the dead larva may be seen within the cell. A larva recently dead of sacbrood is slightly yellow. The color in a few days changes to brown. The shade deepens as the process of decay con- tinues, imtil it appears in some in- stances almost black. Occasionally for a time during the process of decay the remains present a grayish appear- ance. In sacbrood, during the process of decay, the body wall of the dead larva (figs. 7 and 9) toughens, permit- ting the easy removal of the re- mains intact from the cell. The content of the sachke remains, dur- ing a certain period of its decay, is watery and granidar in appearance. Much of the time the form of the remains is quite similar to that of a healthy larva. If the dead larva is not removed, its surface through evaporation of its watery content, becomes wrinkled, dis- torting its form. Further drying results in the formation of the Fig. 9.— Looking into a cell containing a larva dead of sacbrood. The stage of decay is about the same as in figure 8. (Original.) Bui. 431, U. S. Dept. of AgricuHuro. Plate v_WS««V5fy O '^ >% > ■JW*V J^if^ft^ f^^»5^?i^ r» -> '^ > •^<&(i|0' If - . d-^^aa- ■%m Fig. 1.— Marked Sacbrood Infection. Size Slightly Less than Natural. (Original.) mm^m^^^0iijp^^^^^ :-A^^ ^K'iskv^m^^ iwiilam. ^m^mm^m FiQ. 2.— Heavy Sacbrood Infection, Showing a Number of Different Stages OF Decay of Larv/e. Eggs, Young Larvae in Different Stages of Develop- ment, and Diseased Larv/E in Same Area. Natural Size. (Original.) SACBROOD PRODUCED BY EXPERIMENTAL INOCULATION. Bui. 431, U. S. Dept. of Agriculture. Plate II. oo y k □ Q s t u- l> r"^""n W X y z WW JCX !/¥ zz Comparison of a Healthy Larva and the Remains of Larv/e Dead of Sacbrood. a, A cap of a healthy larva; &, c, d, c, and /, caps over larvos in first, second, third, fourth, and fifth stages of decay, respectively; g, a healthy larva, end view; h, i, j, k, and I, an end view of the five stages of decay; m, a healthy larva viewed from above; n, o, p, q, and r, cor- responding view of the five stages of decay; s and y, healthy larva removed from the cell; t, u, V, w. and s, larval remains in different stages of decay removed from the cell; WW, a larva recently dead of sacbrood with the anterior third removed by the bees; x, a scale removed from the cell; xx, larval remains from which a small portion has been removed by bees: i/.v. almost a pupa; cz, a pupa dead of sacbrood which had only recently transformed. (Onglnal.) SACBROOD. 13 "scale" (tigs. 22, 23; PL II, I, r, and x). This scale is not adherent to the cell wall. In sacbrood the brood combs may be said to have no odor. Larvae midergoing later stages of decay in the disease, however, when crushed in a mass and held close to the nostrils are found to possess a disagreeable odor. From a superficial or casual ex- amination alone of a case of sac- brood it may be mistaken for some other abnormal condition of the brood. A careful study of the post- mortem appearances of larvae dead of the disease, however, will make it possible to avoid any such confusion. A more carefid study of the dead larvse is therefore justified. Fig. 10. — Cap of cell contaming the remains of a larva dead of sacbrood. The cap is slightly suTLken and bears two perforations made by the bees. (Original.) APPEARANCE OF LARV^ DEAD OF SACBROOD. No signs in a larva dying of sac- brood have yet been discovered by which the exact time of death may be determined. As the larvse in this disease usually die during the time when they are motionless, lack of movement can not be used as an early sign of death. I-n this descrip- tion it is assumed that the larva is dead if it shows a change in color from bluish-white to yellowish or indications of a change from the normal turgidity to a condition of flaccidity. The appearance of a larva dead of sacbrood varies from day to day, changing gradually from that of a living healthy larva to that of the dried residue — the scale. A de- FiG. 11.— End view of cell containing a laiva scrip tion that WOuld be COrrect for deadofsacbrood.withacapwhichhasthe _■ , ^avva nn r,r\P flnv there- appearance of never having been com- ^ dead larva On One uay, inere pieted. (Original.) forc, may and probably Would be incorrect for the same larva on the following day. Moreover, all larvro dead of the disease do not undergo the same change in appear- ance, causing another considerable range of variation. For con- venience of description, this gradual and contmual change in appear- ance is here considered in five more or less arbitrary stages. As the 14 BULLETIN" 431, U. S. DEPAETMENT OF AGEICULTTJEE . same plan will be followed and similar terms will be used in describing these stages as were employed in tbe description of a healthy larva of the same age, the interpretation of the description wiU be aided if the appearance of a healthy larva as described above is borne in mind. FiEST Stage. Uncapping a larva showing the first symptoms of the disease, it win be observed that it has assumed a slightly yellowish appearance. Fig. 12.— First stage: Larva showing first symptoms of sacbrood and presenting tlie dorsal view of the anterior third. Cap removed artificially. (Original.) This shade deepens somewhat during the stage, but does not become a deep yellow. Anterior third. — The lateral margins and extreme cephahc end of the an- terior third (fig. 12; PI. II, &, Ti) may have assmned, and frequently do as- sume, a more or less transparent ap- pearance (represented in the figure by shading). The position and the sur- face markings of the anterior third are approximately those of the normal larva. When a change in the position is observed, however, the extreme anterior end of the larva — the apex of this cone-like third — having settled somewhat, does not approach so near the roof of the cell as does that of a healthy larva. It is sometimes found also that this cone-hke third is deflected more or less to one side or the other. Middle and posterior thirds. — The changes from the normal that have taken place in these two thirds are similar and can, therefore, be described together. The yellowish tint is here observed. The trans- verse ridges and furrows are still well marked (fig. 13). The trans- FiG. 13. — First stage: Ventral view of larva dead of sacbrood as seen from above and at an angle, giving a ventral view of all three thirds. Cap torn across. (Original.) SACBROOD. 15 verse trachess under slight magnification may be distinctly seen. The narrow, somewhat transparent area present along the ventral median line of the healthy larva is still to be seen in this stage of the decay. The lateral and posterior margins are stiU deeply notched and are frequently found to appear quite transparent. This appear- ance is due to a watery looking fluid beneath the cuticular portion of the body wall. Sometimes only the remnant of a larva (fig. 14; PI. II, ww) dead of sacbrood is found in the cell. Such remnants vary in size. The ' Fig. 15. — Second stage: Dorsal view of an- terior third of a larva dead of sacbrood. (Original.) surface left from the removal of tissues is somewhat roughened, indicating that the removed portion has been taken away piecemeal, and is more or less transverse to the larva. Consistency of the larva in the first stage. — The cuticular portion of the body wall, which chiefly constitutes the sac that characterizes the disease sac- brood, is less easily broken at this time than in the healthy larva. When the body wall is broken the tissues of the larva, which constitute the contents of the sac, flow out. This fluid tissue mass is less milky in appearance than that from a, normal larva. The granular character of the con- tents of the sac which is marked in later stages of decay is already m evidence. By microscopic exammation the granular appearance is found to be due chiefly to fat cells. Condition of the virus in the -first stage.— When larvae of this stage are crushed, suspended in sirup, and fed to healthy bees, a large Fig. 14.— First stage: Portion of a larva dead of sacbrood, showing a more or less transverse roughened surface from which the bees have removed a portion of the larva piecemeal. (Original.) 16 BULLETIN 431, V. S. DEPAETMENT OF AGKICULTUEE. amount of sacbrood is readily produced, showing that the larval re- mains in this stage are particularly infectious. This is an important fact, as it is the stage of decay at which the larva is frequently re- moved piecemeal from the cell. Second Stage. The color of the decaying larva has changed from the yellowish hue of the first stage to a brownish tint. The yellow, however, has not Fig. 17.— Third stage: Dorsal view of an- terior tliird of larva dead of sacbrood. (Original.) yet in all cases entirely disappeared. Anterior third. — The shade of brown is deeper in the anterior third (fig. 15; PI. II, i) as a rule than in the other two thirds. On the ventral surface of the anterior third there are sometimes present minute, very dark, nearly black areas, appearing httle more than mere points. Upon dissecting away the molt skin, these areas are found to be associated with the developing head and thoracic appendages of the bee. The position of the anterior third in this stage has changed only shghtly from that observed in the preceding one. The apex is farther from the roof of the cell (PL II, i). The deflection is more marked and is seen ia a greater number of larvae. The surface markings have not changed materially. Middle and posterior thirds. — ^The changes that have occurred in each of these two thirds are still similar and can, therefore, again be described together. Fig. 16. — Second stage: Larva dead of sacbrood, ventral view. tOriginal.) SAOBEOOD. 17 The ventral surface of these two thirds (fig. 16, PI. II, o) is less con- vex from side to side. The ridges and furrows, representing the seg- ments, are less pronounced. The lateral margins are stiU deeply notched. The prominent angle seen on the ventral side of a healthy larva, at the jimcture of the middle and posterior thirds, has given place to a wider one in this stage of decay. The clear subcuticular fluid frequently observed at the lateral and posterior margins of lar- vae dead of this disease is here increased in quantity. Consistency of the contents of the sac. — The cuticular sac is now more readily observed and less easily broken. The decaying contents con- sist of a more or less granular-appear- ing mass suspended in a watery ap- pearing fluid, the mass possessing a slightly brownish hue. The micro- scopic examination shows that the granular appearance is due to the presence of decaying tissue cells, chiefly fat cells, which are changing slowly as the decay of the larva goes on. Condition of the virus. — The results of inoculations show that the remains of larvae at this stage of decay are still in some instances infectious. The amount of infection produced when such larvae are used in making in- oculations is very much less, how- ever, than when larvae in the first stage are used. Third Stage. Fig. 18. — Third stage: Larva dead of sacbrood, ventral view. (Original.) The color of the dead larva of this stage is quite brown, that" of the an- terior third being a deeper shade than that of the other two thirds. An indication that the remains are drying is observed in the wrinkling of the surface that is beginning to be in evidence. Anterior third. — The color of the anterior third is a deep brolwn. This third still preserves its coneHke form (figs. 17 and 9; PI. II, j), the distance of the apex from the roof of the ceU being still further increased. This may equal one-fourth or more of the diameter of the mouth of the cell. The surface markings are still quite similar to those of a healthy larva with the exception that evidences of drying are present. 58574°— BuU. 431—17 3 18 BULLETIN 431, V. S. DEPARTMENT OF AGEICULTUEE. Middle third.— While the color of the middle third is similar to and often approaches in its shade that of the anterior, very frequently it is considerably Hghter. The ventral surface of this third (figs. 18 and 7) is less convex from side to side than ui the preceding stage, and the segmental markings, while stiU plainly visible, are less pro- nounced. The notches along the lateral margins are* also less pro- nounced. Posterior third. — ^The color of the posterior third (figs. 18 and 7; PI. II, p) equals or exceeds in depth of shade that of the middle third and sometimes equals that of the anterior third. The surface markings are stUl pronounced and much resemble those of the normal larva. That the watery content of the sac is being lessened through evapo- ration is evidenced by the diminution of the quantity of the watery- FiG. 19.— Third stage: Larva dead of sacbrood, lateral view. (Original.) appearing substance seen at the lateral margins of the middle and posterior thirds and by the wrinkling of the cuticular sac. These wrinkles are small and numerous. The lateral view of the larva in the third stage (fig. 19) shows that it stni maintains, in a general way, the form and markings of the normal larva (fig. 4). The turgidity is gone, although the position in the ceU is very much as it is in the healthy larva. Consistency of the sac and its contents. — It is the appearance of the remains of the larva in the third stage of the decay that best character- izes the disease, sacbrood. The cuticular sac is now quite tough, permitting the removal of the larva from the cell with considerable ease and with httle danger of its being torn. The content of the sac is a granular mass, brownish in color and suspended in a comparatively small quantity of a more or less clear watery-appearing fluid. Upon microscopic examination the mass is found to consist of decaying tissues, chiefly fat cells. Condition of the virus in the third stage. — When the larval remains in this stage of decay are crushed and fed in sirup to healthy colonies no sacbrood is produced, indicating that the dead larvae at this stage SACBBOOD. 19 are not infectious. The status of the virus in this stage is not defi- nitely known, but the facts thus far obtained indicate that it is probably dead. Fourth Stage. The brown color of the larval remains has further deepened, the anterior thh-d being much darker as a rule than the other two-thirds. The marked evidence of drying now present might be said to charac- terize this stage. Anterior third.— The color is a very deep brown, often appearing almost black. As a result of drying, the apex of this conehke third Fig. 20.— Fourth stage: Remains of larva dead of saebrood. (Original.) is often nearer the roof of the cell in this stage than in the preceding one. As a result it has also been drawn inward from the mouth of the cell. The surface markings seen in the normal larva are in this stage (fig. 20; PI. II, Ic) of decay almost obhter- ated through the wrinkling of the surface, due to drying. Middle third. — This third is de- cidedly brown, but lighter in shade than the anterior third. The ventral surface (fig. 21; PI. II, q) is slightly concave from side to side. The segmental markings are still to be seen, but are not at all prominent. The notched lateral mar- gins extend upon the side walls of the cell. The subcuticular fluid so noticeable in some of the earlier stages has disappeared through evaporation. The effect of drying is very noticeable, causing a marked wrinkling of the surface. Posterior third. — The posterior third (PI. II, q) may or may not be darker than the middle third, but it is not darker than the anterior Fig. 21. — Fourtli stage: Remains of larva dead of saebrood, ventral view. ( Original.) 20 BULLETIN 431, U. S. DEPAHTMENT OF AGEICULTUEE. third. The effect of the drying on this third is quite perceptible also. The surface markings and notched margin of the normal larva are still indicated in the decaying remams, but are much less pronounced. The subcuticular fluid is no longer in evidence. Consistency of the contents of the sac— Upon tearing thesac, the contents are found to be less fluid than in preceding stages. The decaying tissue mass is stiU granular in appearance. As the drying TiQ. 22.— Fifth stage: Scale, or larval re- mains, in sacbrood as seen on looking into the cell. (Original.) proceeds further the contents of the sac become pastelike in consistency. Condition of the virus in the fourth stage. — As in the preceding stage, the larval remains in the fom-th stage do not seem to be infectious. Fifth Stage. Fig. 23.— Filth stage: Scale, or larval remains, in sacbrood viewed at an angle from above. (Original.) The dead larva in this last stage has lost by evaporation all of its moisture, leaving the dry, mummylike remains known as the "scale." Anterior third. — The anterior third (fig. 22 ; PI. II, Z) through dry- ing is retracted from the mouth of the cell, with the apex drawn still deeper into the ceU and raised toward its roof. This third is greatly wrinkled, and, being of a very dark-brown color, presents often an almost black appearance. Middle third. — The middle third (flg. 23; PI. II, r), is deeply concave from side to side and may show renmants of the segmental markings of the larva. The surface is often roughened through drying. Sometimes both longitudinal and transverse trachese are SACBTiOOD. 21 plainly visible. The margin frequently presents a wavy outline cor- responding to the original furrows and ridges of the lateral margin of the larva. Posterior third.— The posterior third (figs. 23 and 24) extends upon the bottom of the cell, but does not completely cover it. A lateral view of the scale (fig. 24) shows that it is turned upward anteriorly and drawn somewhat toward the bottom of the cell. The ventral surface is concave, often roughenc-d, and directed somewhat forward. This margin, hke that of the middle third, has a tendency toward being irregular. The scale.— The scale can easily be removed intact from the cell. (PL II, aj.) . Indeed, when very dry, many of them can be shaken from the brood comb. When out of the cell, they vary markedly in appearance. The anterior third is of a deeper brown than the the other two thirds as a rule. The dorsal side of the middle and Fig, 24. — Scale, or larval remains, in position in cell out lengthwise, lateral view. (Original.) posterior thirds is shaped to conform to the floor of the cell, being in general convex, with a surface that is smooth and polished. The margin is thin and wavy. The anterior third and the lateral sides of the middle and posterior thirds being turned upward, the ventral sur- face being concave, and the posterior side being convex, the scale in general presents a boathke appearance and could be styled "gondola- shaped." This general form of the scale has been referred to by beekeepers as being that of a Chinaman's shoe. When completely dry, the scale is brittle and may easily be ground to a powder. Condition of the virus in the scale. — The scales in sacbrood, when fed to healthy bees, have shown no evidence of being infectious. The length of time that dead larvae are permitted by the bees to remain in the cells before they are removed varies. They may be removed soon after death, they may remain until or after they have become a dry scale, or they may be removed at any intervening stage in their decay. Not infrequently they are permitted to remain to or 22 BULLETIN 431, TJ. S. DEPAETMENT OF AGKICULTUBE. through the stage described above as the thu-d stage (figs. 7, 9, 17, and 18; PI. II j, p). That the dead larvffi are allowed to remain in the cells often for weeks is in part the cause of the irregularity ob- served ia the appearance of the brood combs (p. 11). (Pis. I, IV.) APPEARANCE OF THE TISSIIES OF A LAKVA DEAD OF SACBROOD. The gross appearance of a larva during its decay after death from sacbrood has just been described.- The sachke appearance o'f the remains, with its subcuticular watery-like fluid and its granular content, can better be interpreted by knowing something of the microscopic structure of the dead larva. A section through a larva (fig. 25, A) dead of sacbrood shows that the fat tissue constitutes the greater portion of the bulk of the body. The fat ceUs (FC) are comparatively large. In the prepared section when considerably magnified (C) they are seen to be irregular in outline, with an irregular-shaped nucleus (Nu). Bodies stained black, more or less spherical in form and varying in size, are found in them. The presence of these cells is the chief cause for the granular appearance of the contents of larvae dead of sacbrood. This appearance has often been observed by beekeepers and is a weU- recognized symptom of sacbrood. In the section (A) may be seen a molt skin (Cj), which is at a con- siderable distance from the hypodermis (Hyp). Another cuticula (Cj) is already quite well formed and lies near the hypodermis. Be- tween these two cuticulae (Cj and C^) during the earlier stages of decay there is a considerable space (" in tercuticular space") (IS). This space is filled with a watery-looking fluid. That the fluid is not water, but that it is of such a nature that a coagulmn is formed in it during the preparation of the tissues for study, is shown by the presence of a coagulmn in the sections. The body (B, A) wall of the larva is composed of the cuticula (Cj), the hypodermis (Hyp) and the basement membrane (BM). The hypodermal cells may be present in the mass content of the larval remains. These cells are comparatively small. Similar ones are to be found in the tracheal walls (Tra). These cells, however, make up only a small portion of the contents of the sac. There are many other cellular elements to be found in the decaying mass of larval tissues, some of which contribute to this granular ap- pearance. Among these are the oenocytes (Oe), cells (D) larger than the fat cells, but comparatively few in number. These are found among the fat cells, especially in the ventral half of the body. The oenocytes in the prepared tissues are irregular in outhne, having a nucleus regular in outline. The cytoplasm is uniformly granular and does not contain the black staining bodies found in the fat cells (C). SACBROOD. 23 Fig. 25.— The tissues of a worker larva after being dead of sacbrood about one week. A, cross section, semidiagrammatic, of the abdomen in the region of the ovaries, showing a recently cast cuticula, or molt sWn (Cj), a newly formed cuticula (Ci), the hypodermis (Hyp), the stomach (St), the ovaries (Ov), the heart (Ht), the ventral nerve cord (VNC), the dorsal diaphragm (DDph), tracheae (Tra), ceno- cytes (Oe), and fat cells (FC). Between the cuticula C2 and the cuticula Ci is a considerable intercu- tioular space (IS). B represents the body wall in this patholofjical condition, showing the cuticula C2 and the cuticula Ci, both bearing spines (SCj and SCi), and theintercutioular space (IS) in which is found evidence of a coagulum formed from the fluid filling the space by the action of the fixing fluids. The remainder of the body wall, the hypodermis (Hyp), and the basement membrane (BM) are also shown. C, fat cell with irregular outline, irregular nucleus (Nu), and deep staining bodies (DSB). Dj oenocyte with uniformly staining cytoplasm, and with a nucleus (Nu) havmg a uniform outline. E, a portion of the stomach wall showing the epithelium (SEpth) during metamorphosis, it being at this time quite columnar in type, and the musculature (M). (Original.) 24 BULLETIN 431, TJ. S. DEPARTMENT OP AGEICULTURE. The molt skin (Cj) is probably the one that is shed nonnally. about three days after the larva is capped. The cuticula (Ci), already quite well formed, is probably the one which normally would have entered into the formation of the molt skin that is cast at the time the larva or semipupa changes to a pupa. The molt skin (Cj) constitutes for the most part the sac which is seen to inclose the decaying larval mass in sacbrood, the cuticula (Ci) probably assisting somewhat at times. The presence of the subcuticular fluid is made more intelli- gible by these facts. Larvae dying of sacbrood at an earlier or later period in their development will present an appearance varying somewhat from that just described. Contrasted with the stomach (midintestine or midgut) of a feeding larva, the stomach (A, St) of a larva at the age at which it dies of sac- brood is small. The cells lining the wall of the organ vary con- siderably in size and shape, depending upon the exact time at which death takes place. In contrast to the low cells of the stomach wall in younger larvse, the cells (E, SEpth) at this later period are much elon- gated. These cells would also at times be found in the decaying granular mass present in the larval remains. The various organs of the body contribute to the cellular content of the decaying larval mass. At the period at which the larva dies of sacbrood, the cellular changes accompanying metamorphosis are particularly marked. This condition introduces various cellular ele- ments into the decaying larval mass. The granular mass from the larval remains in sacbrood is, therefore, a composite affair. Upon examining the mass microscopically, it wiU be found that the granular appearance is due for the most part to fat cells suspended in a liquid. The Mquid portion seems to be chiefly blood of the larva, or, at least, derived from the blood, although augmented most probably by other liquids of the larva and possibly by a hquefaction of some of the tissues present. The granular mass suspended in a watery fluid, as a symptom of sacbrood, is by these facts rendered more easily understood. CAUSE OF SACBROOD. DooUttle (1881), Jones (1883), Simmms (1887), Root (1892 and 1896), Cook (1902), Dadant (1906), and others through their writ- ings have pointed out the fact that there are losses sustained from sacbrood. There has been no consensus of opinion, however, as to the infectiousness of the disease. On this point Dadant (1906) writes: Wliatever may be the cause of this disease (so-called Pickled Brood), and although it is to a certain extent contagious, it often passes off without treatment. But, as colonies may be entirely ruined by it, it ought not to be neglected. SAGBKOOD. 25 In the quotation Dadant expresses the belief that the disease is an infectious one. This view has been proved by recent studies to be the correct one. Since the disease is one of a somewhat transient nature, .often subsiding and disappearing quickly without treatment, and is quite different in many ways from the f oulbroods, it is not strange that some writers should have held that it is not infectious. PREDISPOSING CAUSES. Beekeepers have known for many years certain facts concerning the predisposing causes of sacbrood. Recent studies have added others relative to sex, age, race, climatic conditions, season, and food as possible predisposing factors in the causation of the disease. Age. — The results of the studies suggest that adult bees are not directly susceptible to the disease. Pupse are rarely affected (PI. II, zz). If one succumbs to the disease, it is quite soon after trans- formation from the larval stage. Primarily it is the larvae that are susceptible. When a larva dies of the disease, it does so almost invariably after capping, and usually during the 2-day period immedi- ately preceding the time for the change to a pupa. Sex. — Worker and drone larvse may become infected. Queen larvte apparently are also susceptible, although this point has not yet been completely demonstrated. Race. — No complete immunity against sacbrood has yet been found to exist in any race of bees commonly kept in America. That one race is less susceptible to the disease than another may be said to be probable, although the extent of such immunity has not been established. The question: "What race of bees is there in the diseased colony? " was asked beekeepers sending samples of diseased brood. Out of 140 rephes received from those sending sacbrood samples, 53 reported hybrids, 49 reported Itahans, 21 reported blacks, and 17 reported Itahan hybrids. These replies show that the bees commonly kept by American beekeepers are susceptible, although their relative suscepti- bihty is not shown. The bees which have been inoculated in the experimental work on sacbrood have been largely Italians or mixed with Itahan blood. Blacks have also been used. No complete immunity was observed in any colony inoculated. That the blacks are more susceptible than strains having Italian blood in them is suggested by some of the results. Facts concerning the problem of immunity as relating to bees are yet altogether too meager to justify more definite state- ments. Climate. — Historial evidence strongly suggests that sacbrood is found in Germany (Langstroth, 1857), England (Simmins, 1887), 58574°— Bull. 431—17 i 26 BULLETIN 431, TJ. S. DEPARTMENT OF AGEICTJLTUEE. and Switzerland (Bmri, 1906). Beuhne (1913) reports its presence in Australia, and Bahr (1915) has encountered a brood disorder among bees in Denmark which he finds is neither of the foul broods. He had examined 10 samples of it but had not studied it further. He says it may be sacbrood. About 400 cases of sacbrood have been diagnosed by Dr. A. H. McCray and the writer among the samples of brood received for examination at the Bureau of Entomology. A few of these were obtained from Canada. Whether the disease occurs in tropical chmates or the coldest chmates in which bees are kept has not yet been completely estabhshed. The mountains and coast plain of the eastern United States, the plains of the Mississippi VaUey and the mountains, plateaus, and coast plain of the western portion of the country have contributed to the number of samples examined. It occurs in the South and the North. Its occurrence in such widely different localities is proof that sac- brood is of such a natm-e that it can appear under widely different climatic conditions. The relative frequency of the disease, further- more, is not materially different in the different sections of the country. It must be said, however, that the extent, if any, to which the dis- ease is affected by chmate has not yet been determined. The practical import of these observations regarding climate, of particular interest here, is that the presence of sacbrood in any region can not be attributed entirely to the prevailing chmatic conditions. Season. — It has long been known that sacbrood appears most often and in the greatest severity during the spring of the year. As is shown by the results obtained in the diagnosis of it in the laboratory, the disease may appear at any season of the year at which brood is being reared. In the inoculation experiments sacbrood has been produced with ease from early spring to October 21. While it is thus shown that the brood is susceptible to sacbrood at all seasons, various factors together cause the disease to occur with greater frequency during the spring. Food. — ^Before it was known that sacbrood is an infectious disease the quantity or quaUty of food was not infrequently mentioned by beekeepers as being the cause of the disease. Since a filterable virus has been shown to be the exciting cause of the disease, it is left to be considered whether food is a predisposing cause. The distribution of the disease mentioned above, under the heading "Climate," here again serves a useful purpose. Since it occurs in such a wide range of localities, wherein the food and water used by the bees vary as greatly almost as is possible in the United States, the conclusion may be drawn that its occurrence is not dependent upon food of any restricted character. Furthermore, sacbrood is found in colonies having an abundant supply of food, as well as in colonies having a SACBEOOD. 27 scarcity. It has been produced experimentally in colonies under equally varying conditions in regard to the quantity of food. While it is possible that the quantity or quality of food may influ- ence somewhat the course of the disease in the colony, the r61e played by food in the causation of sacbrood must be slight, if indeed it con- tributes at all appreciably to it. Practically, therefore, for the present it may be considered that neither the quality nor quantity of food predisposes to this disease. EXCITING CAUSE OF SACBHOOD. That sacbrood is an infectious disease was demonstrated by the wi'iter (1913) through experiments performed during the summer of 1912. This was done by feeding to healthy colonies the crushed tissues of larvse dead of sacbrood, suspended in sugar sirup. The experiments were performed under various conditions, and it was found that the disease could be produced at will, demonstrating thereby that it was actually an infectious one. In the crushed larval mass no microorganisms were found either microscopically or culturally to which the infection could be attrib- uted, although the experiments had proved that the larva dead of the disease did contain the infecting agent. This led to the next step in the investigation, which was to determine whether the virus was so small that it had not been observed, and whether its nature would permit its passage through a filter. The first filter used for this purpose was the Berkefeld. The process by which the filtration is done is briefly this: Larvae which have been dead' of sacbrood only a few days are picked from the brood comb and crushed. The crushed mass is added to water in the proportion of 1 part larval mass to 10 parts water. A higher dilution may be used. This aqueous suspension is allowed to stand for some hours, preferably overnight. To remove the fragments of the larval tissues stiU remaining, the suspension is filtered, using filter paper. The filtrate thus obtained is then filtered by the use of the Berkefeld filter ^ (fig. 26) properly prepared. The filtering in the case of the coarser filters especially can be done through gravity alone. To determine whether any visible microorganisms are present in this last filtrate, it is examined microscopically and culturally. When f oimd to be apparently free from such microorganisms, a quan- tity of it may be added to sirup and the mixture fed to healthy colo- 1 The Berkefeld filter consists of a compact material (infusorial earth) in the form of a cylinder. A glass mantel (A) in which is fixed the filter forms a cup for holding the fluid to be filtered. Havmg filtered the aqueous suspension of crushed sacbrood larvae through paper, the filtrate is then filtered by aUowmg it to pass through the waUs of the Berkefeld cylinder (B). The filtrate from this filtration is collected into a sterile flask (F) through a glass tube (D) with its rubber connection (C). In flltermg m this instance gravity is the only force used. 28 BULLETIN 431, U. S. DEPAKTMENT OF AGEICULTUEE. nies. When all this is properly done, sacbrood will appear in the inoculated colonies. This shows that the virus ' of this disease, to a Fig. 26.— Berketeld filter (B) -witli the glass mantle (A), glass tubing (D), a connecting rubber tubing (C), and a flask (F) with a cotton plug (E). (Original.) certain extent, at least, passes through the Berkefeld filter. With this filter the virus is therefore filterable. 1 In referring to the infecting agent in sacbrood, the term "virus" is preferable to the terms "germ" or "parasite." In relation to the disease, however, its meaning is the same as that conveyed by the latter terms. SACBEOOD. 29 In the study of the virus of sacbrood use has been made also of the Pasteur-Chamberland filter ' (fig. 27). This is a clay filter, the pores of which are much finer than those of the Berkefeld used. In using this filter, an aqueous suspension of larvse dead of the disease is prepared as before. This is filtered by the aid of pressure obtained Pig. 27. — A convenient apparatus wmch can be employed in using the Pasteur-CIiamberland, Berkefeld, and other filters. Pasteur-Chamberland filter (b) with a glass mantle (a), arubber stopper (c) through which passes the filter, a connecting rubber tubing (d), glass tubing (e), a perforated rubber stopper (f), a vacuum jar (g), designed by the writer, in which is placed a cotton-stoppered and steril- ized flask, a glass stopcock (h), a vacuum gauge (i), a reservoir (m) with pressure-rubber connections 0), and a vacuum pump (k). (Original,) by means of a partial vacuum in an apparatus devised for this pur- pose. Filtrates obtained from this filter when fed to healthy colonies produced the disease. Since the virus of sacbrood wiU pass through iThe Pasteur-Chamberland filter consists of clay molded in the form of a hollow cylinder and baked. This is used with a glass cylinder (a) fitted with a rubber stopper (c). In the use of this filter, force is employed. This was obtained for these experiments through the use of a jar (g) devised by the writer in which a partial vacuum can be produced. In this jar, is placed a flask plugged with cotton and sterilized. Connections are made as shown in the illustration, the vacuum being produced through the use of the pump (k). In less than half an hour usually a half-pint of filtrate can be obtained with this apparatus. 30 BULLETIN 431, U. S. 0EPAETMENT OF AGKICULTUEE. the pores of the Pasteur-Chamberland filter also, it is therefore fil- terable and is very properly referred to as a "filterable"' virus. In considermg the virus of sacbrood it is suggested that the bee- keeper think of it as a microorganism ^ which is so small or of such a nature that it has not been seen, and which will pass through the pores of fine clay filters. This conception of it wiU at least make it more easily understood. WEAKENING EFFECT OF SACBROOD UPON A COLONY. The first inoculations in proving that sicbrood is an infectious disease were made on June 25, 1912. Two colonies were used, each being fed with material from a different source. The inocu- lation feedings were made on successive days. Sacbrood having been produced in the colonies, the inoculations were continued at intervals throughout July and August. During this period, a large amoimt of sacbrood was present in both colonies. By the end of July these colonies had become noticeably weakened, and by the end of August they had become very much weakened, as a result of the sacbrood present in them. On September 5 one of the colonies swarmed out. The brood (PI. IV) of this colony, large in quantity, was practically all dyiQg of sacbrood. The other colony, when examined on Sep- tember 16, was found to be very weak. At this time, however, most of the dead brood had been removed and healthy brood was being reared. This colony increased in strength and wintered successfully. The results obtained from the inoculation of these two colonies demonstrated not only that sacbrood is an infectious disease, but also that the disease in a colony tends to weaken it. The results indicate also that a colony may be destroyed by 'the disease, or it may recover from it, gain in strength, and winter successfully. Each year since 1912 two or more colonies have been fed sacbrood material at intervals during the brood-rearing season for the purpose of obtaining disease material for experimental purposes. The inocu- lated colonies in aU instances have shown a tendency to become weakened as a result of the inoculations. The death of the worker larvae is the primary cause for the weak- ness resulting from the disease in a colony. Another point to be thought of is that dead sacbrood larvae remaining in the cells for weeks, as they not infrequently do, reduce the capacity of the brood nest for brood rearing, which has a tendency also to weaken the colony. ' In searching the tissues of larvse dead of sacbrood and the filtrates obtained from them nothing has been discovered by the aid of the microscope, or culturally, which has yet been demonstrated as being the infect- ing agent. This being true, the virus could be spoken of tentatively as an "ultramicroscopic virus." It is preferable, for the present, howe^'e^, to refer to it simply as a filterable virus. 2 There is some question whether, in thn case of diseases having a virus which is Alterable, the infecting agent is in every instance a microorganism. The evidence is strong, however, that it is. SAOBEOOD. 31 AMOUNT OF VIRUS REQUIRED TO PRODUCE THE DISEASE, AND THE RAPIDITY OF ITS INCREASE. Assuming the virus of sacbrood to be a very minute microorgan- ism, the number of germs present in a larva dying of the disease must be considered as exceedingly large. Whether a single germ taken up by a larva wiH produce the disease in every instance, or in any instance, is not known. If the disease does result at any time from the ingestion of a single germ, aU of the conditions, it may be assumed, must be especially favorable for the production of the disease. From what is known of diseases of other animals and of man, and from the results thus far obtained in the study of sacbrood, it is well, at present, to assume that the number of sacbrood germs taken up by a larva may be so small that no disease results. It is certain, however, that a comparatively small nxmiber of sacbrood germs ingested by a lai-va about two days old are sufficient to produce the disease. That the few germs thus taken up can increase within the larva during an incubation period of five or six days to such a vast number as is assumed to be present in a larva dying of the disease indicates the extreme rapidity with which the germs are able to multiply. The minimum quantity of virus necessary to produce a moderate infection in a colony has not been definitely determined. It was found by experiments, however, that the virus contained in a single larva recently dead of the disease was sufficient to produce a large amount of sacbrood in a colony. As a very rough estimate, it may be said that the quantity of virus in a single larva dead of sacbrood is sufficient, when suspended in half a pint of sirup and fed to a healthy colony, to produce in- fection in and deaith of afc least 3,000 larvse. Startmg then with the virus contained in a single larva, in less than one week it would easily be possible to have 3,000 larvse dead of the disease, which means that the virus has been increased 3,000-fold within one week. This latter amount of virus would be sufiicient to produce an equal amount of infection in 3,000 colonies, increasmg the amount of virus again 3,000-fold. In less than two weeks, therefore, theoretically it would be possible to produce a sufficient amount of virus to infect 9,000,000 colonies, more colonies probably than are to be found at present in the United States. Carrymg the idea somewhat further, within three weeks, theoretically enough virus could be produced to inoculate every colony in existence. These facts are sufficient to indicate somewhat the enormous rapidity with which the virus of sacbrood is capable of increasing. 32 BULLETIN 431, TJ. S. DEPAETMENT OF AGEICULTTJBE. METHODS USED IN MAKING EXPERIMENTAL INOCULATIONS. The laboratory study of bee diseases being new, it has been neces- sary in many instances to devise new methods. In the experimental inoculations of bees the methods used have undergone revision from time to time. Those now employed have proved quite satis- factory. As the virus of sac- brood has not been cultivated in the lab- oratory artificially, it has been necessary in these investigations to inoculate a large number of colonies. A nucleus of bees that could be accom- modated on from 3 to 6 brood frames was found to serve very satisfactorily the purpose of an ex- perimental colony. The queen should al- ways be clipped. The frames are placed in one side of a 10-frame hive body (fig. 28). Over the entrance to the hive is placed wire cloth, leaving a small space of about 1 inch in length on the side occupied by the brood frames. Petri dishes * (fig. 29) serve well the purpose of a feeder. Both halves of the dish are used as receptacles. These are placed, preferably about four of the halves, within the hive on the bottom board on the side not occu- pied by frames. The hives of the experimental apiary (PI. Ill) are arranged chiefly in pairs, with the entrances of consecutive rows pointing in opposite directions. The space occupied by the apiary should be ' A Petri dish, a much-used piece of apparatus in a laboratory, is simply a shallow, circular, glass dish with a flat bottom and perpendicular sides. It consists of two halves, a bottom and a top. These are very similar. The top half, being slightly larger, fits over the bottom one when the two halves are placed together. Fig. 28.— The hive as it is employed to house and feed a colony used for experimental inoculations. Here are shown four Hoffman frames, a division board, four open Petri dishes as feeders, and the en- trance nearly closed with wire cloth, the opening being on the side of the hive body occupied by the colony. The dimensions indicated are approximate. The angle at which the hive was photographed for this drawing caused its length to appear foreshortened. ( Original. ) Fig. 29.— Petri dish. The top half is slightly raised. Tliosa used here are 4 inches in diameter. (Original.) Plate Bui. 431 , U. S. Dept. of Agriculture. SACBTtOOD. 33 broken up, preferably by trees or shrubbery. By these means, it will be observed, there is a tendency to minimize the likelihood of robbing, swarming, absconding, and accidental straying or drifting of bees to foreign colonies. In preparing the material with which the colony is inoculated, larvse in early stages of the disease are picked from the brood frames, crushed, and added to sugar sirup. The crushed mass from 10 or more sacbrood larvse, sus- pended in somewhat more than half a pint of sugar sirup, has been found to be a suitable quantity of the infective material to use in making an inoculation. The suspension may be fed to the bees as one feeding or more. The inoculation feedings should be made as a rule toward evening to avoid the tendency to rob, which may be noticed during a dearth of nectar. Inocu- lations should not be made when the tendency to rob is at all marked. Before a colony is inoculated it should be deter- mined that its activities are normal. A colony should not be inoculated for several days after it has been made by division, or immediately after its removal from a foreign location. An experimental colony when inoculated should have larvse of all ages, and a queen doing well. Between five and six days after a colony has been inoculated with sacbrood virus, the first symptoms of the disease are to be expected. The finding of capped larvse having a slightly yellowish hue (fig. 12; PI. II, h, h) is the best early symptom by which the presence of the disease may be known. Another method of inoculation may be used and under certain circiimstances is desirable. The method is more direct than the one just described. The crushed tissues of a diseased larva are suspended in a small amount of water or thin sugar sirup. With a capillary pipette (fig. 30) made from smaU glass tubing, a very small amoimt of the suspension is added di- rectly to the food which surrounds the healthy larva in the cell. This is easily done. Having drawn some of the suspen- sion into the pipette, carefully touch the food iu the ceU surround- ing the larva with the point of the pipette. A small amount of the suspension will flow out and mix with the food. Larvse approxi- mately two days of age should be selected for feeding. A dozen Fig. 30— Capillary pipette. A piece of glass tubing drawn to capil- lary size at one end. Keduced to three-fourths of the size nsed. (Original.) 34 BULLETIN 431, TJ. S. DEPARTMENT OF AGRICULTURE. or more should be fed in making an inoculation. The area of brood inoculated may be designated by marking on the brood frame, or by removing the brood from around the area inoculated, thus marking it off. MEANS FOR THE DESTRUCTION OF THE VIRtS OF SACBROOD. Although the virus of sacbrood may increase with great rapidity, fortunately it is quite as readily destroyed. Nature suppUes many means by which this may be accomplished. While theoretically a sufficient amount of virus may be produced within one month to inoculate all the bees in existence, within another month, if left to natiu-al means alone, practically all such virus would be destroyed. This latter fact constitutes one of the chief reasons for the compara-. tively rapid self-recovery of colonies from this disease. It was observed in the experiments that larvse dead of sacbrood when left in the brood comb ceased to be infectious in less than one month after death. HEATING REQUIRED TO DESTROY SACBROOD VIRUS WHEN SUSPENDED IN WATER. Approximate results have been published (White, 1914) relative to the heating that is necessary to destroy the virus of sacbrood when it is suspended in water. In the following table are given some results which have been obtained : Table I. — Effect of heating on the virus of sacbrood suspended in waters Date of inoculation. Temperature. Time of heating. Results of inoculation. Aug. 6, 1913.. Sept. 10, 1913 Sept. 9, 1913., Sept. 18, 1913 June 30, 1915. Sept. 10, 1913 Aug. 28, 1915. Sept. 10, 1913 Aug. 28, 1915, Aug. 26, 1913. Do Do Do °F. 122 131 131 135 136 136 138 140 142 149 158 167 176 MintUes. 3D 10 20 15 10 10 10 15 10 15 15 15 15 Sacbrood produced. Do. Do. Do. Do. No disease produced. Do. Do. Do. Do. Do. Do. Do. 1 Fractions will be omitted in this paper, the nearest whole number being given. It will be observed from Table I that 138° F. (59° C.) maintained for 10 minutes was sufficient to destroy the virus of sacbrood in the inoculation experiments recorded. Technically, in view of the variable factors which must be considered in experiments of this kind, this residt, as representing the thermal death point of the sacbrood virus, should be considered as being only approximate. For practical purposes, however, it is sufficient. SACBROOD. In performing these experiments a crushed mass, representing from 10 to 20 larvse recently dead of the disease, is diluted to about 10 times its volume with tap water. About one-half ounce of this suspension is placed in a test tube (fig. 31), almost filling it. The tube is stoppered with a perforated cork, bearing a short glass tube of small cahber and drawn at one end to capillary size. This is all immersed in water at a temperature to which it is desired that the virus shall be heated. It requires nearly five minutes for the tem- perature of the suspension in the tube to reach that of the water outside. Aftei' reaching the degree desired the 'temperature is maintained for 10 minutes, after which the tube is removed and the contents added to about one-half pint of sirup. The suspension is then fed to a healthy colony. If by such a feeding no sac- brood is produced, the virus is considered as having been destroyed by the heating. On the other hand, if the disease is produced it follows naturally that the virus had not been destroyed. HEATING REQUIRED TO DESTROY SACBROOD VIRUS WHEN SUSPENDED IN GLYCERINE. 35 ■o !i In determining the amount of heating that is necessary to destroy the virus of a disease when it is suspended in a liquid, the results should always be given in terms of at least the three factors, (1) degree of temperature, (2) time of heating, and (3) the medium in which the virus is suspended. With the virus of sacbrood the results vary markedly; depending upon the nature of the liquid in which the suspension is made. To illustrate this point the re- sults of a few inoculation experiments are given here in which the virus was heated while suspended in glycerine. 1 1 a » II Table II. — Effect produced by heating the virus of sacbrood suspended in glycerine. Date of inoculation. Temperature. Time of heating. Results of inoculation. June 25, 1915 'F. 140 149 158 160 163 167 °C. 60 65 70 71 73 75 Minutes. 10 10 10 10 10 10 Sacbrood produced. Do. June 24, 1915 . . . June 25, 1915 Do. Aug. 28, 1915 Do. Do Aug. 7, 1915 Do. w 3 I 36 BULLETIN 431, U. S. DEPAETMENT OF AGEICULTTJRE. In these inoculations it will be observed that a temperature some- what greater than 158° F. (70° C.) maintained for 10 minutes was necessary to destroy the virus of sacbrood when it was suspended in glycerine, while a temperature somewhat less than 140° F. (60° C.) is sufEcient to destroy it when suspended in water (p. 34). The same technique was employed when glycerine was used as the suspending medium as was employed when water was used as the medium. The same strain of virus was used in both instances. The point here illustrated is of special interest in connection with the heating of honey containing the virus of sacbrood. HEATING REQUIRED TO DESTROY SACBROOD VIRUS WHEN SUSPENDED IN HONEY. From the results obtained by heating the virus of sacbrood iu glycerine as given above it might be expected that a higher tempera- ture would be necessary to destroy the virus when it is suspended in honey than when it is suspended in water. In determining the heating necessary to destroy the virus when suspended in honey the technique followed was similar to that employed when water and glycerine suspensions were used. The virus used in the inoculations bearing the date 1915 was of the same strain in all instances. Table III. — Results obtained vhen the virus of sacbrood was heated in honey. Date of inoculation. June 1, 1915. June 11, 1915, Do June 4, 1915. June 24, 1915. Do June 1, 1915. June 18, 1915, July 3, 1915.. Aug. 28, 1915 Aug. 7, 1916. Aug. 28, 1915 June 1,1915. Aug. 7, 1915. June 1, 1915. Temperature. °F. 140 145 149 154 156 158 158 158 160 160 163 163 167 167 176 'C. 60 63 65 68 69 70 70 70 71 71 73 73 75 75 80 Time of heating. Minutes. 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 J Results of inoculation. Sacbrood produced. Do. Do. Do. Do. Do. No disease produced. Do. Do. Do. Do. Do. Do. Do. Do. As shown by the results recorded in Table III, the virus of sacbrood when suspended iu honey was destroyed in 10 minutes at a tempera- ture very near 158° F. (70° C). This temperature is more than 18° F. (10° C.) greater than the temperature required to destroy in the same time the virus when suspended in water and approximately equal to that necessary to destroy it when suspended in glycerine. SAOBROOD, 37 RESISTANCE OF SACBROOD VIRUS TO DRYING AT ROOM TEMPERATURE. In the experiments made for the purpose of determining the amount of drying which the virus of sacbrood will withstand, larvse recently- dead of the disease were used. These are crushed, strained through cheesecloth, and the crushed mass poured into Petri dishes (fig. 32) to the extent of a thin layer for each dish, the material in each being the crushed remains of about 30 larvae. These are placed in a drawer, shielding the larval material from the light. The drying then pro- ceeds at the temperature of the room. This temperature varied greatly from day to day, sometimes being as high as 93° F. (34° C). At intervals, reckoned in days, after the preparation of the virus, colonies are inoculated. An aqueous suspension is made of the drying larvaJ content con- tained in a Petri dish. This is added to sirup, and the sirup suspension is fed to a healthy colony gave the following results : Fig. 32.— Open Petri dish. One-half of Petri dish, either top or bottom. (Original.) The experiments Table IV. — Resistance of sacbrood virus to drying at room temperature. Date of inoculation. Time of drying. Resxilts of inoculatidn. Aug. 8, 1914.. Aug. 14, 1914. Sept. 6, 1915. July 1, 1915. . Sept. 28, 1915 Julys, 1915.. Sept. 3, 1915. Sept. 27, 1915. Oct. 9, 1914.. July 29, 1915. Sept. 3, 1915. Do May 22, 1915. Do 3 days 7days 13 days 16 days 18 days 20 days 22 days 26 days 28 days 28 days 35 days 45 days 7 months 12 days . 7 months 21 day^. Sacbrood produced. Do. Do. Do. Do. Do. No sacbrood produced. Do. No. Do. Do. Do. Do. Do. From the results recorded in Table IV it will be noted that the virus of sacbrood in the experiment referred to withstood drying at room temperature for approximately three weeks. The inoculations made during the third week indicated, by the re- duced amount of sacbrood produced, that much of the virus had already been destroyed. Obtaining negative results from the use of larval material which had been drying more than seven months tends toward eliminating the possibility that the virus possesses a resting stage. 38 BULLETIN 431, XJ. S. DEPARTMENT OF AGEICULTTJEE. Similar prelimiaary experiments made to determine the amount of drying which the virus of sacbrood will withstand at outdoor tempera- ture and at incubator temperature (about 99° F. [37° C.]) gave results approximately those obtained from drying at room temperature, the time being somewhat less in the case of drying at incubator tempera- ture. Prehminary experiments indicate also that when the virus is mixed with poUen and allowed to dry the period for which it remains virulent is iacreased only slightly. RESISTANCE OF SACBROOD VIRUS TO DIRECT SUNLIGHT WHEN DRY. In the experiments made to determine the amount of sunlight which the virus of sacbrood is capable of resisting, Petri-dish prepara- tions similar to those made in the drying experiment were prepared. After drying a few hours in the room the uncovered dish is exposed to the direct rays of the sun. At different intervals, measured in hours, inoculations of healthy colonies are made similar to those in the drying experiments. The following results were obtained: Table V. — Resistance of the virus of sacbrood, when dry, to direct sunlight. Date of inoculation. Time of exposure to sun's rays. Results of inoculation. Sept. 17, 1915. July 29, 1915.. Sept. 17, 1915. Sept. 16, 1915. Do Do Aug. 25, 1915. Sept. 10, 1915. Do Sept. 9,1915.. Do Aug. 19,1915. July 16, 1915. Aug. 20, 1915. Sept. 11, 1915. Hours. 2 2J 6 4 5 7 9 12 13 18 21 Sacbrood produced. Do.. Do. Do. Do. Do. Do. No disease produced. Do, Do. Do. Do. Do. Do. Do. The results recorded in Table V show that the virus of sacbrood in the experiments made was destroyed in from four to seven hours' exposure to the direct rays of the sun. The results obtained also indicate that much of the virus was destroyed in a 2-hour exposure to the sun. It vnR be readily appreciated that the time that the virus will resist the sun's rays will depend a great deal upon the intensity of the rays at the time of its exposure and the thickness of the layer of the infective larval material in the Petri dish. The drying that SACBKOOD. 39 would naturally take place during the exposure to the sun would tend also to destroy the virus, but as the resistance to drying is better given in weeks than days, this factor may be disregarded here. RESISTANCE OF SACBROOD VIRUS TO DIRECT SUNLIGHT WHEN SUS- PENDED IN WATER. In the experiments made for the purpose of determining the resist- ance of the virus of sacbrood to the direct rays of the sun when suspended in water, Petri dishes were again used. About 1^ ounces of the aqueous suspension containing the crushed tissues of 30 larvae is poured into the dish and exposed to the direct rays of the sun. After intervals reckoned in hours the inoculations of healthy colonies are made. The contents of a single Petri dish are added to about one- half pint of sirup and the suspension fed to a healthy colony. The following results were obtained from the experiments: Table VI. — Resistance of sacbrood virus to the direct rays of the sun when suspended in water. Date of inoculation. Time of exposure to sun's rays. Results of inoculation. Sept. 10, 1915. Aug. 20, 191S. Sept. 14, 1915. Aag.24,igi5. Aug. 18, 1915. Sept. 9, 1915.. Sept. 10, 1915. Aug. 24, 1915. Do Aug. 16, 1915. Sept. 8, 1915.. Do Sept. 9, 1915.. Do Aug. 25, 1915. Aug. 20, 1915. Jtdyia, 1915. Aug. 26, 1919. Sacbrood produced. Do. Do. Do. Do. Do. Do. " Do. No disease produced. Do. Do. Do. Do. Do. Do. Do. Do. Do. From Table VI it wiU be seen that when suspended in water the virus of sacbrood was killed in from four to six horn's. The aqueous suspensions in the Petri dishes in these experiments did not reach by several degrees the temperature 138° F. (59° C.) at which the virus "is destroyed readily by heating (p. 34). Naturally experiments of the natm-e of those in this group will vary in all cases with the intensity of the sun's rays to which the virus is exposed. The exposures were made in these experiments between 9 and 4 o'clock, the sun's rays toward the middle of the day being most often used. 40 BULLETIN 431, U. S. DEPARTMENT OP AGBICULTtTEE. RESISTANCE OF SACBROOD VIRUS TO DIRECT SUNLIGHT WHEN SUS- PENDED IN HONEY. The crushed and strained tissue mass of larvae dead of sacbrood was susj)ended in honey and exposed to the direct rays of the sun. To prevent robbing by bees, closed Petri dishes were used. At intervals reckoned in hours healthy colonies were inoculated, each with the virus from a single Petri dish. The exposures were made during the day between 9 and 4 o'clock, preference being given to the hours near midday. The group of experiments conducted on this point gave the following results : Table VII. — Resistance of the sacbrood virus to direct sunlight when suspended in honey. Date of inoculation. Time of exposure to sun's rays. Besults of inoculation. Aug. 24, 1915. Do Aug. 18, 1915. Sept. 9, 1915.. Sept. 10, 1915. Aug. 24, 1915. Aug. 16,1915. Aug. 25, 1915. Sept. 8, 1915.. Do Sept. 9, 1915.. Do Aug. 25, 1915. Sept. 11, 1915. Aug. 26, 1915. Sept. 11, 1915, Hours. 1 2 4 4 4 5 5 5 5 6 7 8 10 12 13 18 Sacbrood produced. Do. Do. Do. Do. Do. No disease produced. Do. Do. Do. Do. Do. Do. Do. Do. Do. From the results of the experiments recorded m Table VII it wUl be observed that the virus of sacbrood when suspended in honey was destroyed by the direct rays of the sun in from five to six hours. These figures represent the time for destruction of all of the virus used in each experiment. The results obtained from the experi- ments indicate, however, that much of it was destroyed earlier. LENGTH OF TIME THAT SACBROOD VIRUS REMAINS VIRULENT IN HONEY. In devising methods for the treatment of sacbrood it is of particular interest to know the length of time that the virus will remain vindent when it is ia honey. Experiments have been made to gain data on this point. Larvae recently dead of sacbrood are crushed, strained, and suspended ia honey. About one-half piat of the suspension, representing the virus from about 30 dead larvae, is placed in each of a number of glass flasks. These are allowed to stand at room temper- ature, being shielded from the light by being placed in a closed cabinet. SAOBEOOD. 41 After periods reckoned in days inoculations of healthy colonies are made. The following results have been obtained : Table VIII. — Length of time the virus of sacbrood remains virulent in honey. Date of Inoculation. June 17, 1915. June 4, 1915.. Oct. 2, 1915... Sept. 3, 1915.. July 29, 1915.. June 30, 1915. Do July 17, 1915.. Oct. 21, 1915.. Sept. 8,1915.. May 13, 1915.. May 6, 1915... May 4, 1915... May 18, 1915.. Sept. 3, 1915.. Time virus was in Results of inoculation. honey. Mos. Days. 20 Sacbrood produced. 23 Do. »() Do. 24 No disease produced. 29 Do. 33 Do. 35 Do. 3H Do. 49 Do. 70 Do. 17 10 Do. 7 20 Do. 8 2 Do. 8 21 Do. 12 1 Do. I Tlie dead brown liuval remains were not cruslied before being introduced into the honey. The experiments recorded in Table VIII show that the virus of sacbrood when suspended in honey at room temperature remained virulent for three weeks, but was entirely destroyed before the end of the fifth week. It is most likely that the virus in most instances is destroyed by the end of one month at this temperatiire. The experiments in which the virus had been allowed to remain in the honey for more than seven months suggest that there is prob- ably no resting stage of the virus to be considered in this connection. The facts tend to indicate that the vkus does not receive any marked amount of protection by being in honey. From the dates of the experiments in this group it wiU be noted that the virus was sub- jected to summer temperature. The evidence at hand indicates that it remains virulent somewhat longer when the temperature is lower. RESISTANCE OF SACBROOD VIRUS TO THE PRESENCE OF FERMENTA- TIVE PROCESSES. Fermentation and putrefaction ^ are other means by which the virus of sacbrood may be destroyed in water. A crushed and strained mass of tissue from larvae recently dead of the disease is suspended m a 10 per cent sugar (granulated or cane sugar) solution. 1 "Fermentation" has reference here particularly to the breaking up of carbohydrate substances by the growth of microorganisms, the sugars in honey being naturally the carbohydrates especially of mterest in these discussions. The process results in the formation of a large number of suhstances-acids, alcohols, etc. The odor accompanying such a process could not be called offensive. By the term "putrefaction" is meant the breaking up of nitrogenous organic substances by microorganisms. These have a chemical composition quite different from the carbohydrates. When broken up the resulting substances are more often alkaline in nature. The odor from a suspension in which putrefactive processes are gomg on is usually distinctly offensive. 42 BULLETIN 431, U. S. DEPARTMENT OP AGRICTJLTTJRE. A small quantity of soil is added to inoculate the suspension further. This is then distributed in test tubes (fig. 33), the quantity in each tube representing the virus from about 15 larvse. These suspensions are allowed to remain at room temperature, shielded from the Ught. Under these conditions fermenta- tion goes on rather rapidly. After intervals reckoned in days colonies free from the disease are inoculated, each with the suspension from a single tube. Results from such inoculations are given in the following table: Table IX. — Resistance of sachrood virus to fermentation in a 10 per cent sugar solution at room temperature. v_y Date of inoculation. Period of fermen- tation. Results of inoculation. Sept. 9, 1915. Days. 1 2 3 4 3 5 5 7 9 13 34 51 85 87 90 244 Sacbrood produced. Sept. 11, 1915.. Do. Do Do. Sept. 13, 1915 . Do. July 14, 1916 No disease produced. July 22, 1916.... Do. Sept. 14, 1915 . Do. Sept. 22, 1916 Do. July 10, 1916.... Do. June 10, 1915. . Do. July 7, 1914 ' Do. Aug. 27, 1914.. . Do. Do Do. Do Do. Do Do. Do Do. Fia. 33.— Test tube bearing a cotton plug, used in testing the ef- fect of fermentation, putrefaction, and dis- infecting agents on the virus of sacbrood. (Original.) 1 The resultsrecorded for 1914 were obtained with a suspension of crushed larvse, in various stages of decay, in sirup made from about equal parts water and sugar. From the results of experiments recorded in Table IX it win be noted that the virus of sacbrood was destroyed in from three to five days in the presence of fermentation in 10 per cent cane sugar (saccharose) at room temperature. As the rapidity of fermentative processes varies with the temperature present, it is natural to sup- pose that the time required for the destruction of the virus will vary. From experiments it is found that at incubator temperature the time is slightly less, and at outdoor temperature it is somewhat greater than at room temperature. RESISTANCE OF SACBROOD VIRUS TO FERMENTATION IN DILUTED HONEY AT OUTDOOR TEMPERATURE. Employing the egg test * as used by beekeepers in diluting honey for the purpose of making vinegar, it is found that it requires about ' This test is applied in the following manner: Water is added to honey until an egg placed in the mixture Is nearly submerged, the surface remaining above the liquid being only about as large as a 10-cent piece. SACBEOOD. 43 fom- volumes of water to one of ripened honey to obtain the strength recommended. The honey solution by volmne, therefore, is about 20 per cent honey. A suspension of the virus of sacbrood in such a solution is dis- tributed in test tubes placed in an empty hive body and allowed to ferment at outdoor temperature. After periods reckoned in days colonies are inoculated as was done in case of the sugar solutions described above. The following results were obtained from the experiments performed : Table X. — Resistance of sacbrood virus to fermentative processes in a 20 per cent honey solution at outdoor temperature. Date of inoculation. Results of inoculation. Sept. 11, 1915. Sept. 13, 1915. Sept. 14, 1915. Aug. 4, 1915.. Sept. 15, 1915. Sept. 14, 1915. Sept. 22, 1915. Sept. 17, 1915. Sept. 8, 1915.. Sacbrood produced. Do. No disease produced. Do. Do. Do. Do. Do. Do. In the presence of fermentative processes taking place in a 20 per cent honey solution at outdoor temperature it wiU be observed that the virus of sacbrood in the experiments recorded in Table X was destroyed in six days. The outdoor temperature during these experiments was quite warm. Had it been cooler, the time for the destruction of the virus would have been somewhat increased. In the making of vinegar it may be concluded that the virus of sacbrood, should it be present in the honey used, would be destroyed in a com- paratively short time as a result of fermentation. RESISTANCE OF SACBROOD VIRUS TO THE PRESENCE OF PUTREFACTIVE PROCESSES. Larvse containing the virus of sacbrood are crushed and suspended m water. A small quantity of soil is added. The suspension is stramed and distributed in test tubes. These are allowed to stand at room temperature in a state of putrefaction. After periods reckoned in days colonies free from the disease are moculated, each with the contents of a single tube added to sirup. From experiments of this kind the results following have been obtained. 44 BULLETIN 431, V. S. DEPARTMENT OP AGBICULTUEE. Table XI.— Resistance of sacbrood virus to putrefaction. Aug. Aug. Aug. July Sept. Sept. July July May Sept. Aug. Sept. Sept. iept uly Date of inoculation. 6,1914.. 7,1914.. 10,1914. 20, 1915. 13, 1916 14, 1915 22,1915. 8,1915.. 22,1915. , 22, 1916. 18, 1915. , 16, 1914. , 25, 1914. 1,1915.. Results of inoculation. Sacbrood produced. Do. Do. Do. Do. Do. Do. Do. Do. No disease produced. Do. Do. Do. Do. From Table XI it will be noted that the virus of sacbrood was destroyed m. the experiments recorded in from 7 to 10 days. As in the case of fermentation, so in the case of putrefaction, it is to be expected that the time for the destruction of the virus will vary appreciably with the temperature at which the putrefactive processes take place. RESISTANCE OF SACBROOD VIRUS TO CARBOLIC ACID. Larvae recently dead of sacbrood are crushed and strained. This larval mass is diluted with carbolic acid in aqueous solution. About 10 parts of carboUc acid to 1 part of the larval mass is used. This suspension is distributed in test tubes and allowed to stand at room temperature. Each tube contains the virus from about 15 larvae. After periods, reckoned in days, colonies free from disease are inocu- lated, each with the contents of a single tube added to sirup. Carbolic acid solutions of J, 1, 2, and 4 per cent were used in mak- ing the suspensions. The following results were obtained from the experiments : Table XII. — Resistance of sacbrood virus to carbolic acid. Date of inoculation. Strength of car- Time in bolic acid used. Per cent. Bays. ,^ 1 1, 10 y 24 38 4 50 i 50 i 2.38 1 16 25 38 50 50 261 Results of inoculation. Sept. 3, 1914.. Sept. 18, 1914 . Sept. 3, 1914.. Sept. 17, 1914. Aug. 12, 1916. Aug. 20, 1916. May 14, 1915- . Sept. 3, 1914.. Sept. 18, 1914 . June 23, 1915.. Sept. 17, 1916. Aug. 12, 1916., Aug. 21, 1915.. June 4, 1915... Sacbrood produced. Do. Do. No disease produced. Do. Do. Do. Sacbrood produced. Do. Do. No disease produced. Do. Do. Do. SACBROOD. 45 Table XII. — Resistance of sacbrood viriis to carbolic acid — Continued. Date of inoculation. Strength ot car- bolic add used. Sept. 3, 1914.... Sept. 18,1914... June 23, 1915... Sept. 17, 1915... Aug. 12, 1915... Aug. 21, 1915... June 23, 1915.. July 1,1915... June 23, 1915. Aug. 12, 1915. Per cent. 2 2 2 2 2 2 Time in suspen- sion. Days. 1 16 25 38 42 50 Hmirg. Days. 25 50 Besults o( inoculation. Saobrood produced. Do. Do. No disease produced. Do. Do. Sacbrood produced. Do. No disease produced. Do. From the preliminary results recorded in Table XII it wiU be observed that the virus of sacbrood shows a marked resistance to the disinfecting power of carbolic acid. Under the conditions of the experiments the virus resisted its action for more than three weeks in ^, 1, and 2 per cent aqueous solutions. These results lead naturally to a consideration of the effect of drugs on the virus of sacbrood in the treatment of the disease. On this point complete data are yet wanting. While the disinfecting power of a compound, as shown in experi- ments such as those described above for carboHc acid, may indicate something as to the value of the compound as a drug, it does not necessarily prove its value. More definite proof is gained through feeding colonies with the virus suspended in honey medicated with the drug, and then continuing to feed the inoculated colonies with honey similarly medicated daUy thereafter untU the time for the appearance of the disease. To illustrate the nature of experiments which are being conducted to determine the value of drugs in the treatment of sacbrood, experi- ments with quinine and carbolic acid are here referred to. A colony was fed the virus of sacbrood suspended in honey and water, equal parts, to which was added 5 grains of the bisulphate of quinme to one-half pint of diluted honey, and on each of the five days following the inoculation the same colony was fed diluted honey containing no virus, but medicated with quinine in the same way. On the seventh day following the inbciilation with the virus there was found to be a large quantity of sacbrood produced in the colony so inoculated and treated. A s imil ar experiment in which carbohzed honey was used gave like results. These experiments, although not furnishmg conclusive proof, do indicate something of what might be expected from the use of quinine or carbolic acid as a drug in the treatment of sacbrood. 46 BULLETIN 431, U. S. DEPAETMENT OF AGBICULTUBE. Technically the foregoing studies should be thought of as being prehminary. Questions relating to virulence of the virus, resistance of the bees, technique, and many other factors contribute to make results such as these vary. For practical purposes, however, they are sufficiently complete. In estimating the time necessaiy for the destruction of the virus in practical apiculture by any of the fore- going tables of results it should be emphasized that the time element should be somewhat increased, inasmuch as the conditions present in the experiments were more favorable for its destruction than would ordinarily be the case in practice. MODES OF TRANSMISSION OF SACBROOD. The transmission of a brood disease must be thought of as taking place (1) from diseased to healthy brood within a colony and (2) from a diseased colony to a healthy one. The manner in which sacbrood is spread naturally depends directly upon the modes by which the virus of the disease is transmitted. As is shown experimentally, the virus of sacbrood produces the disease when it is added directly to the food of young larvae or when it is mixed with sirup and fed to a colony. From this fact it is fair to assume that sacbrood may result whenever the food or water used by the bees contains the hving virus of the disease. Bees have a tendency to remove diseased or dead larvae from the cells. When the removal is attempted about the time of death, it is done piecemeal. Each fragment removed from such a larva, if fed t(5 a young healthy larva within a week, would most likely produce sacbrood in the larva. Within the hive, therefore, the dis- ease may be transmitted to healthy larvae more or less directly in this way. Just what becomes of these bits of tissue removed from the dis- eased larvae, however, is not known. If it were the rule that the tissues of the dead larva after being removed in fragments were fed unaltered to the young healthy larva? within two weeks after its removal, it would seem that the disease would increase rapidly in the colony as a result. Such an increase, however, is unusual, the tendency in a colony being in most cases toward a recovery from the disease. This fact leads one to think of other possibilities regarding the destiny of the infected tissues removed as fragments from the dis- eased larvie. If the infective material were fed to the older larva;, death probably would not result. Should it be used by adult bees as food for themselves, the hkehhood of the transmission of the dis- ease under such chcumstances would apparently be very materially reduced. If the infective material were stored with the honey and SACBEOOD. 47 did not reach the brood within a month or six weeks, it is not prob- able that the disease would be transmitted mider such cu-cumstances (p. 41). Should the dead larva or any fragments of them be car- ried out of the hive, the virus would have to be returned to the hive, as a matter of course, before further mfection of the brood could take place from such infective material. It is left to be considered in what way the infective material if removed from the hive might be returned to the brood and infect it. Should any material containing the virus reach the water sup- ply of the bees, or the flowers visited by the bees, it is within the range of possibUity that some of the hving virus might be returned to the hive and reach healthy young larvae. While out of the hive, however, the virus must withstand certain destructive agencies in nature. Under more or less favorable cir- cumstances it would withstand drying alone for a few weeks (p. 37), but if exposed to the sun it might be destroyed in a few hours, (p 38). If the virus were subjected to fermentation it might be destroyed within a week (p 43), and if subjected to putrefaction, within two weeks (p. 44). The experimental evidence indicates that the virus, once out of the hive and freed from the adult bees removing it, during the warmer seasons of the year, at least, has but little chance of being returned to the hive and producing any noticeable infection. In the experimental apiary (PI. Ill) a large number of colonies have been heavily infected with sacbrood through experimental inoculation, and no infection was observed to have resulted in the uninoculated colonies. If throughout the main brood-rearing season the usual source of infection were the flowers or the water supply, a quite different result would be expected. Tentatively it may be concluded, therefore, that the probability of the transmission of the virus of sacbrood by way of flowers visited by bees, practically considered, is quite remote, being, however, to a limited extent theoretically possible. It would seem that there is a greater likeUhood of the water supply being a source of infection than flowers. The chances for infection from this source, should it occm- at all, would be greater in the spring, as at such a time the quantity of infective material in dis- eased colonies is greater, increasing the chances that some of it might be carried to the water supply and contaminate it, and fur- thermore, the destructive agencies in nature are at this time less efficient. Bees drifting or straying from infected colonies to healthy ones must be thought of as possible transmitters of the disease. That the disease is not spread to any great extent in this way is evidenced 48 BULLETIN 431, TJ. S. DEPARTMENT OF AGEICULTUBE. by the fact that colonies in the apiary that were not inoculated experimentally remained free from disease, although many colonies in the apiary were heavily infected at the time. Sacbrood has a tendency to weaken a colony in which it is present. Frequently this weakness is noticeable and often marked. Kob- bing, which occurs not infrequently at such a time, results in the transmission of the virus, to some extent at least, directly to healthy colonies. Kobbing, therefore, must always be considered as a prob- able means of transmission. The modes of transmission of sacbrood within the colony and from colony to colony, as will be seen, are not by any means completely determined. In what way the sacbrood virus is carried over from one brood-rearing season to another is one of the many problems con- cerning this disease that are yet to be solved. The foregoing facts, accompanied by the brief discussions, it is hoped, wiU throw some light upon this important phase of the study — the transmission of this disease — and will serve as an aid to later researches. DIAGNOSIS OF SACBROOD. The diagnosis of sacbrood can be made from the symptoms already described (p. 10). The colony may or may not be noticeably weak- ened. The adult bees are normal in appearance. Scattered here and there on the brood frame among the healthy brood are found dead larvae in the late larval stage. Usually there are only a few of them, yet sometimes there are many. These larvae may be in capped or uncapped ceUs. When found in uncapped cells, however, the cap- pings had already been removed by the bees after the death of the larvae. The cap over a dead larva in a cell may be found punctured or not. The brood possesses no abnormal odor, or practically none. The post-mortem appearances of larvae dead of the disease are espe- cially valuable in making the diagnosis. The larva is found extended lengthwise in the cell and on its dorsal side. Throughout the period of decay it will be found to maintain much of the form and markings of a healthy larva of the age at which it died. Soon after death the larval remains are slightly yellow. After a period they assume a brownish tint. Since the brown color deepens as the process of decay and drying takes place, the remains may be foimd having any one of a number of shades of brown. They may appear at times almost black. After death the cuticular portion of the body wall becomes tough- ened, permitting the easy removal of the larva intact from the cell. When removed, the saclike appearance of the remains becomes easily apparent. Upon rupturing the cuticular sac the contents are found to be a brownish, granular-appearing mass suspended in a compara- SACBROOD. 49 livoly small quantity of more or less clear liquid. The scales formed by the drying of the decaying remains are easily removed from the cells. After becoming quite dry many of them indeed can be shaken from the brood comb. Upon crushing larvse which have been found dead for some time but not yet dry, a marked unpleasant odor will be noticed if the crushed mass is held near the nostrils. Microscopically no microorganisms are to be found in the decay- ing remains of the larviie. Cultures made from them are also neg- ative. Differential diagnosis. — Sacbrood must be differentiated from the- other brood diseases. American f oulbrood may be recognized by the peculiar odor of the brood combs when the odor is present. The body wall of the larval and pupal remains is easily ruptured, and the decaying mass becomes viscid, giving the appearance popularly referred to as "ropiness." The scale adheres quite firmly to the floor of the cell. The presence of BaciUus larvse in the hrood dead of the disease is a positive means by which it may be differentiated from sacbrood. European foulbrood may be recognized by the fact that the larvae as a rule die while coiled in the cell and before an endwise position is assumed. In the majority of instances, therefore, death takes place before the cells are capped. The sachke appearance characterizing the dead larvse in sacbrood is absent. The granular consistency of the decaying mass is absent also. Microscopically, a large number of bacteria are found in larvae dead of European foulbrood, but are absent in larvse dead of sacbrood. The presence of Bacillus pluton is a positive means by which European foulbrood may be recognized. Bacillus alvei and other species may also be present. Sacbrood must also be differentiated from other conditions re- ferred to as chilled brood, overheated brood, and starved brood, which occasionally are encountered. This can be done by a compar- ison of the symptoms presented by these different conditions with the symptoms of sacbrood, and the history of the cases. Some of the larvse dead from these conditions will be found to have died while yet coiled in the cell. 'This fact suggests some condition other ^;han sacbrood. When dying later, the sachke remains characterizing sac- brood are not present in conditions other than sacbrood. PROGNOSIS. The tendency in a colony affected with sacbrood is to recover from the disease. Colonies which during the spring months show the pres- ence of more or less disease, by midsummer or earlier may, and very 50 BULLETIN 431, U. S. DEPARTMENT OF AGEICTJLTUEE. frequently do, contain no diseased brood. Experimentally it is pos- sible to destroy a colony by feeding it repeatedly the virus of sac- brood, and beekeepers report that the disease sometimes destroys colonies in their apiaries. The percentage of colonies, however, that actually die out as a direct result of the disease is small. The weak- ening of the colony in the spring of the year not only reduces or entirely eliminates the profits on it for the season, but may also cause it to be in a weakened condition on the approach of winter. Whether a larva once infected ever recovers from the disease is not known. Reasoning from what is known of the diseases of other ani- mals and man, one would expect that a larva may recover from sac- brood infection. It is known that many larvae, both worker and drone, do die. From the information thus far obtained it does not appear that a queenless colony would be likely to remain so as a con- sequence of the disease. As to the prognosis of the disease in a colony it may be said, there- fore, that it is very favorable for the continued existence of the colony. As to the economic losses to be expected from the disease, the present studies suggest that they may vary from losses that are so light as not to be detected upon examination to losses that may equal the entire profits of the colony for the year. Indeed, at times the death of the colony takes place as a result of the disease. RELATION OF THESE STUDIES TO THE TREATMENT OF SACBROOD. An earher paper (White, 1908) contains a brief general discussion of the relation existmg between the cause of bee diseases and the treatment of them. The general remarks made in it apply also to sacbrood. No doubt the beekeeper in studying the results given here has already observed relations existing between them and points which should be incorporated in methods for treatment. Mention- Lag a few of them here may serve to suggest still others. That the weakness resulting in a sacbrood colony is due to the death of worker larvae; that adult bees are not susceptible to the disease; that queenlessness is rarely to be expected as a sequence of the disease; that the disease may be produced with ease at any time of the year that brood is being reared; that it occurs at all seasons, but is more frequently encoimtered in the spring; that it is ioxmd in localities differing widely as to food and climatic con- ditions; and that no complete racial [immunity to the disease has yet been foimd are facts concerning the predisposing causes of sac- brood which beekeepers will at once recognize as bearing a cIosq rela- tion to the methods by which the disease should be treated. As sacbrood can not occur m the absence of its exciting cause (a filterable virus), a knowledge of this cause is of special importance in the treatment of the disease. SAOBEOOD. 51 That sacbrood is very frequently encomitered ; that it is infectious, but that it is more benign in character than malignant; that it does not spread rapidly from one colony to another; that colonies manifest a strong tendency toward self-recovery from the disease; that this tendency is stronger after midsummer ; that the disease may so weaken a colony during the early brood-rearing season that the profits from it may be much reduced, or even rendered nil; and that the disease may mdeed destroy the colony arc facts which must be considered in devising logical methods for its treatment. That the virus of sacbrood remains virulent in larvae dead of the disease for less than one month; that it remains virulent Iq honey approximately one month ; that when mixed with pollen it ceases to be virulent after about one month; and that in drying no virulence is to be expected after one month, are facts that accoimt in a large measure for the strong tendency to recover from the disease manifested by the colony and that furnish information concerning the use of combs from sacbrood colonies. From the results it may be concluded that it is better, theoretically, to store combs from sacbrood colonies for one or two months before they are again used, provided such storing entails no particular inconvenience or financial loss to the beekeeper. Further experiments show that brood frames from badly-infected colonies may be inserted into strong, healthy ones, and cause thereby very little infection and consequently only a shght loss. This is especially true after the early brood-rearing season of the year is past. Since this can be done, it is qiute probable that the practical beekeeper wiU find that this disposition of the combs will be the preferable one to make. At any event, it is comforting to know that it is never necessary to destroy the combs from sacbrood colonies on account of the disease. The experimental results here given regarding the destruction of the virus through heating, fermentation, putrefaction, drying, and du-ect sunlight should assist materially in the solution of the problem of the transmission of sacbrood, and should be found helpful in de- vising efficient methods for the treatment of the disease. Toward disinfecting agents it is shown that the vims of sacbrood possesses, in some instances at least, marked resistance. These and other experimental results thus far obtained indicate that' the use of any drug m the. treatment of the disease should not be depended upon until such a drug has been proved to be of value. No fear need be entertamed in practical apiculture that the disease willbetransmittedbythehands or clothing of the operator, by the tools used about the apiary, through the medium of the wind, or by the queen . It would seem at aU tunes superfluous in the case of sacbrood to flame or bum the inside of the hive or to treat the ground about a hive containing an infected colony. 52 BULLETIN 431, V. S. DEPARTMENT OF AGRICULTURE. There is but little danger that the disease will be Iratismitted by way of flowers visited by bees from sacbrood colonies and later from healthy ones. Theoretically, it is possible that the disease may be transmitted through a contamination of the water supply by bees from sacbrood colonies. Whether infection ever takes place in this way, however, is not yet known. If the disease is ever transmitted in this way, it would seem that it is more likely to take place in the spring of the year than at any other season. While there is yet much to be learned about sacbrood, it is hoped that by carefully considering these studies the be~ekeepers will be aided in devising efficient and economical methods for its treatment. SUMMARY AND CONCLUSIONS. The following summary and statements of conclusions seem to be justified as a result of the investigations recorded in this paper: (1) Sacbrood is an infectious disease of the brood of bees. (2) Adult bees are not susceptible to the disease. (3) The infecting agent causing sacbrood is of such a nature that it passes through the pores of a fine clay filter. It is therefore a filterable virus. (4) A colony may be inoculated by feeding it sirup or honey con- taining the virus. (5) The quantity of virus contained in a single larva recently dead of the disease is sufficient to produce quite a large amount of sacbrood in a colony. (6) The period from time of inoculation to the appearance of the first sjonptoms of the disease — the incubation period — is approxi- mately six days, being frequently slightly less. (7) By inoculation the disease may be produced at any season of the year that brood is being reared. (8) The disease is more often encountered during the first half of the brood-rearing season than during the second half. (9) It occurs among bees in locaHties having as wide a range of climatic conditions, at least, as are found in the United States. (10) The course of the disease is not greatly affected by the char- acter or quantity of the food obtained and used by the bees. (11) Larval remains recently dead of the disease prove to be very infectious when fed to bees. Dead larvae which have been in the brood comb more than one month are apparently noninfectious. (12) Colonies possess a strong tendency to recover from the disease without treatment. (13) The vu-us of sacbrood suspended in water and heated to 138° F. (59° C.) was destroyed in 10 minutes. Considering the vary- ing factors which enter into the problem, the minimum temperature necessary to destroy this virus when applied for 10 minutes should SAOBHOOD. 53 be found at all times to lio soinewh0ro between the limits of 131° F (55° C.) and l-i9° F. (65° C). (14) When the virus of sacbrood is suspended in honey it may be destroyed by heating the suspension for 10 minutes at approximately 158° F. (70° C). (15) The virus resisted drying at room temperature for approxi- mately three weeks. (16) The virus when diy was destroyed by the direct rays of the sun in from four to seven hours. (17) The virus when suspended in water was destroyed by the direct rays of the sun in from four to six hours. (18) The virus when suspended in honey was destroyed by the' dii-ect rays of the sun in from five to six hours. (19) The virus when suspended in honey and shielded from direct sunlight remained virulent, for slightly less than one month at room temperature diu-ing the summer. (20) The virus was destroyed in approximately five days in the presence of fermentative processes taking place in 10 per cent sugar solution at room temperature. (21) In the presence of fermentative processes going on in 20 per cent honey solution at outdoor temperature the virus of sacbrood was destroyed in approximately five days. (22) In the presence of putrefactive processes the virus remained virulent for approximately 10 days. (23) The virus will resist | per cent, 1 per cent, and 2 per cent aqueous solutions of carbolic acid, respectively, for more than three weeks, 4 per cent being more efifeotive. (24) Neither carbolic acid nor quinine as drugs should at present be relied upon in the treatment of sacbrood. (25) Varying factors entering into many of the problems discussed in this paper tend to vary the results obtained. In such problems the results here given must be considered from a technical point of view as being approximate only. They are sufficiently exact for application by the beekeeper, but to insure the destruction of the virus in practical apiculture the time element indicated from these experiments as siifficient should be increased somewhat. LITERATURE CITED. (1) Bahe, L. 1915. Sygdomme hos Honningbien og dens Yngel. Meddelelser fra den Kgl. Veterinaer-og Landboh^jskoles Serumlaboratorium, XXXVII, 109 p., 11 fig. (2) Beuhne, F. K. 1913. Diseases of bees. In Jour. Dept. Agr. Victoria, v. 11, pt. 8, p. 487-t93, 4 fig. (3) BuERi, R. 1906. Bakteriologische Uatersuchungen liber die Faulbrut und Sauerbrut der Bienen. 40 p., 1 pi., 1 fig. Aaran, Switzerland. 54 BULLETIN 431, U. S. DEPAKTMENT OF AGEICULTURE. (4) Cook, A. J. 1904. The Bee-Keeper's Guide or Manual of the Apiary, ed. 18, 543 p., 295 fig. Chicago. (5) DOOLITTLE, G. M. 1881. Foul brood. In Gleaniags in Bee Culture, v. 9, no. 3, p. 118-119. (6) Dadant, C. p. 1908. Diseases of Bees. Langstroth on the Hive and Honey Bee. 575 p. (p. 487), 229 fig. Hamilton, 111. (7) Howard, Wm. B. 1896. A new bee disease — pickled brood or white fungus. In Amer. Bee Jour., V. 36, no. 37, p. 577, 6 fig.; also in ABO of Bee Culture, 1903, p. 157-158. (8) . 1898. Pickled brood and bee paralysis. In Amer. Bee Jour., v. 38, no. 34, p. 530-531. (9) Jones, A. D. 1883. Symptoms of foul brood. In The American Apiculturist, v. 1, no. 4, p. 79-80. (10) KuflSTBINEE, J. 1910. Zusammenstellung der Ergebnisse des vom Mai 1903 bis Dezember 1909 unterauchten, faulbrutverdachtigen Wabenmaterials. In Schweizerische Bienen-Zeitung, Yahrg. 33, no. 4, p. 187-189. (11) Langstroth, L. L. 1857. A practical Treatise on the Hive and Honey-Bee. ed. 2, 534 p. (p. 275 ), illus. (12) [Editorial.] 1892. Is it a new bee disease? Something that resembles foul brood, its causes and cure not definitely known. In Gleanings in Bee Culture, V. 20, no. 15, p. 594-595. (13) . 1896. Dead brood — what is it? How distinguished from foul brood. In > Gleanings in Bee Culture, v. 24, no. 16, p. 609-610. (14) Root, A. I. and E. R. 1913. ABC and XYZ of Bee Culture. 717 p., illus. Medina. Pickled brood and its cause, p. 250. (15) SiMMlNS, S. 1887. Foul brood, dead brood. In, British Bee Jour., v. 15, no. 270, p. 371- 372; also in Canad. Bee Jour., v. 3, no. 28. p. 576-577. (16) White, G. F. 1904. The further investigation of the diseases affecting the apiaries in the State of New York. In 11th Ann. Rpt. Comr. Agr. N. Y., 1903, p 103-114. (17) (18) (19) 1908. The relation of the etiology (cause) of bee diseases t(5 the treatment. U. S. Dept. Agr. Bur. Ent. Bui. 75, pt. 4, p. 33-42. 1913. Sacbrood, a disease of bees. U. S. Dept. Agr. Bur. Ent. Circ. 169. 5 p.; Sackbrut. Fine Bienenkrankheit. A translation by Dr. M. Kiistenmacher. Berlin-StegUtz. 1914. Destruction of germs of infectious bee diseases by heating U S Dept. Agr. Bui. 92, 8 p. (p. 4). PUBUCATIONS OF THE U. S. DEPARTMENT OF AGRICULTURE RELATING TO BEE CULTURE. AVAILABLE FOR FREE DISTHIBUTION BY THE DEPARTMENT. Honeybees; Wintering, Yields, Imports, and Kxports of Honey. (Department Bul- letin 325.) Treatment of Boe Diseases. (Farmers' Bulletin 442.) Bees. (Farmers' Bulletin 447.) Comb Honey. (Faimers' Bulletin 503.) Honey and Its Use in the Home. (Farmers' ISulletin 653.) Outdoor Wintering of Bees. (Farmers' Bulletin 695.) FOR SALE BY THE SUPERINTENDENT OF DOCUMENTS, GOVERNMENT PRINTING OFFICE, WASHINGTON, D. C. Destruction of Germa of Infectious Bee Diseases by Heating. (Department Bulletin 92.) Price 5 cents. Temperature of Honeybee Cluster in Winter. (Department Bulletin 93.) Price 5 cents. 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(Hawaii Agricultural Experiment Station Bulletin 17.) Wice 5 cents. 55 ADDITIONAL COPIES OF THIS PUBLICATION MAT BB PEOCUBED FROM THE SDPEKINTENDENT OF DOCUMENTS GOVERNMENT PEINTINO OFFICE ■WASHINGTON, D. C. AT 10 CENTS PEB, COPY Circular No. 79. United States Department of Agriculture, BUREAU OF ENTOMOLOGY, L. O. HOWARD, Entomologist and Chief of Bureau. THE BROOD DISEASES OF BEES. By E. F. Phillips, Ph. D., In Charge of Bee Culture. In view of the widespread distribution of infectious brood diseases among bees in the United States, it is desirable that all bee keepers learn to distinguish the diseases when they appear. It frequently happens that an apiary becomes badly infected before the owner real- izes that any disease is present, or it may be that any dead brood which may be noticed in the hives is attributed to chilling. In this way disease gets a start which makes eradication difficult. There are two recognized forms of disease of the brood, designated as European and American foul brood, which are particularly virulent. In some ways these resemble each other, but there are certain distin- guishing characters which make it possible to differentiate the two. Reports are sometimes received that a colony is infected with both diseases at the same time, but this is contrary to the experience of those persons most conversant with these conditions. While it may be possible for a colony to have the infection of both diseases at the ^me time, it is not by any means the rule, and such cases are probably not authentically reported. Since both diseases are caused by specific bacilli, there is absolutely no ground for the idea held: by some bee keepers that chilled or starved brood will turn to one or the other of these diseases. Experience of the best practical observers is also in keeping with this. For a discussion of the causes of these diseases the reader is referred to Technical Series, No. 14, of the Bureau of Ento- mology, "The Bacteria of the Apiary, with Special Reference to Bee Diseases," by Dr. G. F. White. AMEEIOAN FOUL BEOOD. American foul brood (often called simply "foul brood") is dis- tributed through all parts of the United States, and from the symptoms published in European journals and texts one is led to believe that it is also the prevalent brood disease in Europe. Although it is found in almost all sections of the United States, there are many localities entirely free from disease of any kind. The adult bees of an infected colony are usually rather inactive and do little toward cleaning out infected material. When the larv^ are first affected they turn to a light chocolate color, and in the advanced stages of decay they become darker, resembling roasted coffee in color.- 5947—09 Usually the larvae are attacked at about the time of capping, and most of the cells containing infected larvae are capped. As decay proceeds these cappings become sunken and perforated, and, as the healthy brood emerges, the comb shows the scattered cells containing larvae which have died of disease, still capped. The most noticeable charac- teristic of this infection is the fact that when a small stick is inserted in a larva which has died of the disease, and slowly removed, the broken-down tissues adhere to it and will often stretch out for several inches before breaking. When the larva dries it forms a tightly adhering scale of very dark brown color, which can best be observed when the comb is held so that a bright light strikes the lower side wall. Decaying larvae which have died of this disease have a very characteristic odor which resembles a poor quality of glue. This disease seldom attacks drone or queen larvae. It appears to be much more virulent in the western part of the United States than in the East. EUROPEAN FOUL BROOD. European foul brood (often called "black brood") is not nearly as widespread in the United States as is American foul brood, but in cer- tain parts of the country it has caused enormous losses. It is steadily on the increase and is constantly being reported from new localities. It is therefore desirable that bee keepers be on the watch for it. Adult bees in infected colonies are not very active, but do suc- ceed in cleaning out some of the dried scales. This disease attacks larvae earlier than does American foul brood, and a comparatively small percentage of the diseased brood is ever capped. The diseased larvae which are capped over have sunken and perforated cappings. The larvae when first attacked show a small yellow spot on the body near the head and move uneasily in the cell. When death occurs they turn yellow, then brown, and finally almost black. Decaying larvae which have died of this disease do not usually stretch out in a long thread when a small stick is inserted and slowly removed. Occasion- ally there is a very slight "ropiness," but this is never very marked. The thoroughly dried larvae form irregular scales which are not strongly adherent to the lower side wall of the cell. There is very little odor from decaying larvae which have died from this disease, and when an odor is noticeable it is not the "glue-pot" odor of the American foul brood, but more nearly resembles that of soured dead brood. This disease: attacks drone and queen larvae very soon after the colony is infected. It is as a rule much more infectious than American foul brood and spreads more rapidly. On the other hand, it sometimes happens that the disease will disappear of its own accord, a thing which the author never knew to occur in a genuine case of American foul brood. European foul brood is most destructive during the spring and early summer, often almost disappearing in late summer and autumn. [Cir. 791 TREATMENl' OJ^ INFECTIOUS DISEASES. The treatment for both American foul brood and European foul brood is practically the same. It is impossible to give minute direc- tions to cover every case, but care and common sense will enable any bee keeper successfully to fight diseases of brood. Drugs. — Drugs, either to be given directly in food or to be used for fumigating combs, can not be recommended for either of these dis- eases. Shaking treatment. — To cure a colony of either form of foul brood it is necessary first to remove from the hive all of the infected material. This is done by shaking the bees into a clean hive on clean frames with small strips of comb foundation, care being taken that infected honey does not drop from the infected combs. The healthy brood in the infected combs may be saved, provided there is enough to make it profitable, by piling up combs from several infected hives on one of the weakest of the diseased colonies. After a week or ten days all the brood which is worth saving will have hatched out, at which time all these combs should be removed and the colony treated. In the case of box hives or skeps the bees may be drummed out into, another box or preferably into a hive with movable frames. Box hives are hard to inspect for disease and are a menace to all other bees in the neighborhood in a region where disease is present. The shaking of the bees from combs should be done at a time when the other bees in the apiary will not rob and thus spread disease, or under cover. This can be done safely in the evening after bees have ceased to fly, preferably during a good honey flow. Great care should be exercised to keep all infected material away from other bees until it can be completely destroyed or the combs rendered into wax. Wax from diseased colonies should be rendered by some means in which high heating is used, and not with a solar wax extractor. The honey from a diseased colony should be diluted to prevent burning and then thoroughly sterilized by hard boiling for at least half an hour, if it is to be fed back to the bees. If the hive is again used, it should be very thoroughly cleaned, and special care should be taken that no infected honey or comb be left in the hive. It is frequently necessary to repeat the treatment by shaking the bees onto fresh foundation in new frames after four or five days. The bee keeper or inspector must determine whether this is necessary, but when there is any doubt it is safer to repeat the operation rather than run the risk of reinfection. If repeated, the first new combs should be destroyed. To prevent the bees from deserting the strips of founda- tion the queen may be caged in the hive or a queen-excluding zinc put at the entrance. [Cir. 79] Treatment with bee escape. — The shaking treatment may be modified so that instead of shaking the bees from the combs the hive is moved from its stand, and in its place a clean hive with frames and founda- tion is set. The queen is at once transferred to the new hive, and the field bees fly there when they next return from the field. The infected hive is then placed on top of or close beside the clean hive and a bee esckpe placed over the entrance of the hive containing disease, so that the younger bees and those which later emerge from the cells may leave the hive but can not return. They therefore join the colony in the new hive. Fall treatment. — If it is desirable to treat a colony so late in the fall that it would be impossible for the bees to prepare for winter, the treatnient may be modified by shaking the bees onto combs with plenty of honey for winter. This will be satisfactory only after brood rearing has entirely ceased. In such cases disease rarely reappears. In the Western States, where American foul brood is particularly virulent, it is desirable thoroughly to disinfect the hive by burning the inside or by chemical means before using it again. This is not always practiced in the Eastern States, where the disease is much milder. Some persons recommend boiling the hives or disinfecting them with some reliable disinfectant such as carbolic acid or corrosive sublimate. It is usually not profitable to save frames because of their compara- tively small value, but if desired they may be disinfected. Great care should be exercised in cleaning any apparatus It does not pay to treat very weak colonies. They should either be destroyed at once or several weak ones be united to make one which is strong enough to build up. Recently some new "cures" have been advocated in the bee jour- nals, particularly for European foul brood, with a view to saving combs from infected colonies. The cautious bee keeper will hardly experiment with such methods, especially when the disease is just starting in his locaKty or apiary, but will eradicate the disease at once by means already well tried. In kll cases great care should be exercised that the bee keeper may not himself spread the infection by handling healthy colonies before thoroughly disinfecting his hands, hive tools, and even smoker. Since it takes but a very small amount of infected material to start disease in a previously healthy colony, it is evident that too much care can not be taken. In no case should honey from unknown sources be used for feeding bees. Care should also be exercised in buying queens, since disease is often transmitted in the candy used in shipping cages, Combs should not be moved from hive to hive in infected apiaries. [Cir. 79] PICKLE BROOD. There is a diseased condition of the brood called by bee keepers "pickle brood," but practically nothing is known of its cause. It is characterized by a swollen, watery appearance of the larva, usually accompanied by black color of the head. The larvae usually lie on their backs in the cell, and the head points upward. The color gradu- ally changes from light yellow to brown after the larva dies. There is no ropiness, and the only odor is that of sour decaying matter, not at all like that of American foul brood. In case the larvae are capped over, the cappings do not become dark, as in the case of the contagious diseases, but they may be punctured. So far no cause can be given for this disease, and whether or not it is contagious is a disputed point. Usually no treatment is necessary beyond feeding during a dearth of honey, but in very rare cases when the majority of larvae in a comb are dead from this cause the frame should be removed and a clean comb put in its place to make it unnecessary for the bees to clean out so much dead brood. CHILLED, OVEEHEATED, AND STARVED BROOD. Many different external factors may cause brood to die. Such dead brood is frequently mistaken, by persons unfamiliar with the brood diseases, for one or the other of them. Careful examination will soon determine whether dead brood is the result of disease or merely some outside change. If brood dies from chilling or some other such cause, it is usually soon carried out by the workers, and the trouble disap- pears. No treatment is necessary. Brood which dies from external causes often produces a strong odor in the colony, but wholly unlike that of American foul brood, merely that of decaying matter. The color of such brood varies, but the characteristic colors of the infec- tious diseases are usually absent, the ordinary color of dead brood being more nearly gray. Approved : James Wilson, Secretary of Agriculture. Washington, D. C, October 3, 1906. [Cir. 79] „ o 4- UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 804 Contribution from the Bureau of Entomology L. O. HOWARD, Chief Washington, D. C. PROFESSIONAL PAPER March 16, 1920 A STUDY OF THE BEHAVIOR OF BEES IN COLONIES AFFECTED BY EUROPEAN FOUL- BROOD ^ By Abnold p. Sturtevant Specialist in the Bacteriology of Bee Diseases CONTENTS Page Introduction 1 Procedure 5 Observations 8 Summary of previous experiments — 15 Supplementary observations 17 Study of naturally Infected colonies 17 Behavior of bees in cleaning • contaminated cells 18 Possible infection through queen 19 Page Supplementary observations — Contd. Distribution of introduced in- fected material 20 Age at which larvje are in- fected 21 Microscopical bacteriological observa- tions 24 Summary and conclusions 28 Literature cited 28 INTRODUCTION The brood diseases of bees cause annually large losses of bees and consequently of the honey crop. The predominant attitude among beekeepers has long been how best to eradicate an invading bee dis- ease after the attack has been made. They depend upon this pro- cedure, because little is known with any degree of certainty concerning the natural conditions which might prevent or control the onslaught of the disease. As a result of this attitude, much more importance has been placed on the significance of apiary inspection and police- power laws and of purely remedial treatment, the reasons for which in many cases are imperfectly understood. But the old adage " an ounce of prevention is worth a pound of cure " has yet to be refuted, particularly with regard to b eekeeping. In the reahn of human 'A series of investigations was started in the spring and summer of 1918 by the Office ^BLSiitoe Investigations, Bureau of Entomology, for the purpose of faking m^hitensive study of European foulbrood of bees, primarily from the standpoint of Z^^ZviTZ relation to the disease, correlated with the facts and practical obsfr^aUon" alrA known to the beekeeper. This paper, wMch was submitted f^ pubUcation January 13, 1919, is a preliminary report on the beginning of the In vestigation. 134440°— BuU. 804—20^ 1 2 BULLETIN 804, V. S. DEPARTMENT OF AGKICULTURE medicine, for the last two decades at least, this precept has been gaining strength so that to-day preventive medicine stands on a par with, if not above, most of the other branches of medicine. Why is it not logical to apply this principle to the control of bee diseases? Ever since European foulbrood of bees was first recognized (in 1894) , in New York State, as a distinct brood disease, there have been much' controversy and speculation concerning the etiology of the disease, the means of transmission, the method of spread, and, result- ing therefrom, the question of control. From the laboratory stand- point, the etiology of the disease has been worked out quite definitely bacteriologically (12) i. But as yet Bacillus ■pluton, the accepted cause of European foulbrood, never has been grown in pure culture on artificial media, although it has been definitely identified as the cause of the disease. This precludes any further advance along this line of attack for the time being. From the side of practical experience, there have been recorded large numbers of observations, many of them of a similar nature. These observations have led to many accepted practices, as, for in- stance, the use of Italian bees and strong colonies in combating the disease. Although the weight of numbers tends to give substantia- tion to observations, the scientific explanation of how these things are true never has been studied carefully and coordinated with the practical side into an epidemiological study of the colony under dis- ease conditions in European foulbrood. ' The history of bee diseases has developed mainly along two lines. The scientific side has been concerned principally with determining the causes of the various diseases microbiologically, the method of diagnosis, and conclusively differentiating them. These facts have been described sufficiently in various bulletins of the Bureau of En- tomology and will not be discussed here. From the practical side, countless observations have been recorded, largely in the bee journals, in which various manifestations of the disease and experiences with methods of treatment have been discussed. But in all this literature, particularly with regard to European foulbrood, there are few ob- servations on the disease and on the behavior of the bees in relation to it beyond simple description of symptoms. Early in the experience with European foulbrood it was learned by careful observers that strong colonies are essential in successfully combating the disease. Later the value of Italian bees was dis- covered. "West (11), a New York State apiary inspector, in giving what is one of the best early descriptions of European foulbrood, makes some pertinent observations on the disease. He states that when diseased brood is placed above a strong, healthy colony, with a queen excluder between, so that any healthy brood may emerge, 1 Reference is made by number in parentliesls to " Literature cited," p. 28. BEES IN COLONIES AFFECTED BY EUROPEAN FOULBROOD 3 the diseased larvse are cleaned out as this is taking place. The union with a healthy colony and the strength gained by the emergence of so many young bees gives the colony the stimulus to eliminate the disease. He notes, as have many other beekeepers since, that in August, when the buckwheat honey flow begins, the stronger of the diseased colonies are stimulated to clean up. Alexander (1) published a method of treatment for European foul- brood, the principle of which, after many varying failures and suc- cesses, is now the basis for the present method of treatment most used; that is, requeening with Italian stock. Alexander mentions the need of three factors: First, the necessity of requeening with young yellow Italians, as hybrids of Italian and black bees are prone to contract the disease in the first place and also are more likely to succumb to it ; second, particularly emphasized, a period (at least 27 days, according to Alexander) of queenlessness in which to allow the bees properly to clean up the cells and polish them, preparatory for eggs of a new queen ; third, a factor which is mentioned only casually but which is equally important with the other two, the direction to unite and strengthen diseased colonies before treating. So little em- phasis was placed on this that the majority of beekeepers overlooked it in using Alexander's treatment and therefore condemned the treat- ment as unsuccessful except in rare cases. In an editorial (8) in the same issue of the journal m which Mr. Alexander was writing, the question was raised as to why the period of broodlessness caused by winter, which is much longer than 27 days, does not always prevent a recurrence of the disease. Mr. Alex- ander answered this question by explaining that when the queen stops laying in the fall, the bees do not polish up the cells as they do earlier in the season, and that some of the dried-down material may remain until the next spring. The opinion also is given in this editorial that Italians are more able to resist the disease than hybrids because they do more thorough work in house cleaning and are less inclined to rob. Phillips (6) makes the statement that "European foulbrood is more destructive during the spring and early summer than at other times, often entirely disappearing during the late summer and early autmrni, or during a heavy honey flow," but gives no indi- cation as to how this takes place. The same year Miller (2) pub- lished his theory of the relation of the nurse bees to the spread of European foulbrood. He believes that the nurse bees suck up the juices of a freshly diseased larva which has not become offensive, and then transmit the disease when feeding the healthy larvae. On this supposition he believes that if egg laying ceases for S or 6 days (" the period the larvae remain unsealed in their cells") there will no longer be larvae in the proper condition for nurse bees to feed upon, 4 BULIxETIN 804, tr. S. DEPAETMENT OF AGEICTILTTJKB nor healthy unsealed larvae to receive the infection, and the disease will thereby come to an end. Dr. Miller has been using a 10-day period of queenlessness in his treatment of European foulbrood sincfe his accidental discovery that 10 days were sufficient, but in a later article (3) in enlarging upon his nurse-bee theory he assumes that the larva is fed during a period of 5 days but is not effective as a carrier of infection during the whole time as probably no larvae are torn open until they are 2 or 3 days old; thus making it possible to shorten the queenless period even more. He admits that not all the dead, partially dried larvae will be cleaned out, but believes that it is only the fresh yellow ones which are infectious. He also states that nurse bees are not inclined to travel far on the combs, a fact which may explain why the disease may be found con- fined to one comb for several days before spreading farther. Dr. Miller seems to have overlooked several important factors which will be discussed later. Quite an extensive piece of investigation was carried on during the summers of 1915 and 1916 by the author at the Massachusetts Agri- cultural Experiment Station upon the effect of requeening diseased colonies with various strains of Italian bees. At that time the im- portance of strong colonies with the requeening had not been em- phasized so strongly and less attention was paid to that factor. The records show, however, that in a total of 50 colonies observed, cov- ering two seasons, of 10 strong colonies only 2 showed recurrence, while 1 was doubtful ; of 20 medium-strength colonies, 10 showed re- currence with 2 doubtful ; of 14 weak colonies, 8 showed recurrence. In all these cases the new queen was not introduced until the colony was nearly or entirely clean. In the case of several of the weaker colonies it was necessary to strengthen them before requeening was possible, in order to save the colony. One or two of these, which were united and requeened with Italian stock, were the best colonies the next spring. Adding some strength to at least part of Miller's theory is a state- ment in a letter by G. C. Matthews, formerly of this bureau, who wrote in February, 1918, concerning his observations in California in 1914. He found that where the hives stood in rows of pairs the disease continued to spread down each row to corresponding members of each pair. This ceased when he rearranged his apiary so that the rows of hives were at least 10 feet apart, and alternate pairs of hives were turned at right angles. No pair was allowed to remain close to another facing the same way. This prevented the drifting of nurse bees, which he believes to be the method of spreading the disease. Furthermore, he found by introducing one Italian queen into the middle colony of an isolated row of hybrid bees that there was considerable drifting of nurse bees. Seven days after the brood BEES IN COLONIES AFFECTED BY EUROPEAN FOULBROOD 5 from the Italian queen began to emerge, yellow bees were found on either side in several of the hybrid colonies. Speaking of uniting weak diseased colonies and requeening, Matthews writes: Alter two or three were put together, each stack of brood was given an Italian cell. When young queens commenced to lay there was still disease in many of those hives, but as the queens increased in laying the bees cleaned out an ever-increasing sphere of comb • for a brood nest until they had the hives free of disease. But in no case, however long a hive might be queenless, did I see the disease cleaned out before a virgin appeared In the hive. In other words, a virgin had to be present before the bees would commence their job of cleaning up. Therefore, I see little to commend the practice of keeping diseased colonies queenless 21 days. A new bulletin by Phillips (7) has been issued recently by the Department of Agriculture. The fundamental idea emphasized is that "in keeping European foulbrood under control it is far more important to prevent the disease from getting a foothold in a colony than it is to eradicate the disease afterward." This bulletin, aside from discussing symptoms and methods of treatment, states concisely for the first time the facts observed in apiary practice on which successful treatment is based, and without an imderstanding of which it is difficult for a beekeeper to use preventive measures with any success. The analysis of these factors of response in behavior to treatment, as stated by Phillips, has been used to some extent as a foundation for the present work on the behavior of the colony in relation to disease, in an endeavor to substantiate, with data obtained under controlled conditions, these facts that are constantly observed in apiary practice and, if possible, to eliminate confusion in methods of treatment. PROCEDURE Shortly after the middle of May, 1918, experiments were started in Ithaca, N. Y., at the Cornell Agricultural College. Through the kindness of Prof. J. G. Needham, head of the department of ento- mology, and others associated with him, the use of a small, isolated yard of bees and also of laboratory facilities was offered for the purpose of carrying on these investigations. This small apiary had been used previously in fruit-pollination studies and had no record of disease. The yard was admirably located in a naturally well- protected hollow beyond the college fruit orchards, about a mile and a half from the main college apiary or other apiaries, with high ground and woods intervening. The author and the Office of Bee- Culture Investigations are under deep obligations to the Cornell authorities for the assistance so cordially extended. Being in the buckwheat district, the general locality was well adapted to the work because of the desire for as late a main honey G BULLETIN 804, xr. S. DEPAETMENT OF AGEICTJLTURE flow as possible in order not to have ihe influence of a heavy honey flow until other factors had been studied. At Ithaca the main honey flow is generally from buckwheat, coming from the 1st to the mid- dle of August. Eather unfortunately for the best results from the experiments, however, the summer of 1918 was unusual in this sec- tion, for the abnormally heavy honey flow from clover necessitated finishing the work earlier than had been planned, owing to the great difficulty of artificially infecting colonies during the heavy honey flow. There were seven colonies in the original experimental apiary. At first it was intended to work on a larger scale, but the trend of the observations soon led to the plan of working in more detail and on a smaller scale. These colonies were moved some distance apart to prevent drifting and robbing. Some were divided and some were strengthened in an effort to make a series of experiments on colonies of different strengths. The colonies were designated by letter and the combs of each colony by number. From time to time some of these colonies were artificially infected with diseased European foul- brood larvae from samples sent to the laboratory for diagnosis. Similar colonies were held intact and uninfected for controls. The infection was made by feeding diseased larvae macerated in sugar solution (about 60 per cent). For the preliminary experiments 10 larvae were fed in about 250 c. c. of sirup. Later, after the heavy honey flow had begun, it was necessary greatly to increase this dose in order to start the infection. The infected sirup was fed to the bees in sterilized glass petri dishes, placed on top of the frames and pro- tected by an empty comb-honey super placed on the regular hive body with the cover on top. At the time of inoculation, the condition of each colony was noted as to age, race, condition and appearance of the queen, proportion of nurse bees to old field bees, the number of frames of brood with ttie amount in each, its age, sealed or unsealed; in other words, the condition of the colony with regard to factors known to be signifi- cant in resisting disease. In two colonies the infected sirup was slightly colored with harmless eosin dye to determine where the fresh sirup was placed and its ultimate disposition. At first daily obser- vations were made to determine the earliest appearance of disease, the period of incubation, the symptoms exhibited, and the rate of increase. By holding up each comb in bright sunlight so that the light shone directly on the larvae, it was easy to detect the first symptoms of the disease. All the healthy larvae had the characteristic firm, well- rounded, pearly-white, glistening appearance. The first effect of the disease, besides an abnormal uneasy movement, was a loss of the BEES IN COLONIES AFFECTED BY EUROPEAN FOULBEOOD 7 glistening character and a slight tinge of grayish or creamy dis- coloration which would not be noticed except in direct sunlight. These larvae showed only Bacillm ^luton present when examined microscopically, as will be mentioned later. Soon after these first symptoms, however, the more noticeable symptoms appeared, such as a larva with its back out, the increase of the light grayish yellow color, and, later, the moist, melting appearance. A statistical record was kept of the number of larvae showing new disease at each observation, the number previously diseased that had been cleaned out in the interval since the previous observation, and those remaining over in the cells uncleaned for more than one period between observations. At various times observations were made of the behavior and types of bees engaged in cleaning up and the fate of the material removed. Great care was necessary in these obser- vations to disturb the colony as little as possible. On good days it was sometimes possible to remove a comb carefully from the hive and to watch the bees continuing at their work, and even to watch the queen laying eggs. An 'eight-frame observation hive containing a strong healthy colony was given a diseased comb from time to time and the bees were observed as they worked on it. One of the difficulties of the work was to find a satisfactory method of recording the desired data for each comb. At first the diseased cells were marked on the comb by a circle of red ceUoidin around the entrance of the cell. Although this dried rapidly, it proved unsatis- factory, as the bees, in their attempt to remove the foreign material, seemed to, remove both diseased and healthy larvae indiscriminately. Next small pins were used, inserted in the cell above the one showing disease. In this case the bees tore down the surrounding cells and completely removed the pins, many of which were found on the bottom board. Finally a method of plotting the diseased cells in a comb was adopted. An empty frame was laid off in inch squares by means of heavy black thread. This, used as a templet superimposed on a comb, aided in the location of the diseased and cleaned out cells, so that they could be recorded on a correspondingly ruled card (fig. 1) . Placing this over the comb, it was easy to locate exactly each cell and to determine how long the diseased material remained, thus aiding in following the course of the disease throughout its various stages. The only difficulty with this method was the tediousness of the obser-_ vations. Therefore, after the disease had become definitely estab- lished, daily observations of each colony were considered unneces-^ sary. Longer periods showed just as well what was happening in the colony. Also, after the disease had developed enough so that it could be definitely predicted whether the colony would recover or gradually^ be exterminated, observations of behavior under treatment were 8 BULLETIN 804, V. S. DEPAKTMENT OF AGBICULTUKE started, the method and degree of house cleaning being watched after the colony had been dequeened, strengthened, and requeened with good Italian stock. Note was also njade of any recurrence of disease and ^ /■^c S/V/f y X -^ ^ /va, v^> ' ^ W/V. P/^ 7ZI.. TAf y y^ "' ' ' \ ^ <' 'tO A O// /^/V c r \Zx L^/. o o V ■^ \ ^> OOi ) 1 o ^ ^ ) ^ ' O o- o- r n~ o- \ / - /=■ \ \ r O \ / G V \ / / /y ^ 1 C /*? S -^ ^ e- T & . 3 /O // /^ /S /'i^ /^ /g' /y^A? Fio. 1. — Method of plotting the location and history of diseased bee larvae in the combs. Freshly diseased larvas. O- Cells that have been cleaned out. (j)-Cells that have been cleaned out and filled with nectar. (J) Larvse remaining in the cells more than one observation period. The area of sealed brood was the amount present at the time of infection of the colony. apparent reason therefor. In other words, a complete study was made of the cycle of the disease and of the activities of the bees during its course. OBSERVATIONS COLONY Race. — Hybrid. Queen. — 1917, dark and poor. Bees. — ^Workers and drones very dark, almost black, very excitable. Condition of colony at time of infection. — ^Brood iu four frames, a little less than half sealed, besides two frames of eggs. Bees covering about eight frames, medium strength. Slightly more field bees than nurse bees, because of having divided this colony, old bees returning from the division. Date of first infection. — ^May 28, 1918. Material used. — ^Ten diseased larv£e from sample No. 5863, macerated in 250 c. c. of a 50 per cent sugar sirup. First appearance of disease noted. — ^May 31, 1918, three day.3 after inocu- lation. Age of larvw first attacked. — ^Three to four days after hatching from the eggs. Colony G (fig. 2 ) , hybrids, soon succumbed to the infection, the first diseased larva appearing three days after infection, the gross diagnosis being confirmed by the finding of Bacillus pluton on microscopic ex- amination. The spread of the disease was rapid, the disease being present in only one comb on the third day and in seven combs on the seventh day. All of this early spread took place in brood unsealed BEES IN COLONIES AFFECTED BY EUROPEAN FOULBEOOD 9 at the time of infection. The first high peak of the disease coming on the nineteenth day was followed by a slight improvement, when for a time the house cleaning exceeded the occurrence of fresh dis- ease. This was probably due to the stimulus of the increasing honey M M M M § M M M ^ ^ c^iwdis'ss'o ^t^d't// ya ly-^ffA^^^ flow. But as soon as the next series of eggs hatched, the disease^ again gained the upper hand, reaching another higher peak on the thirty-first day, at which time it was deemed necessary to start treat-^ ment. It had become evident that the colony was being overrun by: the disease. More and more dead larvae were being allowed to re-^ 134440°— Bull. 804—20 i 10 BTILLETI3Sr 804, IT. S. DEPARTMENT OF AGRICULTUKE main in the cells for several days without being cleaned out. Also' more larvse nearly ready for pupation were being affected. Most of these instead of remaining coiled were inclined to extend on the lower side wall in a brownish gray, slimy mass and exhibited a ten- dency to be viscid. At this stage of decomposition, when a stick is inserted the mass forms a coarse granular band for a short distance and then breaks so as to form droplike masses, but does not stretch out in a fine thread. These larval masses dried down to rubbery dark brown scales something like American foulbrood scales in ap- pearance, but different in consistency. These scales could be removed quite easily and would bend like a piece of partially granular old rubber. They also lay irregularly placed in the cells, often spirally extended, while American foulbrood scales are uniformly on the lower side wall. The bacteriological explanation for this abnor- mal characteristic will be discussed later under bacteriological observations. The predominance of these rubbery masses and scales increased as the disease progressed and the bees seemed to make little attempt to clean them out, even after the queen was caged on the thirty-first day, thus shutting off any increase of fresh larvse, or even after the queen and all queen cells were removed on the thirty-seventh day. On the thirty-ninth and also on the forty-first day, five and four frames, respectively, of emerging brood and Italian bees were united with this colony, but it was not until a new Italian queen, confined in a cage, had been hung in on the forty-fifth day that a final com- plete cleaning up was made. This new queen was not accepted, however, and a young queen was raised from the brood that was added to this colony, so that fur- ther observations wer6 ended here although the virgin queen was killed and another Italian queen introduced. This colony was re- ported healthy, however, about the middle of August. The hybrid bees seemed to lack ambition to fight the disease. When combs were removed from the colony, the bees never were ob- served to be working in the cells, and paid little attention to ma- terial partially drawn from the cells and crushed. COLONY r Race. — Italian, possibly with some slight hybrid blood. Queen. — 1917, fairly good condition. Bees. — Workers, good color, fairly quiet, drones inclined to be darker. Condition of colony at time of infection. — Brood in three frames, a little more than one-third sealed. Bees covering about six frames. Build- _. Ing up well. Proportion of field bees to nurse bees about equal. Date of first infection. — May 31, 1918. .Material used. — Ten diseased larvse from sample No. 5874, macerated in 250 c. c. of a 50 per cent sugar sirup. First appearance of disease noted. — June 4, 1918, four days after in- fection. ■^sejyf larvw first attacked.— Vowr days after hatching from the egg. BEES IN COLONIES AFFECTED BY EUROPEAN FOULBBOOD 11 Colony F (fig, 3), which was the next one to be infected, although not as strong as colony G, was of Italian stock and did not show the appearance of disease until one day later. On the fourth day one cell appeared in each of two combs. It was not until the twenty- N \ .^ .-' \ ^ — -" ,,' \ , ''' / ,,- " ^^^ / ^,- " ^^ ^ ,-' "' / ^ y :?=' \ \ / ^ "^ ^ / ^' y \ ^x y' ^ N 'x y • \ ^x y \ y \ y ^N \ '. / 'x \ '\ '■- ->^ \ \ ^^^^ \ \ -^^ N ~~~ \ 0,. V ^^ \ ^^ \ ?!' ?^ ^^ \ ^ ,5^, \ fl ^S 1 ^ \ v^ V- N $ M^ '^p^ S^'^ V .^ ? \ ^>^? j:^..* •M ~>-/ r \ K n? ^^ .^,^ ;.^H >1^ ;|»i ') ^' ^ n bi "?; ?^'^ ?li=iS j;^^ ■?x \ N. tl n^ ^^ s^ ?;'^^ H^ :ij^ \ 1(1 '^^ ^t^ T«)^ Qk, i^N s-i; ^1 V- *» 1 1 ^ > ^ <- «! tj i i «i \ 1 1 ^ r \ \ can' i/^Sff' » su ■iiW7 5 JO A'. ?KV^/ 1 ^ 15 »^ ^ ^ ^ ^ t I 2 fifth day that the disease ha4 spread to seven combs, the total number of diseased larvai being, as a whole, less than in the hybrid colony. There was not brood in all seven combs at the time of infection, but the brood increased faster than the disease spread. After the twenty-fourth day a permanent improvement began to be manifest. 12 BULLETIN 804, U. S. DEPABTMENT OF AGRICULTURE This improvement continued after the queen was caged and became more marked after she was removed from the colony. These bees were better house cleaners as well; the appearance of larvae remaining over more than one observation period did not be- come evident until after the ninth day, compared with the sixth day in colony G. At no time were there as many of the larvae nearly ready to pupate that were gummy or rubbery. Even though this colony was on the average weaker than colony G all the time, it handled the disease much better. It was 14 days before colony G had cleaned up to such an extent that it was deemed safe to intro- duce a new queen, while in colony F, with the Italian bees, the combs were so nearly cleaned of everything but a few old scales that a five-frame nucleus with a nfew Italian laying queen was united with this colony after a 10-day queenless period and in 9 days more everything was absolutely clean and the queen was laying in the combs that had had disease in them. When an observation was made nine days after the new queen's eggs were first noted, it was found that there was a slight recurrence of disease in three of the combs. But, unfortunately, at the same time, queen cells and no eggs were found, denoting that for some reason this queen had not been accepted. Therefore the queen cells were all removed and a new queen was introduced. Although the author's observations ended of necessity soon thereafter, it was reported to him that this colony was doing nicely later in August and was perfectly healthy. If the first new queen had not disap- peared, it is quite probable that as soon as a sufficient number of her bees had emerged they would have cleaned up the recurring disease in the same manner as was done in colony J, which will be mentioned later. Several times in this colony, during the cleaning-up process, bees were watched in the act of sucking up juices of diseased larvae that had been partially removed' from the cells with the aid of forceps. COLONY H Race. — Hybrid, a division of Colony G, hybrid. Queen. — 1918. Of their own raising. Poor. 5ees.— Dark hybrids, almost black, excitable. Condition of colony at time of infection. — Brood in three frames, a few eggs in one, only one small patch sealed, the remainder from eggs up to 4-day larva. Bees covering about five frames. Fairly good proportion of nurse bees. Date of first infection. — July 1, 1918; second infection, July 8, 1918. Material used.— 20 old, dried, rubbery, diseased scales from sample No. 5898, macerated in 250 e. c. of a 50 per cent sugar sirup, colored with eosin. First appearance of disease noted. — July 5, doubtful. Positive July 8, 7 days after infection. Age of larvm first attacked. — Four days after hatching from the egg. BEES IN COI^ONIES AFFECTED BY EUROPEAN FOULBROOD 13 Colony H (fig. 4) was treated as a double experiment. The infec- tion of this colony was not started until after the honey flow had come on quite heavily. Also, instead of freshly diseased larvae, old brown rubbery scales were used that showed Baxdllus pluton present micro- scopically, but were heavily overgrown by Bacillus alvei. It was de- sired to learn whether these scales were still infectious, so that nurse bees working on them, cleaning them out, might get infective material on their feet and mouth parts which could be carried to healthy larvaj. This was noted later in the observation hive, where, under the magni- fying glass, bees were seen trying to remove some of these rubbery scales, first moistening them with their tongues and then pulling at them with the mandibles and front feet. This colony, which was marked hybrid and weak, was slow in developing the disease, partly because of the diluting effect of the e 3 ^ ^ I £SS — — - — — ■^ ~~ soc A- 1 •IS '■£> 4r^ •.o 1^ ■>c vr " 3 MS. /^ »• ■ f ' c V£ or. f^ r7> w 7. 7yp ■/A r Si- v/» 1 ^ \, ■ \/ss S .34 O W7 f J •.y 7 S£ \i 1 \ - / ■t \ G sr »* RS o ■Pta ^ 1 \ '^ ^ 1 / r \ \ 1 1 / \ \ .^ ^.a? C / / ' \ \ \ A / / ^- ■'" -<■ ^ — — ^ ^ = — ^ :~ ^ ,_^ v^ r Pig. 4. — The course of lEuropean foulbrood in colony H. heavy honey flow and probably partly because there was a smaller number of infectious organisms present in the scales than in fresh larvaB. This is explained by the fact that the secondary putrefactive invading organisms would tend to kill off the primary organism, be- cause of the accumulation of the products of the putrefactive action. On the seventh day before the disease was first noted, a second infection of scales macerated in sugar sirup was given this colony to counteract the effect of these retarding factors. However, later on the seventh day, diseased larvae were found, and from then on .the disease started to spread and increase irrespective of the heavy honey flow, exhibiting all the symptoms and tendencies shown in colony G, of which this colony was a division before infection. On the seventeenth day it was necessary to remove the queen and start treatment, but what was taking place was evident. This removal of the queen did not seem to have a very marked effect on the house 14 BtJLLETIN 804, XT. S. DEPAETMENT OF AGEICTJLTUKE cleaning until the colony was united with colony I, a slightly dis- eased Italian colony. They then began cleaning the H combs, and the combined colony was reported clean in August. COLONY A Race. — Italian with some possible slight hybrid blood. Queen. — 1918. Of their own raising. Bees. — ^Workers, good color; fairly quiet. Drones, some slightly darker than pure Italians. Condition of colony at time of infection. — Brood in seven frames about half sealed. Bees covering about nine frames with a good proportion of young nurse bees. Colony strong and building up. Date of first infection. — July 2, 1918. Second infection, July 6, 1918. Material used. — ^First, 20 diseased larvae from sample No. 5937 macer- ated in 250 c. c. of a 50 per cent sirup, colored with eosin, ab- normally heavy infection; second infection, 20 diseased larvse from sample No. 5953 in 250 c. c. of uncolored sirup. First appearance of disease noted. — July 8, 1918, in drone brood, six days after infection. Age of larvw first attacked. — Four days after hatching from the egg. Colony A (fig. 5) was a fairly strong colony of Italians. Like col- ony H, it was infected after the heavy honey flow had started and was ^^ss^a^.^ ^ ^1 so' r>'0 V. :y.5 y Aw TM '■-v. / ^ s 'f s e ^'is & /o // /^ xff/-^ /s/e- /^A? /^pop/ ^ip£3 ^^^^pe^^as£&.30J/.s^Jtss* Fig. 5. — The course of European foulbrood In colony A. given twice the amount of infective material colonies F and G received. Nothing having appeared on the fourth day, a second infection of the same amount was given. On the sixth day 6 diseased larvae were seen in three combs. This colony, however, was so strong that the disease obtained very little foothold, and from the fourteenth day began to decline, or at least failed to make further gains. As a side experiment in this colony a comb of eggs laid by an Italian queen was placed in between two combs showing disease. If there is anything in the belief that Italian stock is more resistant to disease, the larvse in this comb should not have developed the disease, or at least not so soon. However, on the sixth day one or two larvae showed disease, increasing slightly in numbers for a few days until the obser- vations were of necessity stopped. It was intended to perform this experiment with several variations, such as placing eggs laid by an Italian queen in a diseased hybrid colony and placing eggs from BEES IN COLONIES AFFECTED BY EUROPEAN FOULBBOOD 15 a hybrid queen in a diseased Italian colony, but the presence of the heavy honey flow made it impracticable to carry the matter further. This colony A cleaned up readily after removal of the queen and was reported all healthy in August. Although a new queen was given to them it is probable that a period of queenlessness and the re- turning of the same queen would have answered just as well. COLONY I i?oce.— Italian. Queen.~lQl7, fairly good condition. Condition of colony at time of infection.— Se\en frames of emerging brood well covered with young bees. A strong 8-frame colony. Date of first infection. — July 10, 1918. Material Msed.— Thirty diseased larvse from sample No. 5959, macerated In 250 c. c. of a 50 per cent sugar sirup. This was abnormally heavy Infection of diseased material. First appearance of disease no«e(J.— July 15, 1918, five days after infection. Age of larvw first attacked.— Four days after hatching from the egg. This colony was infected during the heavy honey flow, but although given a heavy infection it had sufficient strength, aided by the heavy honey flow, to prevent the disease from spreading. On July 15 there were a few diseased larvae in two combs. On July 24, 14 days after inoculation, there were only a few diseased larvae in three combs. This was after the queen had been removed on July 18 and the colony had been united with colony H on the 20th. An interesting observation was that under the magnifying glass the methods of the nurse bees in sucking the juices from dead dis- eased larvae and the pulling of the skins out to carry them away could be noted. No bee worked very long at s. time on one larva. One after another worked until all was completed. SUMMARY OF PREVIOUS EXPERIMENTS Table I gives a partial summary of the data thus far described. Table I. — STiotoing the first appearance of disease noted after infection. Also the number of combs showing infection and the spread of the infection from comb to comb in the various colonies under observation Col- Date infected. Days after iuleotion. ony. 1 2 3 4 6 6 7 8 10 11 12 13 14 16 16 17 18 19 20 21 22 23 24 25 26 27 28 1918 May 28 May 31 /July 1 July 8 July 2 July 6 July 10 Number of combs. G>.... •• 1 3 2 3 2 4 3 5 2 4 '4 1 7 8 Fi.... 6 S 7 ... HI.... ... 2 4 ( A>.... ... ... 3 Dtf 5 4 P fir stt 1 Experiments started before the beginning of the heavy honey flow. > Experiments started after the beguming of the heavy honey flow. 16 BULLETIN 804, U. S. DEPARTMENT OF AGRICULTURE In the first group, colonies G and F, it is quite apparent that the Italian bees, colony F, made the better showing, even though the hybrids were the stronger colony in the beginning. As may be seen from a comparison of the two plots in figures 2 and 3, in colony G wherever there was a lag in the house cleaning there was marked increase in the number of larvae remaining over more than one observation period, and these increased until strengthening treatment was started. On the other hand, in colony F these were removed almost entirely by the time the strengthening treatment was started. The Italians did not allow the disease to appear as soon or to spread as rapidly, cleaned house better, left fewer larvae to dry down to the brownish rubbery scales, and responded to the increased honey flow and treatment much more readily. In the second group, colonies H, A, and I, the Italian colonies A and I again made the best showing. With the added diluting effect of the honey flow, they allowed the disease to gain no foothold whatever, while the hybrids, though aided by the honey flow, soon succumbed and allowed the disease to gain on them. It is evident that the Italian bees are much more vigorous house cleaners. In several instances, toward the end of the egg laying of the old queen, and well along in the progress of the disease, cells were noted on these diagrams which had previously contained diseased larvae, but which had been cleaned out, and then in which disease had reap- peared after other eggs had been laid and hatched in them. They were cells in which fresh nectar had not been placed between the two series of larvae. It was also noted that as the honey flow increased and as the brood became more scattered from the effects of the disease, more and more fresh nectar was placed in the brood nest in cells from which dead larvae had been removed. Most of this nectar, however, was moved up later, particularly after the bees began preparing the brood nest for a new queen in the process of treatment. That the ad- vent of a heavy honey flow was effective in controlling the disease is evident, particularly in the length of time between the infection and the first appearance of disease. The data, however, show little dif- ference in the resistance to infection, or so-called immunity, being slightly in favor of the Italians, if there is any difference at all. Disregarding the effect of the honey flow, the period of incubation of the disease is apparently between 3 and 4 days. However, it was noted that after Bacillus pluton was first observed it was anywhere from 24 to 48 hours before many characteristically diseased larvae were observed. Therefore, the actual period of incubation is prob- ably from 24 to 48 hours. BEES IN COLONIES AFFECTED BY EUROPEAN FOULBKOOD 17 SUPPLEMENTARY OBSERVATIONS STUDY OP NATURALLY INFECTED COLONIES As a supplementary study to the preceding artificial infection ex- periments, some observations were made upon the behavior of nat- urally infected colonies undergoing treatment. Through the kind- ness of W. L. Bean, of McGraw, N. Y., it was possible to make a series of such observations. In his apiary of about 30 colonies, all hybrids, the majority were diseased when observed June 8, 1918. Soon thereafter Mr. Bean kindly loaned two of these diseased colo- nies to be carried to Ithaca for closer observation. Mr. Bean at once started treating his bees, requeening with Italian stock by the method of introducing a queen cell almost ready to emerge. Appar- ently, this method was successful, for in the latter part of July Mr. Bean reported all treated colonies healthy and some 800 pounds of surplus honey. COLONY J Race. — ^Hybrid. Queen. — Queenless at time of arrival at Ithaca. Was poor hybrid of own raising, probably reared while disease was present in the colony. Strength in spring. — Weak. Strength, at time of treatment.— Sc&ttered brood in eight frames. Weak in bees, particularly in nurse bees. Approximate date of disease first noted. — ^May 31, 1918. Date of start of treatment observations. — June 16, 1918. This colony made no effort to clean up, even though they had lost their queen shortly before being brought to Ithaca. On the 18th of June six frames of Italian bees and emerging brood were placed on top of it. At once house cleaning started, a reduction of 50 per cent being noted in the fresh, moist, melting larvae within 24 hours. In this colony it was interesting to watch the bees doing the house clean- ing, particularly when diseased larvae in various stages of decomposi- tion were partially withdrawn from the cell with a pair of forceps. With the aid of a powerful hand magnifying glass it was easy to watch them suck up the juices of the dead larvae, even those which had decomposed to the extent of being a coffee brown in color and viscid in consistency. No bee would work long on a larva but would back off and wipe her tongue thoroughly with her front feet. It is conceivable that this might contaminate her, making possible car- riage of the infection to the next larva fed, even though the juices of the diseased larva were not actually fed to the healthy one. The majority of bees engaged in this work were the Italians. From these and other observations of a similar nature there is no doubt that the contamination of the mouth parts is the primary method of spread- ing the disease inside the oolony. 18 BtTLLETIN 804, U. S. DEPAKTMENT OF AGRICULTURE On June 25 an Italian queen was introduced in a cage with candy even though' a few scales were still present. This was fully 10 days after the colony had lost its queen, if not a little longer. On June 27 the queen was out and laying in one comb. Eight days later, on July 5, a recurrence of disease was noted, one larva being discolored and sunken, showing Bacillus pluton on microscopic examination. From that time on, for about 20 days after the first eggs of this queen were noted, one or two new diseased larvae appeared at each observation, the number decreasing, however, until about the twenty- sixth day when they had all disappeared. As the new young Italian bees increased, the disease decreased, until a point was reached where they were in the predominance and had eliminated the disease by their activity. This was also observed in colony F. COLONY K Race. — ^Hybrid. Queen. — 1917. Dark hybrid of their own raising, probably from diseased stock. Strength in spring. — ^Weak. Strength at time of treatment. — Eight frames of scattered brood and hardly enough bees to cover them. Approximate date of disease first noted. — ^May 31, 1918. Date of start of treatment observations. — June 20, 1918, at which time the queen was removed. Colony K, when it was brought to Ithaca, was so weak that it would soon have died. The bees made no attempt to clean out larvae that had been partially pulled out of the cells with forceps and crushed. On June 26, six days later, they were still showing freshly diseased, moist, melting larvae from eggs laid by the old queen, just before removal. At this time five frames of emerging brood and Italian bees were given this colony. On the 27th a new Italian queen was hung in with the cage closed. The presence of the new queen, however, seemed to give added impetus to the house cleaning so that by July 1, 11 days after removal of the queen, they were prac- tically cleaned up and the cage was opened with candy in the open- ing. V Further observations on this colony were ended because they refused to accept this queen. By the time another queen finally was accepted and was laying on July 18, it was too late, as the season's work was closed by the 23d. BEHAVIOR OF BEES IN CLEANING CONTAMINATED CELLS On June 6, 1918, a sample was received for diagnosis (No. 5898), consisting of an entire brood comb, containing quite an area of capped honey. About one-half of each side of the comb contained a large number of dead and diseased European foulbrood larvse, in stages varying from the yellowish, moist, melting larvae to dried rubbery scales of which there was quite a large proportion. This was BEES IN COLONIES AFFECTED BY EUROPEAN FOULBKOOD 19 the same sample from which infectious dried scales were used to in- fect colony H. After this comb had remained in the laboratory, wrapped in paper for about 3 weeks, it was placed in the strong colony in the observation hive. The frame was first placed in the middle of the hive for about an hour and was then removed to the outside, where the work of the bees on it could be watched. A large number of what appeared to be young nurse bees were already hard at work on the dried diseased material. The bees, working on the dried gummy masses, would wet the mass with their tongues for a while and then tear at them with their mandibles, at times removing pieces large enough to be seen from the outside. Often these small pieces were apparently dropped to the bottom board. No one bee worked long at one place. Those bees working particularly on the fresh, moist material, when leaving, would carefully wipe their tongues with their front feet, thereby transferring some of the in- fection to them. Other bees were at work carrying away the larger, more easily removable dead masses. The entrance also was watched to see if any of this material was carried out. Several bees were observed carrying out portions of dead larvae or pupae. One bee carried a piece about 2 yards before dropping it. Others dropped what they were carrying soon after leaving the entrance, but on ex- amining the surface of the ground about the entrance, very little material could be distinguished, so that apparently most of the ma- terial removed must have been carried some little distance before being dropped. After about an hour's work it was apparent that consider- able progress had been made. This comb was removed before it was entirely cleaned and later placed in another healthy colony for obser- vation. It was quickly cleaned up and quite a bit of nectar placed in it and, eventually, several square inches of brood. Observations, however, had to be stopped before any appearance of recurrence was noted. This same observation hive was given one or two other dis- eased combs to clean, but with the repeated probable infection from these sources the colony was so strong that no disease was noted in it during the entire season of observations. POSSIBLE INFECTION THROUGH QUEEN Colony M was a small nucleus made to receive the old queen from diseased colony K from McGraw, N. Y. The queen was introduced on June 20, 1918. For a while she laid fairly well, it being neces- sary to add one or two more combs. But later her brood became more and more scattered. Finally, on July 8, there was observed one dead larva, which looked suspicious, but which, on microscopic ex- amination, proved to be negative. On July 10, however, one definite cell appeared and several other slightly yellowish, abnormally colored larvse. This dead larva contained BaciUus pluton. From then on until this queen was killed and the colony united with another dis- 20 BULLETIN 804, TJ. S. DEPARTMENT OE AGKICULTTJRB eased colony, more discolored larva? appeared, showing definitely the •development of the disease. As far as could be seen the only source of infection was the queen which had come from a diseased colony. This occurrence had been observed previously by the author while employed at the Massachusetts Agricultural Experiment Station. During the summer of 1916 eight queens taken from diseased Euro- pean f oulbrood colonies were introduced into isolated, healthy nucleus colonies. Of these eight nuclei three developed European foulbrood, two were doubtful, and three remained healthy. Several such in- stances have been mentioned in the literature of beekeeping. w • • • • • < n ? o — r- • • • • • • • 1 *** • • • • ••• ^ •, • ^D c • a ' a •• o • •• < • • •• ••• c • D Q , o a .'A . •■ • "•' o • ,■** A .• a *■■•. • , /::> 0° ' 1 e • A L • • a > ;° D ^ ,;.: • ^ n »?« A • 1 •• • > • J^ n /*- °n< A. •. • o • D • ' 1 • • •• d? : . C ) 00 °. ■°j A 1 ° p □ D ^b .■'•° • y/ ••• ..0.. «.... +1-H „y.. uw... °.-\i 1 • y ^ ^ 'i' ^ tff- 7- ,s s> /o /'/ /^ /^ /-^ z.:^ /<^ y^y^ Fig. 6. — Distribution of cells containing infected sugar sirup and subsequent spread of tbe disease in a comb taken from colony H Area covered by brood at time of infection, mostly unsealed. • Location of cells con- taining infected colored sugar sirup on July 3, 1918. ® First positive diseased larvse noted, 2 on July 8. Q Number of new diseased larvaj (4) on July 10. A Number of diseased larvae (13) on July 12. D Number of diseased larvae (39) on July 16. O Number of diseased larvse (52) on July 19. DISTRIBUTION OF INTRODUCED INFECTED MATERIAL An interesting experiment was carried out with sugar sirup, colored by a small amount of a harmless anilin dye, eosin, used as an indi- cator, which gave to the sirup a bright red color. The object of this experiment was to determine where the sirup, or, more important, where fresh nectar is first placed in the hive and combs. On May 27 two colonies were fed this colored sirup from above some time before the heavy honey flow from clover started. The results were striking, for in nearly every case the colored sirup was easily discernible in the cells and the greatest part of the sirup was located in quite a definite area. These colored cells were either scattered among the cells con- taining the larvse or were placed in a ring of cells adjacent to the brood area toward the top of the comb, little being placed with the solicJ stores (fig. 6). Furthermore, for nearly 36 hours after the feeding practically all the young nurse bees showed a marked pinkish discoloration of the anterior end of their abdomens, denoting the dis- BEES IN COLONIES AFFECTED BY EUROPEAN FOULBEOOD 21 tention of the honey stomachs by the retention of the colored sirup therein. About half the bees in the hives were discolored in this manner. After a day or so, however, this begfn to disappear. Also the number of cells showing the pink discoloration began to disappear. Evidently the sirup had been moved up, worked over, and mixed with other nectar or consumed. Later, some time after the heavy honey flow had started, shortly after July 1, two more colonies were fed colored sirup, this time infected with diseased larvae macerated therein. In these cases the discolored abdomens were noted about as before, but the colored cells were less numerous and the color less striking. The location of the colored cells was similar to that in the former ex- periment ; that is, mainly in the brood area or just contiguous to it and mostly above. The outside combs, containing considerable honey, showed scarcely any of the colored cells. This time these colored cells disappeared sooner, showing that the infected material must have been much diluted quite soon after being taken up from the feeding dishes. Figure 6 shows the method of plotting the location of diseased larvae in the combs and also the location of the/cells containing the colored sugar sirup. As will be noted, a fairly large proportion of these cells are located within the area of brood at the time of feed- ing. It is interesting to note the tendency of diseased brood to form concentric circles, showing the two series of larvae occurring between the dates noted. The spreading was from two cells at first to quite a large number at the last observation shown. AGE AT WHICH LAEV^ ARE INFECTED In previous observations it was constantly noted that the larvae affected by European foulbrood were regularly at least 4 days old, the age at which the coiled larvae completely fill the bottom of the cells. Occasionally a slightly younger and smaller larva would become diseased, but this was not the common occurrence. Further- more, in the cases where the colored sirup was fed the bees, within 24 to 36 hours quite a number of larvae averaging 4 days old could be seen discolored from having been fed this sirup, while it was notice- able that the younger larvae under 3 days old never showed the dis- coloration. These colored larvae were examined in a smear under a microscope, but the infecting organisms, being comparatively few in number, had not increased sufficiently at that time to be apparent. The question now arises as to the age at which the larvae first are fed nectar or infected material. There has been much controversy over the subject of composition and source of the larval food, but as yet no conclusive scientific evidence has been presented. Irrespective of the question whether the food at various stages originates from glands or is regurgitated, it is apparent from these observations that 22 BULLETIN 804, V. S. DEPABTMENT OF AGRICULTURE there must be a difference between the food which larvae younger than approximately 3 days old receive and that fed to older ones. Otherwise the younger larvae would also show the pink coloration. Von Planta (9) by chemical analyses, of questionable exactness, how- ever, makes a division in the feeding of the larvae at the age of 4 days, at which time the high protein and low sugar content change to lower protein and higher sugar content. These analyses would tend to coincide with the above observations, only it is probable that the change begins earlier. Additional data upon this subject are recorded in Tables II and III, although the observations were primarily for another purpose. In order to obtain further information relating to a possible difference in resistance to disease between Italian and hybrid bees, a careful record was made of the time when eggs were first noted in empty combs after the infection of the colony and when larvae first showed disease thereafter. In the case of comb Special No. 2, the eggs were laid by an Italian queen in a healthy colony and then placed in a diseased colony. Colonies F, A, and I were of Italian stock while colonies G, H, and J were hybrid. In the recurrence of disease all were given new Italian queens. As has been mentioned before, as soon as the bees of the new Italian queens emerged in sufficient numbers the disease disappeared. Table II THE FIRST APPEAEANCE OF DISEASE IN COMBS IN WHICH EGGS WERE LAID AFTER THE COLONY WAS INFECTED Colony and comb No. Number of dajB after eggs were first noted in comb. 1 2 3 4 5 6 7 8 9 10 11 )2 13 14 X 6 X 07 X G8 X X F3 F4 F7 X H3 X X H5 A2 X X A 4 A 6 X A7 A 8 X X il.\. ::::;;:::::::::::::::::::::::::::::: X EECUREENCE OF DISEASE AFTER EGGS OF A NEW QUEEN WERE FIRST NOTED IN THE COMBS J 2 X X J 3 J 4 X J 5 X J 6a X J 6b X J 7 X J 8 X F3 X X X F4 F? BEES IN COLONIES AFlfECTED BY EUROPEAN FOULBROOD 23 Table III. — Average time, under various conditions, in which disease hecomes apparent in a colony after infection with Europecm foulbrood. (Averages taken from Table II) Before the heavT honey now. Colony G, hybrid.... Colony F, Italian.... Average of these two, Days. 7i During the heavy honey flow. Colony H, hybrid Colony A, Italian Colony I, Italian Average of these three Days. 8i 9 8{i Becurrence of disease, after treat- ment, dur- ing honey flow. Colony J, hybrid originally Colony F, Italian originally. Average of these two Days. 9i The data shown, particularly in Table III, tend to disprove the theory that Italian bees have a natural immunity or resistance. If a larger number of observations could have been made, the variation would have appeared less. The effect of the honey flow is evident, however. When it is a question of the age at which the larvae are fed material that contains infection, these figures are significant. In the life history of the bee, 3 days are spent in the Qgg and from 5 to 6 days as larva before capping, making a period of 9 days in all. After 3 days in the egg and after having been fed predigested food for 3 days, with the additional 24 to 48 hour period of incubation, as was observed earlier in this paper, the larva ought to show disease from the fourth to the fifth day after hatching, or the seventh to eighth day of its existence, if Von Planta's assumption is correct. From actual observation this was found to be true and from observa- tion of the averages in Table III it is seen that the first appearance of disease occurs between the seventh and ninth days, varying with the conditions of the honey flow. Eeferring to Dr. Miller's theories, it is hard to believe that there is not plenty of highly infectious material left in the colony after a 5 or 6 day period of queenlessness. Aside from actual observations of moist, yellow, melting larvae present more than 6 days after the 24 BULLETIN 804, V. S. DEPARTMENT OF AGEICITLTUEE queen has been removed, the juices of which the workers sucked up with avidity, the final eggs laid will be just at the stage where the dis- ease first appears; that is, 3 to 4 days after hatching, at the end of a 6-day period. Furthermore, even though the nurse bees do not feed to healthy larvae the material that is taken up in cleaning out the cells in varying stages of decomposition, infection, even from scales, may be carried on the feet, mouth parts, and tongue, particularly, as was definitely shown with colony H, since these scales are in- fectious. The period of queenlessness and the consequent house cleaning are absolutely dependent on the strength of the colony. A strong colony cleans up rapidly, particularly after the introduction of the new queen in a cage plugged with candy. A weak colony, on the other hand, has not sufficient bees to clean even after complete introduction of a queen, and the disease soon appears again. Under average conditions, therefore, it would appear unsafe to allow less than a 10-day period of queenlessness in treatment of European £oul- brood. MICROSCOPICAL BACTERIOLOGICAL OBSERVATIONS A large number of microscopic examinations were made of larvae under various conditions for the positive presence of the characteris- tic groups of Bacillus pluton. These examinations were made mainly as a check on the gross observations of the first appearance of the disease. Cover glass smears were made of crushed larvae, stained with carbol f uchsin and mounted in Canada balsam. These examina- tions were made at regular intervals after the colonies were infected, larvae of all ages being examined. It was found in the smears of those larvae showing the first slightly abnormal symptoms that Bacillus pluton was the only organism present. This substantiates White's (12) observations that before the disease could be detected by gross examination, by a histological study of sections of larvae during the period of incubation it was demonstrated that " in the production of the disease Bacillus pluton was the first invader of the healthy larvae." As the disease advanced in the various colonies, observations were made of larvae in various stages of decomposition. The bacterial con- tent was found to vary with the change of appearance of the larvae during decomposition. The presence of these secondary invaders easily explains the atypical appearance of certain types of European foulbrood that heretofore have been very confusing to the bee- keeper. For a short time after the death of the larva, the color remains a moist, creamy-grayish yellow. This is during the period when Bacil- lus pluton and such occasional secondary invaders as Streptococcus apis or Bacterium eurydice and other organisms, which do not form BEES IN COLONIES AFFECTED BY EUROPEAN FOULBKOOD 25 spores, are predominant as described by White (12) and McGray (4). Soon the putrefactive spore- forming organisms increase in number, BaciUus aZvei^ being the one most commonly found. This is seen particularly in the case of the more mature larv83, which when dying extend more or less irregularly in the cells, becoming the gray- ish brown slimy masses which develop into the dark brown granular rubbery scales. This fact has been observed for a long time in the many samples which have been received for diagnosis. A partial description of these scales and of the presence of Bacillus alvei in them is given by McCray and White (5), but the experimental observations described in this paper added to diagnostic observations show that this condition is generally much more pronounced and common than described by these writers from laboratory observa- tions. The rapid increase and peculiar process of decomposition of BaciUus alvei, after the death of the larva, often to the exclusion of all other organisms, accounts for this abnormal appearance. In the case of American foulbrood, almost never is any other organism found associated with the disease but Bacillus larvae, the cause of the disease. This accounts for the constancy of the symptoms as compared with the variation of symptoms in European foulbrood where there may be several secondary invaders. Furthermore, in making the smears of the diseased larvae upon cover glasses, the peculiar whitish saclike extrusion of the larval in- testines was often noticed on crushing the larvae preparatory to smear- ing, which White (10) describes as a gross diagnostic character. When this sac was removed and smeared separately, it was always found to be heavily loaded with Bacillus phiton. Therefore it is safe to assume that the intestinal tract is the primary focus of infection, while the secondary putrefaction takes place mostly in the body tis- sues of the dead larva. Coincident with the microscopic examination of larvae, several ex- aminations were made of the contents of the ventriculus, rectum, and in a few cases of the honey stomach and mouth parts of bees. These bees were presumably nurse bees taken from diseased combs, some in the very act of sucking up the juices of dead diseased larvae. Al- though insufficient observations were made to give conclusive evidence, some interesting information was obtained. As may be seen from Table IV, the number of cases where Bacillus pluton or other organisms associated with infectious material were f oimd in the intestinal contents is not very large. However, of more ^Badllus alvei originally was supposed to be the primary cause of European foulbrood, but has been proved by White and others to be only a common secondary invader. Bacil- lus alvei has purely putrefactive functions. From its cultural and biochemical character- istics, Bacilhia alvei apparently belongs to the common Baoillus suitiUs (hay bacllIuB) group of Bpoie-toiming organisms, all having mainly putrefactive functions. 26 BULLETIN 804, U. S. DEPAKTMENT OF AGRICULTURE importance probably, BacUhis pluton was found in a smear made from the mouth parts of a nurse bee and also in the contents of the honey stomach of another. If these observations had been carried out systematically, instead of only casually, it is expected that much more positive data might have been obtained along these lines, owing to what is known already of the habits of house-cleaning bees working on diseased material. Table IV. — The results of the microscopic bacterial examination of the contents of the intestinal tracts of nurse tees taken from diseased colonies Microscopic £iiidiiigs. G. F. H. A. I. J. K. Total. Positive inciZZtts pZ«i07i,.. 1 11 2 24 3 2 18 1 i 9 9 17 12 6 7 97 Baalim alvei or doubtful Bacillus pluton... n SUMMARY AND CONCLUSIONS In arriving at the following conclusions an effort has been made to state them in a manner which will indicate the substantiation of previous observations made both in the laboratory and in the apiary. It may be noted that many of these conclusions are similar to some of the statements made in Farmers' Bulletin 975 in the summary of facts which apiary practice has brought out. 1. European foulbrood is an infectious disease. BaciUvs plioton was found to be the primary invader, appearing in the intestinal tract of larvae before death, contemporary with the first slightly apparent symptoms. 2. The variation in the appearance of the diseased larvae after death is due to the presence or absence of secondary invaders. 3. The period of incubation for European foulbrood was found to be from 36 to 48 hours, although the gross symptoms usually do not become apparent in less than 3 or 4 days, varying with conditions of honey flow and strength of colony. 4. It has been noted in apiary practice that the first brood of the year usually escapes with little loss. During the first 5 to 7 days the spread of the disease in the colony after infection is slow, after which the increase is rapid under favorable conditions. The critical time, therefore, to detect the disease and start treatment is early in its course, thus making conditions unfavorable. 5. The evidence tends to confirm the theory that one of the ways the disease is spread in the colony is by the house-cleaning bees, and from colony to colony by their drifting. It is quite probable that the infective organisms are carried on the mouth parts and pedal appen- dages. The question of infection from intestinal contents or from BEES IN COLONIES AFFECTED BY EUBOPEAN FOULBROOD 27 the source of larval food at various stages needs further substantia- tion. 6. Irrespective of strength of colony, the Italian bees were found to resist infection much better than hybrids and showed more ability to overcome the disease. 7. This apparent resistance of the Italian bees was observed to be largely due to the more vigorous house-cleaning characteristics rather than to a natural resistance or immunity to the disease. There was very little difference in the apparent period of incubation between the Italian and hybrid colonies, possibly a slight difference in favor of the Italians. Furthermore, it was noted that often there may be a slight recurrence of disease in the brood of the new Italian queen until a sufficient number of her bees have emerged to eliminate the infection by house cleaning. Apparently, infection is not always entirely removed by a period of queenlessness. 8. As a rule, requeening is necessary in the treatment of European foulbrood, except possibly in the strongest Italian colonies, which show only slight infection. Where a considerable quantity of dis- ease is present, sufficient to require treatment, it was found unsafe to use a period of less than 10 days' queenlessness, due to the infec- tious condition of the diseased material remaining and the accom- panying behavior of the colony. 9. The stronger the colony in Italian bees, the more rapid was the recovery. 10. A heavy honey flow tends to prevent infection from gaining a foothold. It also tends to eliminate the disease if present before the start of the heavy honey flow. This was found to be due to the effect of dilution on the infection because of the influx and direct feeding of the fresh nectar to the larvae. 11. European foulbrood is a disease of weak colonies. It was found to be difficult effectually to infect any but the very weak colonies during the heavy honey flow. Therefore, colonies kept strong up to the time of the honey flow run very little danger of contracting European foulbrood. This and others of the facts ob- served are in exact harmony with facts already observed in apiary practice. LITERATURE CITED (1) Alexandbb, E. W. 1905. How to rid your apiary of black brood/ In Gleanings in Bee- Culture, V. 33, p. 1125. (2) MnxEE, C. C. 1911. Fifty years among tlie bees. (3) 1918. European foulbrood and its treatment. In American Bee Journ., V. 58, no. 7, p. 232-234. July. (4) McCeat, a. H. 1917. Spore-forming bacteria of tbe apiary. In Jour. Agr. Research, V. 8, no. 11, p. 399-420, pi. 93-94. (5) and White, G. F. 1918. The diagnosis of bee diseases by laboratory methods. U. S. Dept. Agr. Bui. 671, 15 p., 2 pi. (6) Phillips, E. F. 1911. The treatment of bee diseases. U. S. Dept. Agr. Farmers' Bui. 442. May 6. (7) 1918. The control of European foulbrood. V. S. Dept. Agr. Farmers' Bui. 975. July. (8) [Root, E. R.] 1905. (Editorial.) In Gleanings in Bee-Culture, v. 33, p. 1126. Nov. 1. (9) VoN Planta, a. 1888. TJeber den Futtersaft der Bienen. In Zeit. f. Phys. Chemie von Hoppe-Seyler, v. 12, p. 327-354. (10) 1889. Ueber den Futtersaft der Bienen. In Zeit. f. Phys. Chemie von Hoppe-Seyler, v. 13, p. 552-561. (11) West, N. D. 1899. Foul and other forms of diseased brood in the State of New York. In Gleanings in Bee-Culture, v. 27, p. 828. Nov. 15. (12) White, G. F. 1912. The cause of European foulbrood. Clr. 1.57, Bur. Ent., U. S. Dept. Agr. May 10. » " Black brood " Is an old name for European foulbrood. 28 ADDITIONAL COPIES OF THIS PUBLICATION MAT BE PKOCUHED FBOM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHmOTON, D. C. AT 5 CENTS PER COPY laauerl May 6, 1911. U. S. DEPARTMENT OF AGRICULTURE. FARMERS' BULLETIN 442. THE TREATMENT OF BEE DISEASES. BY E. F. PHILLIPS, Ph. D., In Charge of Bee Culture, Bureau of Entomology. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. 442 LETTER OE TRANSMITTAL. U. »S. Depaktment of Agriculture, Bureau or Entomology, Washington, D. G., February 2k-, 1911. Sir: I liav^e the honor to transmit herewith a manuscript entitled " The Treatment of Bee Diseases," by E. F. Phillips, Ph. D., in charge of bee culture in this bureau. In the preparation of this paper, which is intended to supersede Circular 79, of this bureau, the aim has been to give briefly the information needed by the beekeeper who has disease in his apiary. No discussion of the cause or distri- bution of these diseases has been included. I recommend the publica- tion of this paper as a Farmers' Bulletin. Respectfully, L. O. Howard, Eniomologist and Chief of Bureau. Hon. James Wilson, Secretary of Agriculture. 442 2 CONTENTS. Page. Introduction 5 The brood diseases of bees 5 Nature of the diseases 7 Names of the diseases 7 Symptoms 8 American foul brood 8 European foul brood 10 The so-called " pickle brood " 12 Brood dead of other causes 12 " Bald-headed brood " 12 Methods of spread 12 Precautionary measures 13 Treatment for both infectious diseases 13 Shaking treatment 14 Time of treatment 14 Preparation 14 Operation 14 Saving the healthy' brood 16 Saving the wax 16 Cleaning the hive 16 Disposal of the honey 17 The second shake 17 The cost of shaking 17 Treatment with bee escape 17 Fall treatment 18 Drugs 18 Treatment for European foul brood 18 Introduction of Italian stock 19 Dequeening 19 Inspection of apiaries 19 Examination of samples of diseased brood ' 20 The diseases of adult bees 20 Dysentery 20 The so-called paralysis 21 Isle of Wight disease 21 Spring dwindling 21 Publications of the Department of Agriculture on bee diseases 22 442 3 ILLUSTRATIONS. Page. , Fig . 1. Work of tho larger wax moth 6 2. American foul brood 8 3. The ropiness of American foul brood 9 4. American foul-brood comb 9 5. European foul brood 11 6. Apparatus for the shaking treatment 15 7. Gasoline torch 16 442 4 THE TREATMENT OF BEE DISEASES. INTRODUCTION. The diseases which attack the honey bee may be divided into two classes, namelj', tliosu att'ecting the brood and those to which the adult bees are subject. The diseases of adult bees have not been in- \estigated sufficiently to make it possible at the present time to recom- mend methods for their treatment. In the present bulletin, tlierefore, only a brief statement concerning these diseases will be made, mainly for the purpose of indicating the present state of knowledge on these subjects. Concerning the diseases of the brood more is known, and this is particularly fortunate since they are far more destructive in American apiaries than are the diseases of the adult bees. The causes of bee diseases will not be discu.ssed here. For informa- tion on this phase of the subject the reader is referred to other pub- lications of the Bureau of Entomology, which are listed at the end of this bulletin. The aim of this bulletin is to give information that can be used by the practical beekeeper in combating bee diseases. THE BROOD DISEASES OF BEES. The brood diseases of the honey bee are already widely distributed in the United States and seem to be spreading rather rapidly. The loss to the beekeepers of the country, owing to the actual death of colonies by disease, is estimated conservatively at $1,000,000 annually. This does not include the loss of crops, resulting from the destruction of colonies, or the discouragement to the beekeeper which often causes him to give up the business. A considerable part of this loss is due to the indifference of the beekeepers to these diseases and a lack of knowledge concerning them. It frequently happens that colonies in an apiary become infected before the owner realizes that disease is present. He may errone- ously attribute the losses observed to'some other cause. In this way the disease gets a start which makes eradication difficult when once the cause of the loss has been discovered. In view of the widespread distribution of these diseases, it is most desirable that all beekeepers learn to distinguish the diseases when they appear and to know how to keep them under control. It is often a matter of surprise to beekeepers to learn that bees are subject to disease. The most frequent source of confusion is the 442 5 e TREATMENT OF BEE DISEASES. placing of the blame for loss of colonies on some cause other than disease. The poorer class of beekeepers attribute their losses simply to " bad luck," but even well-informed beekeepers err in this matter. Fig. 1. — Work of the larger wax moth in a brood comb. (Original.) The wax moths (see %. 1) are most frequently blamed for the death of colonies, whereas they do no damage to strong, healthy colonies, properly cared for, but enter only when the colony is weakened by queenlessness, lack of stores, disease, or some other cause. In the 442 TREATMENT OP BEE DISEASES. 7 majority of the reports of wax-moth depredations received by this department which can be investigated it is found that the trouble is actually an outbreak of a brood disease. The spraying of fruit trees while in bloom is possibly injurious to bees, and there exists among beekeepers a strong feeling against the jjractice. Since no entomologist now recommends that fruit trees be sprayed during the blooming period, this is probably rarely done by progressive fruit growers. However, it is frequently reported by beekeepers that they are losing bees by poisoning due to spraying. A number of cases of the death of colonies, reported as caused by poisoning due to spraying while trees were in bloom, have been found to be in reality outbreaks of European foul brood, which is particu- larly prevalent in the spring and early summer. Other circumstances to which is often attributed the death of brood or of the colony are chilling, fumes from coke ovens, and malicious poisoning. The wise attitude on the part of the beekeeper is first to suspect diseases as being the cause of any losses which he may sus- tain, and to be sure that there is no infectious disease present before looking elsewhere for a cause. NATURE OF THE DISEASES. There are two recognized infectious diseases of the brood of bees, now known as American foul brood and European foul brood. Both diseases weaken colonies by reducing the number of emerging bees needed to replace the old adult bees which die from natural or other causes. In neither case are adult bees affected, so far as known. The means used by the beekeeper in deciding which disease is present is the difference in the appearance of the larva; dead of the two diseases. That the diseases are entirely distinct can not now be doubted, since they show certain differences in the age of the larvae affected, in their response to treatment, and in the appearance of the dead larvae. This is made still more certain by a study of the bacteria present in the dead larvae. Reports are sometimes received that a colony is infected with both diseases at the same time. While this is pos- sible, it is not by any means the rule, and such cases are usually not authentically reported. There is no evidence that chilled or starved brood develops into an infectious disease or that dead brood favors the development of a disease. NAMES OF THE DISEASES. The names American foul brood and European foul brood were applied to these diseases by the Bureau of Entomology, of this de- partrnent, to clear Up the confusion in names which formerly existed. By retaining the words " foul brood " in each name the disease- inspection laws then in force could be interpreted as applying to 442 8 TEEATMEXT OF BEE DISEASES. both diseases. These names were in no way intended to designate geographical distribution, since both diseases did exist and do now exist in both Europe and America, but were chosen primarily because they were convenient and easily remembered names. Their only significance is in indicating where the diseases were first seriously investigated. It was particularly desirable to change the name of the disease now known as European foul brood, since " black brood "" entirely fails to be descriptive and is misleading. SYMPTOMS. The presence of a particular disease in a colony of bees can be ascertained most reliably by a bacteriological examination, since the symptoms are somewhat variable. It is possible, however, to describe the usual manifestations of the diseases, and the usual differences, so that the beekeeper can in most cases tell which disease is present. American Foul Brood. American foul brood is frequently called simply " foul brood." It usually shows itself in the larva just about the time that the larva fills the cell and after it has ceased feeding and has begun pupation. Fig. 2. — American foul brood : o, 5, J, normal sealed cells ; c. ;. sunken cappings, showing perforations ; g^ sunken oappin::: not perforated; 7i. I, m, n, g, r, larvae affected by disease ; e, i, p, /<, scales formed from dried-down larvse ; (?, o, pupfe affected by disease. Three times natural size. (Orisrinal.) At this time it is sealed over in the comb (fig. 2. a, h, /). The first indication of the infection is a slight brownish discoloration and the loss of the well-rounded appearance of the normal larva (fig. 2. I). At this stage the disease is not usuallj' recognized by the bee- 442 TREATMENT OF BEE DISEASES. 9 keeper. The larva gradually sinks down in the cell and becomes darker in color (fig. 2, /i, m), and the posterior end lies against the bottom of the cell. Frequently the segmentation of the larva is clearly marked. By the time it has partially dried down and has became quite dark brown (coffee col- ored) the most typical character- istic of this disease manifests itself. If a match stick or tooth-pick is in- serted into the de- caying mass and ^vithdrawn the larval remains adhere to it and are drawn out in a thread (fig. 3), which sometimes extends for several inches before breaking. This ropiness is the chief characteristic used by the bee- keejjer in diagnosing this disease. The larva continues to dry down and gradually loses its ropiness until it finally becomes merely a Fig. 3. — The ropiness of American foul brood. (Original.) Pig. 4. — American foui-brood comb, showing irregular patches of sunken cappings and scales. The position of the comb indicates the best way to view the scales. (Original.) scale on the lower side wall and base of the cell (fig. 2, e, p, s). The scale formed by the dried-down larva adheres tightly to the cell and can be removed with difficulty from the cell wall. The scales can best be observed when the comb is held with the top inclined toward the observer so that a bright light strikes the lower si'de wall (fig. 4). 83568°— Bull. 442—11 2 10 TEEATMEXT OP BEE DISEASES. A very characteristic and usually penetrating odor is often iiotice- able in the decaying larvae. This can perhaps best be likened to the odor of heated glue. The majority of the larva? which die of this disease are attacked after being sealed in the cells. The cappings are often entirely re- moved by the bees, but when they are left they usually become sunken (fig. 2. g, c, j) and frequently perforated (fig. 2, e, j). As the healthy brood emerges the comb shows the scattered sunken cappings covering dead larvae (fig. 4) , giving it a characteristic appearance. Pupse also may die of this disease, in which case they, too, dry down (fig. 2, 0, d). become ropy, and have the characteristic odor and color. The tongue frequently adheres to the upper side wall and often remains there even after the pupa has dried down to a scale. Younger unsealed larvae are sometimes affected. Usually the disease attacks onh" worker brood, but occasional cases are found in which queen and drone brood are diseased. It is not certain that race of bees, season, or climate have any effect on the virulence of this disease, except that in warmer climates, where the breeding season is pro- longed, the rapidity of devastation is more marked. European Foul Brood. European foul brood was formerly called " black brood " or " New York bee disease." The name " black brood " was a poor one, for the color of the dead brood is rarely black or even very dark brown. European foul brood usually attacks the larva at an earlier stage of its development than American foul brood and while it is still curled up at the base of the cell (fig. 5, ;•). A small percentage of larv'se dies after capping, but sometimes quite young larvae are attacked (fig. 5, e. m). Sunken and perforated cappings are sometimes observed just as in American foul brood (fig. 2, c, g, j). The earliest indication of the disease is a slight yellow or gray discoloration and uneasy movement of the larva in the cell. The larva loses its well-rounded, opaque appearance and becomes slightly translucent, so that the tracheae may become prominent (fig. 5, &), giving the larvae a clearly segmented appearance. The larva is usually flattened against the base of the cell, but may turn so that the ends of the larva are to the rear of the cell (fig. 5, p), or may fall away from the base (fig. 5, e, g, 1) . Later the color changes to a decided yellow or gray and the translucency is lost (fig. 5, q, h). The yellow color may be taken as the chief characteristic of this disease. The dead larva appears as a moist, somewhat collapsed mass, giving the appearance of being melted. "Wlien the remains have become almost dry (fig. 5, c) the tracheae sometimes become conspicuous again, this time by retaining their shape, while the rest of the body content dries around them. Finally all that is left of the larva is a grayish-brown scale against 442 TREATMENT OF BEE DISEASES. 11 the base of the cell (fig. 5, /, h), or a shapeless mass on the lower side wall if the larva did not retain its normal position (fig. 5, n, o). Very few scales ai-e black. The scales are not adhesive, but are easily removed, and the bees carry out a great many in their efforts to clean house. Decaying larvae which have died of this disease are usually not ropy as in American foul brood, but a slight ropiness is sometimes observed. There is usually little odor in European foul brood, but sometimes a sour odor is present, which reminds one of yeast fer- mentation. This disease attacks drone and queen larvae ^ almost as quickly as those of the workers. Fig. 5. — European foul brood: a, j, h, normal sealed cells; l>t Cj dj ej g, i, X, nij p, q, larvae affected by disease ; r, nar- mal larva at age attacked by disease ; f, h, n, o, dried-down larvie or scales. Three times natural size. (Original.) European foul brood is more destructive during the spring and early summer than at other times, often entirely disappearing during late summer and autumn, or during a heavy honey flow. Italian bees seem to be better able to resist the ravages of this disease than any other race. The disease at times spreads with startling rapidity and is most destructive. Where it is prevalent a considerably larger per- centage of colonies is affected than is usual for American foul brood. This disease is very variable in its symptoms and other manifesta- tions and is often a puzzle to the beekeeper. 1 The tendency of this disease to attack queen larvae is a serious drawback in treat- ment. Frequently the bees of a diseased colony attempt to supersede their queen, but the larvte in the queen cells often die, leaving the colony hopelessly queenless. The colony is thus depleted very rapidly. 442 12 TEEATMENT OF BEE DISEASES. Tlie So-Called " Pickle Brood." Id addition to the two infectious diseases just described, brood dead from other causes is often observed. The most common disease of this kind is what is known among beekeepers as " pickle brood."' This name is seemingly applied to a great many different appear- ances and nothing is known of the cause or methods of spread. The most typical form kills the larva when it has extended itself in the cell. The larva usually lies on its back with the head turned upward. The color varies, but is frequently light yellow or brown, and the head is often almost black. The body is swollen and the contents watery, and the head may be quite hard. There is no ropiness. In case the larva are sealed before djdng the cappings are usually normal. The name usually applied to this condition was unwisely chosen, and for the present and until more is known con- cerning the disease it is sjDoken of as the " so-called pickle brood." This trouble does not appear to be infectious and is usually not serious, except that in the aggregate it may cause loss by weakening colonies. Xo treatment is necessar}', as the trouble usually soon dis- appears. The most serious aspect of this disease is that it is often mistaken for one of the infectious diseases, and the colony is need- lessly treated. Brood dead of other causes. Many different external factors may cause brood to die. If brood is killed by chilling in the spring or fall, or by overheating in ex- tremely hot weather, or in shipping colonies of bees, or by starvation, the loss is often mistaken^ attributed to an infectious disease. Such dead brood is soon removed by the bees. When the cause is removed the trouble then soon disappears. Allien a considerable quantity of brood is killed a disagreeable odor is usually present. " Bald-headed brood.'' It sometimes happens that unsealed or only partially sealed pupae, known as "bald-headed brood,'' are observed in the hive, and fre- quently beginners mistake such a condition for disease. The par- tially built capping is often mistaken for the punctured capping of American foul brood. If, on examination, the pupae are normal no fear need be entertained. METHODS OF SPREAD. Both American foul brood and European foul brood spread from colony to colony and from apiary to apiary in much the same way. The common means of carrying the virus is in honey which has be- come contaminated. The disease may be carried when bees rob a hive in which a colony has died of disease or may be transmitted by 442 if) ' UJ m^ < 1 ■•^ _l (/) < _l < I (J) hi UJ m a. o O O O O h. a: UJ o 3 Q z < > LJ z < J UJ a a 4- >- UJ z z o I o a. < o I o CD < • UJ on D D Q. < u tn 3 o •H lU m 10 e o •o rH a> (0 u a V M a> TS S3 o o •H 3 !^ O" o o< « a CO cc S o Sh c L. •H «Q O >. n sJ a 6 0) 13 oa +> 0) D* -(J » c <>-i 0) a o 13 +> OS O s +J a> jS ■T3 c a> ^ •H a> 3 +> P. s^ o* o ta «* +> O o ^ Ot +> « ^ 3 o no CO CD to "-1 iniri III—-" I iii-iitTiii nftaifa-^maffBlirr "O u (U •«> o •a p< o CO +9 iS e OS TREATMENT OF BEE DISEASES. 13 the use of honey from diseased colonies for feeding bees. It is not ahvays necessary that bees be intentionally fed for them to get dis- ease from contaminated honey. Discarded honey receptacles which have contained honey from a contaminated colony, if not thoroughly cleaned, may contain enough honey to carry disease to a healthy I'.piary. This may occur in the vicinity of bakeries or confectionery shops, or may even occur when empty honey bottles are thrown out from private houses. It is also possible to introduce disease into a colony in introducing queen bees purchased from a distance, probably due to the use of contaminated honey in making the candy to supply tlie queen cages. Precautionary Measures. In combating diseases it is much better to prevent disease from getting a foothold than it is to eradicate it after it has begun its work. All beekeepers, wherever located, should practice the fol- lowing precautionary measures : (1) If a colony becomes weak from any cause, or if disease is suspected, contract the entrance to prevent robbing, and if robbing is imminent close the entrance entirely. (2) Never feed honey purchased on the open market. In case of doubt as to the source of honey feed sugar sirup. (o) If within the range of possibility, see that no honey that comes from diseased apiaries is sold in the neighborhood. This may some- times be accomplished by cultivating the home market so that there will be no incentive for bringing in other honey. (4) In introducing purchased queens, transfer them to clean cages provided with candy known to be free from contamination, and destroy the old cage, candy, and accompanying Avorkers. Of course, if it is certain that the queen comes from a healthy apiary this is not necessary. (5) Colonies of bees should never be purchased unless it is cer- tain that they are free from disease. (6) The purchase of old combs or second-hand supplies is dan- gerous, unless it is certain that they came from healthy apiaries. TREATMENT FOR BOTH INFECTIOUS DISEASES. The treatment of an infectious bee disease consists primarily in the elimination or removal of the cause of the disease. It is definitely known that American foul brood is caused by a bacillus named Bacillus larvce. In treating this disease, therefore, the aim of the manipulation is to remove or destroy all of the bacteria of this species. It should be remembered that the effort is not to save the larvse that are already dead or dying, but to stop the further de- 442 14 TREATMENT OP BEE DISEASES. vastatioii of the disease by removing all material capable of trans- mitting the cause of the trouble. The cause of European foul brood is not definitely known, but the same principles of treatment doubtless apply in this disease also. In all of the operations great pains should be taken not to spread the disease through carelessness. After handling a diseased colony the hands of the operator should be washed with water to remove any honey that may be on them. It does not pay to treat colonies that are considerably weakened by disease. In case there are several such colonies they should be united to form strong, vigorous colonies before or during treatment. In discussing treatment it is assumed that hives with movable frames are in use. Box hives are a menace in regions where disease is present. These may be treated for disease by drumming the colony into another box and then hiving it like a SAvarm in a hive, but box hives are not profitable and are especially to be condemned where disease is present on account of the difficulty in inspecting and treating. Shaking Treatment. The shaking treatment consists essentially in the removal of all infected material from the colony, and in compelling the colony to take a fresh start by building new combs and gathering fresh stores. This is done by shaking the bees from the old combs into a clean hive on clean frames. Time of treatment. — The shaking treatment should be given during a flow of honey, so that other bees in the apiary will not be inclined to rob. If this is not possible the operation may be performed under a tent made of mosquito netting. The best time is during the middle of a clear day when a large number of bees are in the field. It is sometimes recommended that shaking be done in the evening, but this is impossible if many colonies are to be treated. The colony can be handled more quickly when the field force is out of the hive. Preparation. — All implements that will be needed, such as queen and drone trap, hive tool, and lighted smoker, should be in readiness before the operation is begun. A complete clean hive with frames is provided, as well as a tightly closed hive body in which to put the contaminated combs after shaking. An extra hive cover or some similar apparatus should be provided to serve as a runway for the bees as they enter the new hive. The new frames should contain strips of comb foundation from one-fourth to 1 inch wide. Full sheets are not desirable, and if combs built on full sheets of founda- tion are desired they may be built later. Operation.— The old hive containing the diseased colony (fig. 6, .4) is now lifted to one side out of the flight of returning field bees and the clean hive (B) set exactly in its place. The cover ((?) is 442 TREATMENT OP BEE DISEASES. 15 now taken off and a few frames {E) removed from the center of the hive. If unspaced frames are used, those remaining in the hive should be pushed tightly to either side of the hive, thus making a barrier beyond which the bees can not crawl as they move to the top of the hive after shaking. This largely prevents them from getting on the outside of the hive. If self-spacing frames are used, a couple of thin boards laid on the top bars on either side will accomplish the same result. The runway {D) is put in place in front of the entrance. The old hive is now opened for the first time. The frames are removed one at a time, lowered part way into the new hive, and with a quick downward shake the bees are dislodged. The frames are then put into the extra hive body (C) and immediately covered to prevent robbing. After all the frames are shaken the bees remaining on the sides of the old hive {A) are shaken out. Fig. 6. — ^Apparatus for the shaking treatment : A, Hive containing diseased colony (for- merly in position of B) ; B, clean hive ; G, empty hive to receive combs after shaking ; D, hive cover used as runway ; E, frames removed from B to give room for shaking ; Ft queen and drone trap; 0-, cover tor clean hive, B. (Original.) If honey is coming in freely, so that thin honey is shaken out of the combs, cover the runway (Z>) with newspapers and shake the bees in front of the new hive {B), leaving all frames in place and the cover on. After the operation the soiled newspapers should be de- stroyed. In shaking in front of the entrance the first one or two frames should be so shaken that the bees are thrown again, t the entrance, where they can locate the hive quickly. They thei fan their wings and. the others follow them into the hive. If this is not done the bees may wander about and get under the hive or in some other undesirable place. After the bees are mostly in the new hive a queen and drone trap {F) or a strip of perforated zinc is placed over the entrance to prevent the colony from deserting the hive. The queen can not pass through the openings in the perforated zinc and the workers will not leave without her. By the time that new combs are built and new brood is ready to be fed, any contaminated honey carried by the bees into their new hive will have been consumed and the 442 16 TREATMENT OF BEE DISEASES. disease will rarely reappear. If it should, a repetition of the treat- ment will be necessary. Saving the healthy brood. — The old combs are now quickly removed. If several colonies are being treated at one time it may pay to stack several hive bodies containing contaminated combs over a weak diseased colony to allow most of the healthy brood to emerge, thereby strengthening the weak colony. After 10 or 12 days this colony is treated in turn and all the combs rendered into wax. If only one or two colonies in a large ajjiary are being treated it will not pay fo do this. Saving the wax. — Any but a very small apiary should have in- cluded in its equipment a wax press for removing wax from old combs. After the contaminated frames are taken to the honey house the combs should be kept carefullj' covered, so that no bees can reach them until the Avax can be ren- dered. This should not be de- layed Aery long or the comb^ may be ruined by wax moths. The slumgum or refuse remaining after the wax is removed should be burned. Contaminated combs should not be put into a solar wax extractor for fear of spreading the dis^^ase. The wax from contami- nated combs may safely be used for the manufacture of comb foundation. Cleaning the hive. — The hive which has contained the diseased (Origiaai., ^^^j^^^^. ^j^^^j^j ^^ thoroughly cleaned of all wax and honey, and it is desirable that it be care- fully disinfected by burning out the inside with a gasoline blue- flame torch (fig. T). If this piece of apparatus is not available several hive bodies ma}' be piled together on a hive bottom and some gasoline or kerosene poured on the sides and on some straw or excelsior at the bottom. This is then ignited and after burn- ing for a few seconds a close-fitting hive cover is placed on top of the pile to extinguish the flames. The inside of the hive bodies should be charred to a light brown. The careful cleaning and dis- infection of frames always costs considerably more in labor than new frames would cost, but these also may be carefully cleaned and used again. Frames may be cleaned by boiling in water for about half an hour, but this frequently causes them to warp badly. The disinfection of hives and frames with chemicals is not recommended, as the ordinary strengths used are valueless for the purpose. 442 Fig. -Gasoline torch. TREATMENT OP BEE DISEASES. l7 Disposal of the honey. — If there is a considerable quantity of honey m the contaminated combs it may be extracted. This honey is not safe to feed to bees without boiliii"-, but it is absolutely safe for human consumption. If there is a comparatively small quantity it may be consimied in the beekeeper's family, care being taken that none of it is placed so that the bees can ever get it. To put such honey on the market is contrary to law in some States. There is always danger that an emptied receptacle will be thrown out where bees can have access to it, thus causing a new outbreak of disease. It can be safely used for feeding to bees, provided it is diluted with at least an equal volume of water to prevent burning, and boiled in a closed vessel for not less than one-half hour, count- ing from the time that the diluted honey first boils vigorously. The honey will not be sterilized if it is heated in a vessel set inside of another containing boiling water. Boiled honey can not be sold as honey. It is good only as a food for bees, and even then should never be used for winter stores, as it will probably cause dysentery. The second shake. — Some beekeepers prefer to shake the bees first onto frames containing strips of foundation as above described, and in four dajs to shake the colony a second time onto full sheets of foundation, destroying all comb built after the first treatment. This insures better combs than the use of strips of foundation, but is a severe drOjin on the strength of the colony. Since it is desirable to have combs built on full sheets, the best policy is to replace any ir- regular combs with full sheets of foundation or good combs later in the season. The cost of shaking. — If the treatment just described is given at the beginning of a good honey flow, it is practically equivalent to arti- ficial swarming and results in an actual increase in the surplus honey, especially in the case of comb-honey production. The wax rendered from the combs will sell for enough to pay for the foundation used if full sheets of foundation are employed. Since a colony so treated actually appears to work with greater vigor than a colony not so manipulated, the cost of treatment is small. If treatment must be given at some other time, so that the colony must be fed, the cost is materially increased. In feeding, it is best to use sugar sirup, or honey that is known to have come from healthy colonies. Treatment with. Bee Escape. As a substitute for the shaking treatment just described, the bees may be removed from their old combs by means of a bee escape. The old hive is moved to one side and in its place is set a clean hive with clean frames and foundation. The queen is at once transferred to the new hive and the field bees fly there on their return from the 442 18 TREATMENT OF BEE DISEASES. field. The infected hive is now placed on top of or close beside the clean hive and a bee escape placed over the entrance, so that the younger bees and those which later emerge from the cells may leave the contaminated hive but can not return. They therefore join the colony in the new hive. If desired, the infected hive may be placed above the clean hive and a tin tube about 1 inch in diameter placed from the old entrance so that the lower end is just above the open entrance of the new hive. The bees follow down this tube and on their return enter the new hive. When all of the healthy brood has emerged from the infected combs the old hive is removed. This treatment induces less excitement in the apiary and is preferred by many experienced beekeeperb. Care should be taken that the old hive is absolutelj- tight to prevent robbing. The old hive and its contents of honej' and wax are treated as indicated under the shak- ing treatment. Fall Treatment. If it is necessary to treat a colony so late in the fall that it would be impossible for the bees to prepare for winter, the treatment may be modified by shaking the bees onto combs entirely full of honey so that there is no place for any brood to be reared. This will usually be satisfactory only after brood rearing has entirely ceased. Unless a colon}' is (juite strong it does not paj' to treat in the fall, but it should be destroyed or united to another colony. In case a diseased colony dies outdoors in the winter there is danger that other bees may have opportunity to rob the hive before the beekeepers can close the entrance. In case bees are wintered in the cellar it is more ad- visable to risk wintering before treatment, for if the colony does die the hive will not ^3e robbed. Drugs. Many European writers have in the past advocated the use of various drugs for feeding, in sugar sirup, to diseased colonies, or the fumigation of contaminated combs. In the case of American foul brood, of which the cause is known, it has been found that the drugs recommended are not of the slightest value and no time should be wasted in their use. TREATMENT FOR EUROPEAN FOUL BROOD. European foul brood is a very peculiar disease and its cause has not yet been satisfactorily determined. It is, therefore, impossible to discuss the treatment of this disease as definitely as that of American foul brood. From the experience of many careful beekeepers it is, however, possible to suggest some additional manipulations which may be tried by experienced beekeepers. The treatments given pre- viously are strongly recommended for this disease. 442 TREATMENT OF BEE DISEASES. 19 Introduction of Italian Stock. Since, as stated previously (p. 11), Italian bees seem to be better able to withstand European foul brood than are other races, it is recommended that apiaries in rejiions where this diseas^e is prevalent be requeened with young, vigorous Italian queens of good stock. This should be done whether or not the shaking treatment is given. Dequeening. It has been found that tlie removal of the queen and the keeping of the colony queenless for a period often results in the disappearance of European foul brood. The length of time that this should be done is in dispute. ^Ir. E. "W Alexander, who advocated this method,^ recommended that the colony be kept queenless (by cutting out all queen cells at the end of 9 days) for a period of 20 days, at which time a cell containing a queen of Italian stock ready to emerge is to be given the colony. The young queen will thus begin to laj' in about 27 daj's after the old queen has been removed, or in at least 3 days after the last of the drone brood has emerged. Other writers have advocated a shorter time. The dequeening treatment is not alwaj's successful, and it is there- fore recommended that care be exercised in trying it. Since there is a considerable percentage of successful results, this would indicate that there is an important princijDle involved. It should not be for- gotten, however, that European foul brood often disappears in the late summer of its own accord if the case is not severe (p. 11), and it is probable that in many of the cases of dequeening reported as suc- cessful the disease would have disappeared without the treatment. This treatment is suggested only for the experienced beekeeper. IWSPECTION OF APIAKIES. Several States have passed laws providing for the inspection of apiaries for contagious disease and creating the office of apiary inspector. The men holding these offices are usually practical bee- keepers, capable of giving excellent advice regarding disease, and it is desirable, when disease exists in a community, that the owners of apiaries take steps to learn who the inspector is and to notify him of the existence of disease. The Bureau of Entomology of this department can usually give information concerning the inspector and is always glad to be of service in bringing the beekeepers and inspectors in touch with one another. Apiary inspection has proved beneficial to the beekeeping industry in spreading information concerning the nature, symptoms, and ^ Alexander, E. W. — How to rid your apiary of black brood. Gleanings in Bee Culture, vol. 33, pp. 1123-1127. 1905. 442 20 TKEATMEXT OF BEE DISEASES. treatment of the contagious diseases and particularly in compelling negligent and careless beekeepers to treat their diseased colonies. It is quite possible for the individual beekeeper to clean up his own apiary by following the directions given in this bulletin, but unless all of the beekeepers in the neighborhood do the same thing there will probably be a recurrence of the trouble due to infection from outside apiaries. It is therefore manifestly to the advantage of the beekeepers that they cooperate with the inspectors in the fight against diseases. 1 EXAMINATION OP SAMPLES OP DISEASED BROOD. The Bureau of Entomology of this department is prepared to assist in the diagnosis of disease in cases where the beekeeper is unable to tell whether or not disease is present, or to determine which disease is in his apiary. Samples of brood comb about 5 inches square containing diseased or dead larvae should be sent by mail in a strong wooden or tin box. The comb should not be wrapped in paper or cotton, but should be cut to fit the box closelj'. It is not possible to diagnose from empty combs, and no honey should be included in the sample, as it is valueless in diagnosis and will prob- ably spoil the sample as well as other mail matter. The name of the sender must always appear on the package, and any available data should be sent in a separate letter. Xever inclose a letter in the box with the sample. THE DISEASES OF ADULT BEES. The diseases affecting adult bees are but imperfectly known. At present four are known to beekeepers by name. "\A'liether these are entirely distinct or whether mider each name one or more diseases are included is not known. As stated in the introduction, these diseases have not been sufficiently investigated to give much help to the practical beekeeper. DYSENTERY. Dysentery affects bees only in the winter and is manifested by a distension of the abdomen, due to an accumulation of fecal matter in the intestine. When a day warm enough for flight occurs the bees fly from the hive to cleanse themselves, and the hive and sur- roundings are spotted with yellow excreta. After a good cleansing flight the trouble usually disappears, but if the bees are unable to fly they often die in great numbers. It is generally believed that dysen- tery is due to improper winter stores, the honey containing- too high a percentage of indigestible matter. Honeydew honey almost always produces dysentery, while bees wintered on high-class honey or sugar sirup are not affected. From the wide experience of many bee- 442 TREATMENT OF BEE DISEASES. 21 keepers in this matter it is safe to assume that this explanation of the disease is the correct one, and consequently great care should be exercised that the colonies are provided with good stores for winter. Recently it has been claimed that there are two types of dysentery, one form as above described and another form which is infectious. American beekeepers are not familiar with an infectious dysentery, and in practical manipulations it is necessary to consider only the type above described. THE SO-CALLED PARALYSIS. It is quite possible that under the name "paralysis" are included several distinct diseases. This is indicated by the variety of symp- toms reported by beekeepers and the number of different seasons and conditions under which the disease is supposed to occur. The usual manifestation described is that the worker bees are seen crawling in front of the hive with their abdomens trembling. The abdomens are also frequently distended. The bees often climb grass blades and on attempting to fly from the top fall again to the ground. Frequently the bees so affected are almost hairless. The same trem- bling motion may often be observed on opening the hive. The colony is often depleted very rapidly. There is no evidence that the disease is infectious. The cause of this peculiar trouble is unknown, and no remedy can be recommended. It is claimed by some writers that a salt-water spray applied to the combs or salt or sulphur sprinkled on the top bars or entrance is sometimes an effective remedy. ISLE OF WIGHT DISEASE. Recently a supposedly infectious disease of adult bees has deci- mated the bees on the Isle of Wight and is said to be spreading in England. It resembles somewhat the so-called paralysis. No treat- ment other than destruction to prevent the spread of the disease has been recommended. So far as is known no trouble of this kind has been experienced in America. SPRIN,G DWINDLING. It sometimes happens that the adult bees in a colony die off in the spring more rapidly than they are replaced by emerging brood. This dwindling may be diminished somewhat by keeping the colony warm and by stimulative feeding, so that all of the energy of the old bees may be used to the best advantage. This condition is prob- ably due to the fact that the colony goes into winter with too large a percentage of old worn-out bees. To prevent this, brood rearing should be continued as late as possible in the fall; if necessary, by stimulative feeding. 442 22 TREATMENT OF BEE DISEASES. PUBLICATIONS OF THE DEPARTMENT OF AGRICULTURE ON BEE DISEASES. There are several other publications of the Bureau of Entomology of this department which deal with bee diseases. They may be obtained on request to the Editor and Chief of the Division of Pub- lications, Department of Agriculture, and are the following: Circular Xo. 94, " The Ciiuse of American Foul Brood." By G. F. White, Ph. D. 1907. 1 pp. This publication contains a brief account of the investigations which demonstrated for the first time the cause of one of the brood diseases of bees, American foul brood. Bulletin No. 70. " Report of the Meeting of Inspectors of Apiaries, San Antonio, Tex., Xovember 12, 190Li." 19U7. 79 pp., 1 pi. Contains an account of tlie history of bee-disease investigations, the relationship of bacteria to bee diseases, and a discussion of treatment by various inspectors of apiaries and other practical beelieepers who are familiar with diseases of bees. Bulletin Xo. 75. Part II. "Wax ^Motbs and American Foul Brood." By E. F. Phillips, Ph. D. 1907. Pp. 19-212, 3 pis. An account of the behavior of tbo two species of wax moths on combs containing American foul brood, showing that moths do not clean up the disease-carrying scales. Bulletin Xo. 7o, Part III, " Bee Diseases in Massachusetts." By Burton X. Gates. 1908. Pp. 23-32, map. .\n account of the distribution of the brood diseases of bees in the State, with brief directions for controlling them. Bulletin Xd. 7."j. I'art 1\'. "The Kelation of the Etiology (Cause) of Bee Dis- eases to the Treatment." By G. F. White, Ph. D. 190S. Pp. 33-42. The necessity for a linowledge of the causes of bee diseases before rational treatment is possible is pointed out. The present state of linowlodge of tlie causes of disease is summarized. Technical Scries. Xo. 14, " The Bacteria of the Apiary, with Spoc-ial Reference to Bee Diseases." By G. F. White. Ph. D. 19(ir,. ."O pp. A study of the bacteria present in both the healthy and tlie diseased colony, with special reference to the diseases of bees. 442 FARMERS' BULLETINS. Bulletins in this list will be sent free, so long as the supply lasts, to any resident of the United States, on application to his Senator, Representative, or Dologato In Congress, or to the Secretary of Agri- culture, Washington, D. C. Because of the limitnl supplif, applicants are urged to select onltj a few num- (lers, choosing those which are of special interest to them. Residents of foreign countries shoulil apply to the Superintendent of Documents, Government Printing Office, Washington, D. C, who has these bulletins for sale. Price 5 cents each to Canada, Cuba, and Mexico; (i cents to other foreign countries. The bulletins entitlect " Experiment Station Work" give briefly the results of experiments performed by tbe State experiment stations. 22. The Feeding of Farm Animals. 27. Flax for Seed and Fiber. 28. Weeds: And How to Kill Them, 80. Grape Diseases on the Pacific Coast. 32. Silos and Silage. 34. Meats: Composition and Cooicing. 35. Potato Culture. 36. Cotton Seed and Its Products. 44. Commercial Fertilizers. 48. The Manuring of Cotton. 49. Sheep Feeding. 61. Standard Varieties of Chickens. 52. Tbe Sugar Beet. 54. Some Common Birds. 65. The Dairy Herd. 66. Experiment Station Work— I. 60. Methods of Curing Tobacco. 61. Asparagus Culture. 62. Marketing Farm Produce. 64. Ducks and Geese. 65. E.xperiment Station Work— II. 69. Experiment Station Work- III. 73. Experiment Station Work— IV. 77. The Liming of Soils. 78. Experiment Station Work— V. 79. Experiment Station Work— VI. 81. Corn Culture in the South. 82. The Culture of Tobacco. 83. Tobacco Soils. 84. Experiment Station Work— VII. 85. Fisn as Food. 86. Thirty Poisonous Plants. 87. Experiment Station Work— VIII. 88. Alkali Lands. 91. Potato Diseases and Treatment. 92. Experiment Station Work— IX. 93. Sugar as Food. 96. Raising Sheep for Mutton. 97. Experiment Station Work — X. 99. Insect Enemies of Shade Trees. 101. Millets. 103. Experiment Station Work- XI. 104. Notes on Frost. 105. Experiment Station Work- XII. 106. Breeds of Dairy Cattle. 113. The Apple and How to Grow It. 114. Experiment Station Work— XIV. 118. Grape Growing in the South. 119. Experiment Station Work— XV. 120. Insects ASecting Tobacco. 121. Beans, Peas, and Other Legumes as Food. 122. Experiment Station Work— XVI. 126. Practical Suggestions for Farm Buildings. 127. Important Insecticides. 128. Eggs and Their Uses as Food. 131. Household Tests for Detection of Oleomar- garine and Renovated Butter. 133. Experiment Station Work- XVIII. 134. Tree Planting on Rnral School Grounds. 135. Sorghum Sirup Manufacture. 137. The Angora Goat. 138. Irrigation In Field and Garden. 139. Emmer: A Grain for theSemiarid Regions. 140. Pineapple Growing. 142. Nutrition and Nutritive Value of Food. 144. Experiment Station Work— XIX. 145. Carbon Bisulphid as an Insecticide. 149. Experiment Station Work — XX. 150. Clearing New Land. 152. Scabies of Cattle. '154. Home Fruit Garden: Preparation and Care. 155. How Insects Affect Health in Rural Districts. 156. The Home Vineyard. 157. The Propagation of Plants. 158. How to Biuld Small Irrigation Ditches. 162. Experiment Station Work— XXI. 164. Rape as a Forage Crop. 166. Cheese Making on the Farm. 167. Cassava. 169. Experiment Station Work— XXII. 170. Principles of Horse Feeding. 172. Scale Insects and Uites on Citrus T^ees. (I) 173. Primer of Forestry. Part I: The Forest. 174. Broom Corn. 176. Home Manufacture and Use of Unfermented Grape Juice. 176. Cranberry Culture. 177. Squab Raising. 178. Insects Injurious in Cranberry Culture. 179. Horseshoeing. 181. Pruning. 182. Poultry as Food. 183. Meat on the Farm: Butchering, Curing, etc. 185. Beautifying the Home Grounds. 186. Experiment Station Work— XXIII. 187. Drainage of Farm Lands. 188. Weeds Used in Medicine. 190. Experiment Station Work— XXIV. 192. Barnyard Manure. 193. Experiment Station Work— XXV. 194. Alfalfa Seed. Annual Flowering Plants. Usefulness of the American Toad. Importation of Game Birds and Eggs for Propagation. Strawberries. Turkeys. Cream Separator on Western Farms. Experiment Station Work— XXVI. Canned Fruits, Preserves, and Jellies. 204. The Cultivation of Mushrooms. 205. Pig Management. 206. Milk Fever and Its Treatment. 209. Controlling the Boll Weevil in Cotton Seed and at Ginneries. 210. Experiment Station Work— XXVII. 213. Raspberries. 218. The School Garden. 219. Lessons from the Grain Rust Epidemic of 1904. 220. Tomatoes. 221. Fungous Diseases of tbe Cranberry. 222. Experiment Station Work— XXVIII. 223. Miscellaneous Cotton Insects in Texas. 224. Canadian Field Peas. 225. Experiment Station Work— XXIX. 227. Experiment Station Work— XXX. 228. Forest Planting and Farm Management. 229. The Production of Good Seed Com. 231. Spraying for Cucumber and Melon Diseases. 232. Okra: Its Culture and Uses. 233. Experiment Station Work— XXXI. 234. The Guinea Fowl. 235. Preparation of Cement Concrete. 236. Incubation and Incubators. 237. Experiment Station Work— XXXII. 238. Citrus Fruit Growing in the Gulf States. 239. The Corrosion of Fence Wire. 241. Butter Making on the Farm. 242. An Example of Model Farming. 243. Fungicides and Their Use in Preventing Dis- eases of Fruits. 244. Experiment Station Work— XXXIII. 245. Renovation of Worn-out Soils. 246. Saccharine Sorghums for Forage. 248. The Lawn. 249. Cereal Breakfast Foods. 250. The Prevention of Stinking Smut of Wheat and Loose Smut of Oats. 251. Experiment Station Work— XXXIV 252. Maple Sugar and Sirup. 253. The Germination of Seed Corn. 254. Cucumbers. 255. The Home Vegetable Garden. 256. Preparation of Vegetables for the Table. 257. Soil Fertility. 258. Texas or Tick Fever and Its Prevention. 259. Experiment Station Work— XXXV. 260. Seed of Red Clover and Its Impurities. 262. Experiment Station Work— XXXVI. 263. Practical Information for Beginners in Irri- gation. 264. The Brown-tail Moth and How to Control It. 266. Management of Soils to Conserve Moisture. 267. Experiment Station Work— XXXVII. II 269, 270, 271, 272, 273. 274, 276, 276. 277. 278. 279. 280. 281. 282. 283. 284. 287, 288, 289, 290, 291. 292. 293. 294. 295. 296. 298. 299. 301. 302. 304. 305. 306. 307. 309. 310. 311. 312. 313. 314. 316. 317. 318. 320. 321. 322. 323. 324. 325. 326. 328. 329. 332. 333. 334. 336. 341. 342. 343. 346. 346. 347. 348. 349. 360. 851. 352. 353. Industrial Alcohol; Uses and Statistics. Modern Conveniences for the Farm Home. Forage Crop Practices in Western Oregon and Western Washington. A Successful Hog and Seed-corn Farm. Experiment Station Work— XXXVIII. Flax Culture. The Gipsy Moth and How to Control It. Experiment Station Work— XXXIX. Alcohol and Gasoline in Farm Engines. Leguminous Crops for Green Manuring. A Method of Eradicating Johnson Grass. A Profitable Tenant Dairy Farm. Experiment Station Work — XL. Celery. Spraying for Apple Diseases and the Codling Moth in the Ozarks. Insect and Fungous Enemies of the Grape East of the Rocky Mountains. Comparative Value of Whole Cotton Seed and Cotton-seed Meal in Fertilizing Cotton. Poultry Management. Nonsaccharine Sorghums. Beans. The Cotton BoUworm. Evaporation of Apples. Cost of Filling Silos. Use of Fruit as Food. Farm Practice in Columbia Basin Uplands. Potatoes and Other Root Crops as Food. Experiment Station Work — XLI. Food Value of Corn and Corn Products. Diversified Farming Under the Plantation System. Home-grown Tea. Sea Island -Cotton: Its Culture, Improve- ment, aUd Diseases. Corn Harvesting Machinery. Growing and Curing Hops. Experiment Station Work — XLII. Dodder in Relation to Farm Seeds. Roselle: Its Culture and Uses. Experiment Station Work — XLIII. A Successful Alabama Diversification Farm. Sand-clay and Burnt-clay Roads. A Successful Southern Hay Farm. Harvesting and Storing Corn. A Method of Breeding Early Cotton to Es- cape Boll-weevil Damage. Experiment Station Work— XLIV. Experiment Station Work— XLV. Cowpeas. Experiment Station Work — XLVI. The Use of the Split-log Dragon Earth Roads. Milo as a Dry-land Grain Crop. Clover Farming on the Sandy Jack-pine Lands of the North. Sweet Potatoes. Small Farms in the Com Belt, Building Up a Run-down Cotton Plantation. Silver Fox Farming. Experiment Station Work— XLVII. Deer Farming in the United States. Forage Crops for Hogs in Kansas and Okla- homa. Nuts and Their Uses as Food. Cotton Wilt. Experiment Station Work— XLVIII. Harmful and Beneficial Mammals of the Arid Interior.' Cropping Systems lor New England Dairy Farms. Macadam Roads. Alfalfa The Basket Willow. Experiment Station Work- XLIX. The Cultivation of Tobacco in Kentucky and Tennessee. The Boll Weevil Problem, with Special Refer- ence to Means of Reducing Damage. Some Common Disinfectants. The Computation of Rations for Farm Ani- mals by the Use of Energy Values. The Repair of Farm Equipment. Bacteria in Milk. The Dairy Industry in the South. The Dehorning of Cattle. TheTubereulinTestofCattleforTuberculosis The Nevada Mouse Plague of 1907-8. Experiment Station Work— L. 364. Onion Culture. 355. A Successful Poultry and Dairy Farm. 357. Methodsof Poultry Management at theMaine Agricultural Experiment Station. 368. A Primer of Forestry. Partll: Practical For- estry. 359. Canning Vegetables in the Home. 360. Experiment Station Work— LI. 361. Meadow Fescue: Its Culture and Uses. 362. Conditions AflectingtheValueofMarketHay. 363. The Use of Milk as Pood. 364. A Profitable Cotton Farm. 365. Farm Management in Northern Potato- growing Sections. 366. Experiment Station Work— LII. 367. Lightning and Lightning Conductors. 368. The Eradication of Bindweed, or Wild Morn- ing-glory. 369. How to Destroy Rata. 370. Replanning a Farm for Profit. 371. Drainage of Irrigated Lands. 372. Soy Beans. 373. Irrigation of Alfalfa. 374. Experiment Station Work — LIII. 376. Care of Food in the Home. 377. Harmfulness of Headache Mixtures. 378. Methods of Exterminating Texas-fever Tick. 379. Hog Cholera. 380. The Loco-weed Disease. 381. Experiment Station Work— LIV. 382. The Adulteration of Forage-plant Seeds. 383. How to Destroy English Sparrows, 384. Experiment Station Work— LV. 385. Boys' and Girls' Agricultural Clubs. 386. PotatoCultureon Irrigated Farmsof the West. 387. ThePreservativeTreatmentof Farm Timbers. 388. Experiment Station Work— LVI. 389. Bread and Bread Making. 390. Pheasant Raising in the United States. 391. Economical Use of Meat in the Home. 392. Irrigation of Sugar Beets. 393. Habit-forming Agents. 394. Windmills in Irrigation in Semiarid West. 395. Sixty-day and Kherson Oats. 396. The Muskrat. 397. Bees. 398. Farm Practice in the Use of Commercial Fer- tilizers in the South Atlantic States. 399. Irrigation of Grain . 400. A More Profitable Corn-planting Method. 401. Protection of Orchards in Northwest from Spring Fro.stH by Fires and Smudges. 402. Canada Bluegrass; Its Culture and Uses. 403. The Construction of Concrete Pence Posts. 404. Irrigation of Orchards. 405. Experiment Station Work— L VII. 406. Soil Conservation. 407. The Potato as a Truck Crop. 408. School Exercises in Plant Production. 409. School Lessons on Corn. 4) 0. Potato Culls as a Sourceof Industrial AlcohoL 411. Feeding Hogs in the South. 412. Expeiiment Station Work— LVIII. 413. The Care of Milk and Its Use in the Home. 414. Corn Cultivation. 415. Seed Corn. 416. Cigar-leaf Tobacco in Pennsylvania. 417. Rice Culture. 418. Game Laws for 1910. 419. Experiment Station Work— LIX. 420. Oats: Distribution and Uses. 421. Control of Blowing Soils. 422. Demonstration Work on Southern Farms. 423. Forest Nurseries for Schools. 424. Oats: Growing the Crop. 125. Experiment Station Work— LX. 426. Caiining Peaches on the Farm. 427. Barley Culture in the Southern States. 428. Testing Farm Seeds in the Home and in the Rural School. 429. IndustrialAlcohol: SourcesandManufacture. 430. Experiment Station Work— LXI. 431. The Peanut. 432. How a City Family Managed a Farm. 433. Cabbage. 434. The Home Production of Onion Seed and Sets. 435. Experiment Station Work— LXII. 436. Winter Oats for the South. 437. A Si-stem of Tenant Farming and Its Re- sults. o / FARMERS' BULLETIN 975 THE CONTROL OF EUROPEAN FOULBROOD E. F. PHILLIPS Apieulturist UNITED STATES DEPARTMENT OF AGRICULTURE WASHINarON : GOVERNMENT PRINTINQ OEFICE : IflZl EUROPEAN FOULBROOD is a disease of the brood of bees which has caused great losses to American beekeepers. It was first recognized as a distinct disease in the United States by New York beekeepers in 1894, but it has probably been present in the United States for a long ti'me. It is important that the beekeeper know whether European or American foulbrood is in his apiary, for the two do not respond to the same treatment. In European foulbrood control the most important step is to prevent the entrance of the disease by keeping all colonies strong and by having all stock resistant to the disease. This can be done successfully even though the disease is in the neighborhood. In case, through failure to take all precautions, the disease does enter, there are certain practices by which the disease can be readily eliminated, but all of these must be used with care. The facts about the disease on which the pre- ventive and remedial measures are based are dis- cussed in this bulletin. Contribution from the Bureau of Entomology L. O. HOWAHD, Chief Issued July, 1918 Washington, D. C. Reprint December, 1921 THE CONTROL OF EUROPEAN FOULBROOD. CONTENTS. Page. Diffloulties of control 3 Name of the disease 3 Symptoms 4 Basis of treatment 7 Pag». Preventive measures 10 Bemedial measures 13 Oood beekeeping will eradicate the disease. . . IS DIFFICULTIES OF CONTROL. EUEOPEAN FOULBROOD has caused much trouble in treat- ment and causes more anxiety among beekeepers than does American f oulbrood. It is recognized generally that European foul- brood requires less drastic methods than does American foulbrood, but seemingly one cannot always be so sure of the efficacy of the treatment, and it is often said by beekeepers that European foul- brood " does not fight fair." The difficulty seems to lie in the fact that the course of the disease in the colony has not been sufficiently studied and the features of treatment have not been adequately analyzed. It is not enough simply to know the name of the organ- ism which causes the disease, but it is essential to know the habits of the germ in the colony. European foulbrood was first recognized in New York State in 1894, and previous to that time no adequate diflferentiation had been made between this disease and American foulbrood. Various writers, especially those in Europe, had recorded two types of brood diseases and had differentiated them sufficiently to call one mild and the other virulent. Careful observations of beekeepers, as well as bacteriologi- cal investigations, have shown that the two diseases are entirely dis- tinct, that one does not change to the other, and that in treatment they behave differently. Now that the symptoms of the two diseases have been carefully studied, one can examine the earlier literature and find indications that European foulbrood was rather widespread in the United States before it was recognized as a distinct disease. At any rate it appears certain that all the European foulbrood in the country did not spread from the first recognized outbreak in New York State. New York beekeepers with justice objected to the name " New York bee disease " which was at one time applied to the disease. NAME OF THE DISEASE. When American beekeepers first differentiated this disease the name " black brood " was generally applied to it. When the investi- 79121°— 21— Bull. 976 3 4 FARMEES BXJLLETIN 975. gation of bee-disease control was inaugurated by the Bureau of Ento- mology it was recognized that this name was not well chosen, for black is not the predominating color of the dead larvae. If any color designation were to be used, yellow would be best, but color is not a safe guide, as this is a variable symptom. Any descriptive name seemed unsafe for a disease with such variable manifestations, and the author therefore proposed that the name be changed. After con- sultation with beekeepers and apiary inspectors it was decided to adopt the name European foulbrood. This was first used in a cir- cular ^ of the Bureau of Entomology and the name has been gener- ally accepted by beekeepers throughout the country. The adjective "European" was chosen because it appeared that this disease had first been subjected to bacteriological investigation by European in- vestigators, while the other disease, American foulbrood, had not been investigated carefully until such work was undertaken in Amer- ica. The names obviously are not intended to convey the idea that the diseases originated one in America and the other in Europe, for the honeybee is not native to America. The names were chosen simply that beekeepers might have names which could be used with safety, and which would not lead to confusion by being descriptive. SYMPTOMS. The beekeeper should know whether he has to deal with American or European foulbrood, for they do not respond to the same treat- ment. The symptoms of European foulbrood are simply the out- ward manifestations of the disease, being chiefly the appearance of the larvae after death. The symptoms are therefore variable. The most accurate method of diagnosis is by bacteriological examination, but this is, of course, not possible in apiary practice. In cases of doubt samples should be sent to the Bureau of Entomology for diagnosis.^ In regions where both diseases occur, beekeepers at times experi- ence difficulty in differentiating them, due chiefly to insufficient ob- servation of the symptoms. If European foulbrood appears in an apiary in the spring, and if American foulbrood is then observed later, the beekeeper may erroneously conclude that both types are 1 Phillips, E. F. Tlie brood diseases of bees. tJ. S. Dept. Agr. Bur. Ent. Clrc. 79. 5 p. 1906. = If dead brooiJ is observed and the beekeeper is not able to diagnose it with accuracy, samples may be sent the Bureau of Entomology for examination. A piece of comb con- taining dead larvse about 4 by 5 inches should be cut out and mailed In a heavy paste- board or wooden box. Tin boxes should never be used, as the brood usually molds in transit, making examination impossible. The sample should not be wrapped before being placed in the box. A suitable box for sending samples will be mailed on request. It is not possible to diagnose from empty combs, and no honey should be included in the sample, as it is valueless in diagnosis and will probably spoil the sample as well as other mail matter, 'xne name of the sender must always appear on the package, and any available data should be sent in a. letter. Never inclose a letter In the box with the sample. CONTROL OF ETTROPEAN FOTJLBROOD. 5 I manifestations of one disease, or that European foulbrood changes to American foulbrood. Such is not the case. It is therefore essen- tial that the symptoms be studied with great care, since to treat American foulbrood by methods applicable only to European foul- brood will result in the spread rather than in the eradication of the disease. (1) Age of larvce affectea. — European foulbrood usually attacks the larva at an early stage of its development, while it is still curled up at the base of the cell (fig. 1, E). At the time of the first mani- festation of disease the larva is about three days old, from the hatching of the egg. A very small percentage of larvae die after Fig. 1. — Portion of comb showing the effect of European foulbiood upon the larvae: a,j,k, Normal sealed cells; b,c,d, e, g, i, I, m, p, g, larvse affected by disease; r, normal larva at age attacked by disease- /, liinfOf dried^down larvae or scales. Three times natural size. capping, but sometimes quite young larvae are attacked (fig. 1, E, M) . Sunken and perforated cappings, which are such common symptoms of American foulbrood, are sometimes seen in colonies suffering with European foulbrood. (2) Early sym/ptoms. — The earliest indications of the disease are a slight yellow or gray discoloration and the uneasy movement of the larva in the cell. The larva loses its well-rounded, opaque ap- pearance and becomes slightly translucent, so that the tracheae may become prominent (fig. 1, B), giving the larva a clearly segmented appearance. (3) Position of larnw. — The larva may be flattened against the base of the cell, may turn so that the two ends are to the rear of the cell (fig. 1, P) , or may fall away from the base (fig. 1, E, G, L) . The 6 farmers' bulletin 975. position of the larva is one of the best means of differentiating American foulbrood and European foulbrood. In American foul- brood the larvae almost without exception are found on the lower side wall, while in European foulbrood they may be there, or at the base of the cell, or on any of the side walls, even the upper one. (4) Color. — As the decay proceeds the color changes to a decided yellW or gray and the translucency is lost (fig. 1, Q, H) . When the disease first appears in a region the yellow color of the decaying larvae seems more constant than later, due probably to the fact that as the disease spreads the germ causing the disease is accompanied by other organisms. The yellow color may be taken as the chief characteristic of the disease. The dead larva appears as a moist, somewhat collapsed mass, giving the appearance of being melted. (5) Scale. — When the remains have become almost dry (fig. 1, C), the tracheae sometimes become conspicuous again, this time by re- taining their shape, while the rest of the body content dries around them. Finally all that is left of the larva is a yellow or grayish- brown scale against the base of the cell (fig. 1, F, H), or a shapeless mass on one of the side walls if the larva did not retain its normal position before death (fig. 1, N, O). Very few scales are black. (6) Adhesion to cell. — At no time during the decay does the larva adhere to the wax closely, but is easily removed, and the bees carry out a great many of them in their efforts to clean house. (7) Usual lack of ropiness. — A slight ropiness is sometimes ob- served in the decaying larvae. This is not, however, at all like the fine ropiness observed in larvae dead of American foulbrood, but the decaying mass behaves more like an old rubber band which has lost its elasticity and which breaks when stretched. (8) Odor. — ^There is usually little odor in European foulbrood, but sometimes a sour odor is present which reminds one of yeast fermen- tation. This odor is quite constant in some regions and seems to come from the decay due to organisms other than the one which causes European foulbrood. (9) Sex. — A symptom of the greatest importance is the fact that the disease attacks drone and queen larvae ^ nearly as quickly as those of the workers. (10) Epidemic cMracter. — In regions where the disease occurs a considerably larger percentage of colonies is affected than is usual for American foulbrood. However, not many colonies die of Euro- pean foulbrood, but the chief trouble is that weakened colonies suc- cumb during winter unless well cared for. The disease spreads at 1 The tendency of this disease to attack queen larvse is a serious drawback in treat- ment. Frequently the bees of a diseased colony attempt to supersede their queen, but the larvoi In the queen cells often die, leaving the colony hopelessly queenless. ' The colony Is thus depleted rapidly. CONTROL OF EUROPEAN FOULBROOD. 7 times with startling rapidity, much more rapidly than American foulbrood. (11) Variability. — In all its symptoms European foulbrood is more variable than is American foulbrood. Color is perhaps the most constant symptom. BASIS OF TREATMENT. The confusion in the treatment of the disease is due to a failure to analyze the factors forming the basis of treatment. Various treat- ments have been described in the beekeeping journals as distinct when they were simply modifications of the same treatment. (1) European foulbrood is a disease of weak colonies. While at times one may observe larvae dead of this disease in strong colonies, usually .they are removed before the disease can do much harm. It should be pointed out, further, that it is the colony which is failing to increase in strength in the spring which is most seriously affected, for a small colony which is rich in young and vigorous bees and which is increasing in strength is often able to overcome the disease. It is therefore a disease of weak rather than small colonies. (2) The disease is prevalent in the spring and early summer. While at times it is observed at other periods of the year, this is not usual. Samples of European foulbrood have been received by the Bureau of Entomology in every month of the year, but, as will be seen from Table I, they are far more commonly received in the early part of the active season. These samples are listed according to the date of receipt at the bureau laboratory. The highest number is re- ceived in June and the average date for the removal of these samples from the hives is probably a few days previous to June 15, perhaps June 10. The earliest samples received are regularly those from California, where the season opens early. There is a sudden increase in May and June and almost as rapid- a decline later. The few sam- ples received from October to April may be largely disregarded, as they are almost without exception dried material of unknown age. Table I. — Distril)Ution of European foulbrood by months, including all posi- tively diagnosed samples received by the Bureau of Entomology from 1908 to December, 1917. Month. Total Califor- number. nia. 3 4 3 17- 10 33 17 ISO' 24 334 30 240 20 164 9 9S 8 17 1 7 3 2 1 New York. January... February. . March April May June July August September October... November. December. 2 23 50 41 20 8 3 1 8 FAEMEES' BtTLLETIH" 9^5. (3) The disease disappears later in the summer unless the colony has become so badly weakened that it can not remove the dead larvae. Such weakened colonies usually die in winter or in a time of dearth. Colonies do not as a rule die as a direct result of European foul- brood. There may still remain some dead larvae in the combs, show- ing that the bees have not been able to remove all of them, but in any but the worst cases even these disappear. If conditions which com- monly prevail in early summer again appear there may be a recur- rence of the disease the same season. (4) This disappearance of the disease usually accompanies the beginning of the honey flow. At this time, unless the colony has already reached maximum strength, there is a rapid increase in brood rearing and the colony increases in strength, bringing about conditions unfavorable for the development of the disease. • If the honey flow fails, the disease may continue and under such condi- tions is at its worst. It should be noted that in regions where the early honey flows are uncertain or usually lacking European foul- brood has done the most damage, for in years of failure the disease spreads with such rapidity that the entire region becomes badly infected. European foulbrood is rarely observed in regions where an early honey flow is certain. (5) The .earliest brood of the year usually escapes with little loss. This important fact has been overlooked in previous discussions of this disease, but it is evident from Table I. The scarcity of Euro- pean foulbrood in the early spring was mentioned in the earliest accounts of its prevalence in New York. This in all probability is due to the fact that the colonies have been able to remove most of the disease dui'ing the previous summer and there has been left only a little of the infecting material. (6) Some bees resist the disease more successfully than others. It has been found through the experience of beekeepers generally that the three-banded Italian bees are best for this purpose. These bees have a further advantage in that they give excellent results in all lines of beekeeping activity, and it is therefore safe to recommend them as the best. This does not at all indicate that other races of bees would not give as good results, as far as European foulbrood control is concerned, but that it is easier to get good three-banded Italian than good bees of any other race. The resistance appears to be either a form of immunity or a greater ability to remove the dead larvse completely. (7) European foulbrood is an infectious disease. This was clearly shown by the experience of beekeepers before the disease was investi- gated from a bacteriological standpoint, and these investigations have supported the observations of the beekeeper. The bacteriological CONTROL OF EUBOPEAN FOULBEOOD. 9 work has shown, further, that the disease is caused by an organism * which has never been found in any other brood disease of bees. The cause of the disease is, therefore, a specific organism, and the disease is entirely distinct from American foulbrood. This is an important point, for there has in the past been considerable confusion in that a few beekeepers have claimed that one disease changes to the other. It should be made clear that this supposition is not supported by any careful observation in the apiary, and that it was recognized generally by beekeepers before the bacteriological investigations were made that the diseases were distinct. (8) The organism causing European foulbrood does not seem from observations in the apiary to be so difficult to eradicate as does the one causing American foulbrood. This is partially confirmed by the bacteriological observations also. (9) When a bee larva dies of European foulbrood the decaying mass does not adhere closely to the cell wall at any time in the decay or when it has dried down to a scale in the back or on the side walls of the cell. Dead larvae may therefore be removed easily by the bees if conditions are favorable for this cleaning. (10) The bees are able under suitable conditions of colony strength and resistance to clean the cells so thoroughly that when future larvae are reared in these cells the disease is not contracted. (11) The method of spread of the disease is not well known, al- though there is some evidence that the infection is carried chiefly by nurse bees. It has been observed that under some circurstances it may be transmitted through feeding, but the experience of beekeepers indi- cates that contaminated honey is not the common means of carrying the disease. It is well known that honey from infected colonies may be given to healthy colonies with entire safety provided the healthy colonies are in such condition that they are able to resist the disease. It is therefore not necessary to disinfect the honey from colonies having European foulbrood, as is the case with that from colonies suffering from American foulbrood. (12) It has not been found necessary to disinfect hives, combs, or frames from diseased colonies. This does not indicate that the germ causing the disease is absent from such material, but that if present it does not do any damage. (13) While the disease spreads with great rapidity at times, it does not seem to be so malignant as is American foulbrood, since many colonies exposed to infection fail to contract the disease. These facts concerning the disease have been discovered in the apiary rather than in the laboratory. The facts are supported by repeated observations, and while the records of observation are not as accurately made as are those of the laboratory the correctness of most ^ Bacillus pluton. 1Q PABMEES' BULLETIN" 9^5. of the facts is attested by the experience of hundreds of beekeepers. In certain cases the findings have been corroborated by bacterio- logical investigation.^ The methods of treatment have also all been devised in the apiary. The difficulty in drawing conclusions from practical observations is that too often beekeepers fail to show the ways in which their ex- perience differs from that of others or in what manner the same principles have been applied in a slightly different manner. PREVENTIVE MEASURES. In keeping European foulbrood under control it is far more im- portant to prevent the disease from getting a foothold in a colony than it is to eradicate the disease afterward. This is not true of American foulbrood, for reliable and practicable preventive measures have not been found for that disease. (1) The use of resistant stock is of the greatest importance, other- wise there is no hope of warding off the disease when it enters a re- gion or of eradicating it froir the apiary after it is once introduced. The use of strong, vigorous Italian stock is best from the standpoint of honey-production, and every beekeeper should therefore see that his apiary is provided with such queens even before European foul- brood appears in the unmediate neighborhood or in the apiary. When the disease is absent it is quite permissible for the beekeeper to save any mismated queens which show themselves to be good, but when European foulbrood is near by this course is unsafe, and in no case should a mismated queen be used as breeding stock. The purity of mating of queens then becomes a matter of first importance and this entails more work than is necessary in the ordinary practices of the apiary. It is not enough simply that queens be pure bred and purely mated, however, for it often occurs that a queen will be poor from other causes. Whenever a queen shows signs of failing it is good bee- keeping to replace her with a good queen. When European foul- brood is present this becomes far more important. Not all Italian stock is equally resistant to European foulbrood, and when the disease is nearby it becomes important that the beekeeper find out which stock is best. Not all queens sold as Italians are pure bred. By far the best plan is to buy a few untested Italian queens from each of several queen breeders and after these have been under observation for a short time the beekeeper will be able to choose from the lot those best suited for breeding purposes. It is not so good a practice to buy a breeding queen, for such queens do not ship 1 Bacteriological studies of bee diseases have been useful to practical beekeepers in explaining the reasons for success or failure with various treatments attempted. These studies have been especially important, however, because through them methods of laboratory diagnosis of the different diseases have been wmrked out. CONTROL OF EUROPEAN EOULBEOOD. 11 SO well in the mails, and even a breeding queen of the most resistant stock might allow her colony to become infected simply because she had been so injured in the mails that she could not keep up egg- laying properly. The buying of untested queens is to be advised at all times, for until more accurate work in breeding is done the indi- vidual beekeeper can choose breeding stock as well as most breeders. It would be possible to recommend certain stock as the best were it not for the fact that the stock of the various queen breeders is not constant. The stock which in one year makes the best showing possi- bly can not be duplicated by the queen breeders the next year. The best course therefore is for each beekeeper, or possibly a group of beekeepers, to try out several strains of Italian bees to find which is best. Having done this, they can continue to breed from the best stock obtained, and they can do as well by that means as they can if they continue to buy queens from the queen breeders. (2) Strength of colony is fully as important as resistant stock. Unfortunately too many beekeepers fail to provide conditions neces- sary to the bees in order that the colonies may be at the proper strength in time to combat European foulbrood successfully. It is good beekeeping to have all colonies strong, and nothing leads to large honey crops as does this factor, yet throughout the country there are thousands of beekeepers who annually fail to get half the crop through failure to have strong colonies at the right time. When the honey-flow comes early in the season, as is the case throughout most of the United States, it is important that every colony be at maximum strength early in the spring. Since European foulbrood appears in the spring and early summer, good beekeeping practice again coincides with the requirements for preventing the ravages of this disease. One difficulty arises from the fact that there is no standard for strength of colony and what one beekeeper considers a strong colony may be considered weak by another and better beekeeper. At the opening of the honey-flow every colony from which a full crop is to be expected should be strong enough to have 10 full combs of Langstroth size filled with brood. Of course this brood may be in a larger number of combs, since the bees usually store some honey at the top of each comb, but it is easy to estimate the brood in terms of full combs. If now we accept the same standard for the desired strength of colony for the purpose of resisting European foulbrood, we will have a condition under which (assuming resistant stock) this disease will never get a start in any colony in the apiary. It is of course recognized that such a standard is seldom realized before or at the beginning of the honey-flow, and this fact is the reason for the loss of so much honey as well as the fullexplanation of the rav- ages of European foulbrood in so many places. It is suggested that each beekeeper in a region where European foulbrood exists ask 12 FAEMEES' BULLETIN 975. himself whether his colonies are actually in as good condition at the opening of the year as he has supposed and that he find out how strong the colonies may be made by providing the best of conditions for the development of the colony population. A beekeeper whose colonies do not measure up to this standard should not condemn the standard until he assures himself that it is entirely impossible, under his conditions, to reach it. Obviously the proper wintering of bees becomes a matter of the highest importance in regions where European foulbrood is found. Those who fail to practice good wintering are the ones who first lose so many colonies that they become discouraged and give up bee- keeping, while those whose wintering has been better are able to treat the disease although their standard of colony strength may not be high enough entirely to ward it off. As was pointed out earlier, the first brood of the year usually escapes with little loss. If proper conditions are provided for winter, either in the cellar or outdoors, brood-rearing is delayed, whereas in poor wintering brood-rearing may begin during the coldest period of the winter.^ If then brood-rearing is delayed by protection, it will begin as a reaction to incoming nectar and pollen. The vitality of the bees has not been destroyed by unseasonable brood-rearing and the colony can rear large quantities of brood from the very beginning. This can, of course, occur only when the colony has proper spring protection. The earliest brood will emerge without appreciable loss from disease, the colony is increased in strength at once, and its capacity for brood-rearing is great. Provided the stock is resistant, the colony is then able to ward off the disease. To bring about all the proper conditions with the least labor on the part of the beekeeper and the least waste of effort on the part of the bees, it is desirable to winter outdoor colonies in two hive-bodies, which has been recommended by this department for other reasons also. Good beekeeping, in so far as handling the bees is concerned, con- sists of providing conditions in the fall so that the colony is full of young, vigorous bees for winter; of providing conditions of protec- tion and good stores such that the bees are not depleted in numbers and vitality during the winter by excessive heat-production ; of pro- viding plenty of stores, adequate room for breeding, and abundant protection during the period of heavy brood-rearing in spring; and of preventing reduction in the strength of the colony by swarming. All of these things, and there are no others of importance, pertain to keeping colonies strong. The beekeeper who provides conditions such that the bees can keep up their own strength will not only reap the honey-crop but he will escape the ravages of European foulbrood. To a large degree the failure of American beekeepers to get their colonies strong enough is due to the use of small hives that are in- > The explanation is given in the publications of the Bureau of Entomology on wintering. CONTROL OF EUROPEAN POULBROOD. 13 suiEciently protected during the winter and spring. The single- walled hive was first made as a means of reducing the cost. Such a hive is a good tool for the beekeeper but it is a poor home for the bees. When the 10-frame hive was found too large to be filled with bees in time for them to go into the supers as soon as the honey-flow opened, instead of protecting the hive the use of the 8-frame hive was commonly adopted. This hive is in rather general use through- out the United States, although fortunately it is now being replaced by the 10-frame hive in many localities. In order that the beekeeper may reduce his labor, it would be well to raise the standard of colony strength by providing better protection and more room for the bees. This will to a large degree eliminate the spring manipulations so often practiced, will get better crops, and will make European foul- brood a minor trouble of the apiary. REMEDIAL MEASURES. When strong colonies headed by vigorous queens of resistant stock are present, European f oulbrood will usually make little if any head- way, yet from time to time there may appear cases which require treatment. The shaking treatment used for American f oulbrood^ is often advocated for European foulbrood and is recommended by many inspectors of apiaries. It was recommended in previous pub- lications of this department, but later observations show that other methods are more reliable. If colonies are given young Italian queens at the time of shaking, results will usually be good, but unless this is done shaking is of little or no value. Some beekeepers prac- tice heavy feeding of either honey or sugar sirup when European foulbrood appears. This often gives good results, for it brings about the conditions which are advocated as preventive measures, although as applied it constitutes a remedial measure. The same amount of stores left with the colony the previous fall will usually do more good than heavy spring feeding as a means of disease control. The remedial measures here described should be used only to re- move the disease if it enters the apiary. Preventive measures should then be employed to avoid a recurrence of the disease. (1) The dead larvae are easily removed from the cells, and the re- medial treatment serves to provide conditions such that these may be removed by the bees during a period when no new diseased ma- terial is appearing in the combs. Usually the queen is removed from the colony, and, since a queen whose colony becomes badly infected is rarely of any value, she is killed. In five or six days all queen cells are removed, so that the colony is hopelessly queenless. The workers do not clean out the diseased cells so rapidly unless they have a queen 1 For a description of this treatment the reader is referred to Farmers' Bulletin 442, " The Treatment of Bee Diseases." ]^4 farmers' bulletin 975. or a queen cell. As soon as the dead larvae are removed, which may be easily determined by examinations, the colony is given a young vigorous Italian queen of resistant stock. If only a few diseased cells are observed and if the colony is fairly populous the queen may simply be caged and released later when the dead brood is removed. The length of time necessary for the cleaning out of the dead larvae varies with the strength of the colony, and for weak colonies it may be necessary to wait until all brood has emerged before giving a young queen.^ This method should not be employed unless each colony has enough bees to sustain at least five combs full of brood. Some col- onies seem to clean out dead brood more rapidly than others of the same strength. If the honey-flow comes early it will usually be pos- sible to reduce the period of queenlessness to a few days. A bee- keeper may use the time necessary for cleaning up as an indication of the strength of his colonies, for if he finds a long time needed he may be sure that his colonies, for some reason, are not as prosperous as they should be. If it is certain that there will be no honey-flow until midsummer or later it is not so necessary, from the standpoint of good beekeeping, to have all colonies strong so early in the year, but it is surely an exceptional locality where there is nothing for the bees to get in early summer. Where the beekeeper is dependent on a late honey-flow it is often desirable to move the bees during the early part of the season to some place where nectar may be obtained. This will often be easier and less expensive than treating the colonies. For example, the author was shown a location in the west where European foulbrood caused great annoyance during the spring, while apiaries not many miles away were able to get enough nectar to ward off the disease and at the same time to give the beekeeper enough profit to justify the ex- pense and time of moving. In such a case preventive measures are cheaper and better than the remedial measures here described. Apiary inspectors should exercise judgment in such cases and permit the moving of colonies to such places, provided they are sure that due precautions will be taken. No precautions need be demanded if the new location is already infected. ^ This method of treatment was described in Its essentials in 1905, In an article published in a periodical devoted to beekeeping. The writer of that article advised that the colony be left queenless for three days after all drone-brood has emerged, thus making a queenless period of 27 days. Later other beekeepers tried shorter periods with success. It should be remembered that the apiaries belonging to the writer of the article referred to were located in the buckwheat region of New York, and that he used a small hive, and on account of these conditions It may be safely assumed that at the time when European foulbrood attacks colonies his colonies were unusually weak. Those who have found a shorter time sufficient have been located in regions where the colony strength may be developed earlier because of earlier honey-flows, or perhaps in some cases these beekeepers wintered better, so that in the spring their colonies were in better condition to resist the ravages of the disease. It would be quite possible to refer to apiaries where the wintering is good and where the spring care Is sufficient to elimi- nate entirely the period of queenlessness. CONTROL OF ETJBOPBAN POTTLBEOOP. 15 The methods of requeening and rearing the queens are matters aside from the treatment of European foulbrood, but in many cases the directions have been obscured by including all such details. Usually it is easier to introduce a queencell of the proper age for the queen to emerge and mate by the time egg laying may again proceed safely in the colony. (2) A substitute for the treatment just described introduces no new principle. The colonies found to have European foulbrood are graded according to strength, and half or more of the stronger ones are shaken to dry extracting combs (not comb foundation) at the same time that the old queens are killed and replaced by young, vig- orous stock. No colony too weak to have five frames of brood should be so treated. If there is no honey coming in, the combs may contain some honey, and it is immaterial whether or not it comes from a col- ony having European foulbrood. The removed brood is now stacked on the weaker diseased colonies so that they may be increased in strength. Just as soon as these have reached the degree of strength possessed by the first colonies shaken, they, too, may be shaken to drawn combs containing no brood, and the diseased brood is given to the remaining few diseased colonies. Usually by the time that the last colonies are ready for treatment it will be found that treatment is not necessary, for in many cases the dead brood will have been removed. If necessary, of course, every diseased colony may be treated. This substitution for the more usual method of treatment has cer- tain advantages. No colony is left queenless and, as a result, the total brood reared in the apiary is increased. No brood is wasted, and the colonies which receive the most of the combs containing diseased brood are usually made sufficiently strong to gather a good crop. (3) Another method which is much used is to place all the brood combs of the infected colony except one in the second hive body over a queen-excluder and to place the queen below with the one frame of brood and frames containing foundation or even drawn combs. Others prefer to put the queen and one frame of brood above. Of course only good Italian queens should be used. , It is interesting to note that the methods used in the control of European foulbrood are exactly the same as are used in remedial methods for swarm control.^ Either the queen or the brood is re- moved or the queen and brood are separated within the hive. Such a similarity is probably of significance, but this at present is merely a matter of speculation. GOOD BEEKEEPING WILL ERADICATE THE DISEASE. It can not be emphasized too strongly that the practices of good beekeeping are those which result in the eradication of European ^ See Farmers' Bulletin 503, " Comb Honey." 16 FABMERS' BULLETIN 975. foulbrood. It does not follow that because a beekeeper is troubled with European foulbrood he is a poor beekeeper, for he may have had good results before the disease appeared. With the entrance of the disease, however, he can change his system so as to overcome the trouble and he may do this with assurance that the changes are such as to result in good beekeeping. Unlike American foulbrood, the disease does not make it necessary that anything of value be de- stroyed by the beekeeper, and if the proper system of management for the particular locality can be found it will result, in most circum- stances, in larger crops than are usually obtained. O Reprinted from Journal of Economic Entomology Vol. 27, No. 3, June, 1934. STUDIES ON THE BACTERIA ASSOCIATED WITH EUROPEAN FOULBROOD By C. E. BuRNSiDE, Assistant Apiculturist, Bureau of Entomology, United States Department of Agriculture The etiology of European foulbrood of bees is an unsettled problem, several theories having been advanced regarding the cause of this dis- ease. In 1885 Cheshire and Cheyne (2) described Bacillus alvei, which they claimed was the cause of the brood disease now known as European foulbrood. In 1907 Maassen (9) stated his belief that the etiology of the mild form of foulbrood (European foulbrood) is not uniform but that the disease is caused principally by Streptococcus apis and B. alvei. White (11, 12, 13) was unsuccessful in attempts to produce typical European foulbrood with cultures of B. alvei, S. apis, or Bacterium eurydice and concluded that this disease is caused by a new species. Bacillus pluton White, which failed to grow on artificial media. Bor- chert {1 p. 12) and Lehmann and Newman {4 p. 236), of Germany, have pointed out that uncertainty still exists concerning the etiology of European foulbrood. Wharton (14) reported having cultured B. pluton and producing infection in a colony of black bees by inoculation with cultures derived from primary colonies. Lochhead (5) says that this organism cultured by Wharton "appears to be closely related if not iden- tical with Streptococcus apis described by Maassen." Wharton (14) also says that "cultures of B. pluton have been observed to change to B. alvei form resembling biologically the B. alvei isolated from infected larvae.'' Lochhead (5, 6) reported the origin of a coccoid bacillus in cultures of B. alvei. The coccoid was isolated and stabilized and is said to have "all the appearance of what White calls Bacillus pluton.'' Both Lochhead and Wharton question the secondary-organism theory of White as regards European foulbrood. At the time White conducted his studies on European foulbrood it was generally believed that bacterial species remain constant in mor- phological and cultural characteristics. In recent years evidence has been constantly increasing that bacteria are capable of morphological. June, '34] burnside : bacteria associated with European foulbroou 657 cultural, and biological transformation, and the old doctrine of fixity of bacterial species is gradually giving way before this evidence. Chief among the investigators in this field is Mellon, whose extensive works have demonstrated that many species of bacteria, when cultured under different environments, produce mutants and variants with greater fre- quency than is commonly supposed. Works of Mellon, Hadly {3), Lohnis and Smith {7, 8), and others strongly indicate the existence of life cycles among bacteria similar to life cycles among the fungi. Mellon {10) has aptly stated what seems to be the situation in the following quo- tation: "Thus the analogy is complete, constituting rather formidable evidence for our contention that biologically bacteria may be properly regarded as fungi which have been telescoped down into a state of exist- ence where their life cycles, although much compressed and often abbre- viated, are still not obliterated." In 1928 the writer started observations and experiments on European foulbrood to obtain evidence in support of one or another of the theories regarding the cause of this disease. He repeated experiments of others but sometimes interpreted them differently, and he also performed new experiments. Experimental results were not always so conclusive as might be desired and the significance of observations was not always ap- parent. Observations and experimental results which may aid, directly or indirectly, in arriving at a true conception of the etiology of European foulbrood are reported in this paper. Morphology of Bacteria in Affected Brood. — Wide variation was observed in the morphology of bacteria present in sick or dead brood. (Plate 6, A, B, and C.) In recently infected larvae the bacteria were mostly very short rods occurring singly, in pairs, or m short chains. Medium-long rods were sometimes present, but no distinctly pointed cells were found during early infection. As the disease progressed and bacteria increased in number, variability in their morphology increased. Most frequently coccoid cells predominated, but at times moderately long rods were equally numerous. Cells of the B. pluton type (Plate 6, C) originated from the coccoid cells at about the time multiplication of bac- teria was checked by overcrowding. The pointed condition appeared to be an expression of dormancy, since these cells usually occurred singly in coherent masses with rarely any indication of active division. In very late infection pointed cells usually predominated, but among differ- ent larvae and different colonies the proportion ranged from 10 per cent or even less to nearly 100 per cent. In some larvae coccoid cells pre- dominated (Plate 6, A), in others moderately long rods were most numerous (Plate 6, B), and occasionally long, slender, faintly staining •J K L Bacterial Forms from Larvae Infected with European Foulbrood (x 1,500) A, B, and C, Smears from the stomach of different larvae in an advanced stage of infection, showing difference in morphology of the bacteria. In A only a few June, '34] burnside : bacteria associated with European foulbrood 659 rods were present in small numbers (Plate 6, C). Thus it is apparent that the morphological forms encountered in sick larvae present a com- plex and variable picture. When bacterial growth occurred after death of larvae, it usually con- sisted almost entirely of moderate-sized rods, of which a variable per- centage formed spores of B. alvei. (Plate 6, D.) Occasionally coccoid bacilli indistinguishable morphologically from bacilli that grow in the di- gestive tract of sick larvae caused decay of the body tissues after death. In still other larvae decay was caused by both the rod and the coccoid form. Cultures from Sick or Dead Brood Yielded Different Morph- ological Forms. — Rough inoculation of bouillon agar slants from sick or dead brood most frequently yielded cultures of B. alvei which sporu- lated promptly. Many cultures, particularly those prepared from the digestive tract of larvae in an early stage of infection, yielded a coccoid organism in apparently pure culture which morphologically and cul- turally closely resembled 5". apis, and there seems to be little doubt that it is identical with the form described by Maassen (P) in 1908 and later studied by White (^11, 12, 13), Wharton {14), and Lochhead (5). On egg-yolk agar many cells of this form become lancet-shaped and were of the cells are still dividing; the majority are coccoid with rounded ends, while some are more or less pointed. In B the coccoid cells, short rods, and medium long rods are about equally numerous. In C most of the cells have pointed ends and are typical of the type known as Bacillus pluton; two long, slender rods, such as occur in small numbers in infected larvae, are also seen. D, Spores of Bacillus alvei from the decayed remains of a larva dead of European foulbrood. E, Pure culture of Streptococcus apis from agar culture containing unheated egg yolk. In some cultures 50 per cent or more of the cells become more or less pointed and are indistinguishable morphologically from Bacillus pluton. F, Bacillus alvei and Streptococcus apis from an agar culture prepared directly from a sick larva. (2 days at 36° C.) G, Streptococcus apis from a culture prepared directly from a sick larva in brood filtrate. (2 days at 36° C.) H, Asporogenic agar culture of Bacillus alvei, which morphologically closely resembles Bacterium eurydice. (5 days at 20° C.) /, Threadlike rods from an asporogenic agar culture of Bacillus alvei. (17 days at 20° C.) /, Rods from an asporogenic bouiUon-agar culture of Bacillus alvei with beaded and granular protoplasm. (S days at 36° C.) K, Culture of Streptococcus apis in bouillon broth, showing rods of B. alvei which appeared after 6 days at 36° C. L, Pure culture of Streptococcus apis from bouillon agar to which 10 per cent honey was added. (,36° C.) 660 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 27 indistinguishable morphologically from B. plitton. (Plate 6, E.) Many cultures yielded both B. alvei and 6". apis. (Plate 6, F) In some cul- tures the cells of the coccoid form were observed to be dissociated. Oc- casional cultures of B. alvei prepared from sick or dead brood — the rela- tive number varied in different samples of infected brood comb — grew slowly and sporulation was delayed and incomplete. In a few instances rough inoculation from affected brood yielded cultures of rods which did not form spores at all when cultivated at room temperature. Cul- tures of asporogenic rods were also obtained, some of which closely re- sembled B. enrydice, by plating directly from sick larvae at room temperature. In a few cultures on bouillon agar or egg-yolk agar prepared with bacteria from the digestive tract of sick larvae no growth was detected. From the same larvae, however, prompt and abundant growth was ob- tained in dilute sterile filtrate prepared from macerated honeybee larvae. In filtrate medium a coccoid organism resembling .S". apis (Plate 6, G) was usually obtained, but some cultures yielded also small or moderate- sized rods. It is evident that failure to obtain growth on ordinary nu- trient agar does not prove the absence of culturable bacteria. Cultures from healthy-appearing larvae from different infected colo- nies yielded in a variable percentage of the tubes apparently one or an- other of the same forms obtained in cultures from sick or dead larvae {B. alvei or S. apis). When combs of brood were removed from colonies shortly after infection had subsided and were kept either at room temperature or at 36° C, none of the larvae dying of starvation or chilling were noticeably decayed by B. alvei, even though this organism was found by cultural tests to be present in the digestive tract of more than 90 per cent of them. Bacteria Present in Honey from Infected Colonies. — Bacillus alvei was found to be abundant in honey and pollen from the brood chamber of infected colonies. In advanced cases inoculations of nutrient agar with a single loopful of honey (about 0.001 cc) practically always yielded B. alvei, while a few also yielded 5". apis. In early or mild cases part of the cultures prepared with honey or pollen yielded B. alvei. Bacteria Found in Colonies with European Foulbrood Not Present in Healthy Colonies. — In striking contracts to the preva- lence of bacteria in larvae from infected colonies is the complete absence of these forms in healthy colonies. The writer has made microscopical examinations of and prepared cultures numbering well into the thousands from larvae dead of American foulbrood, sacbrood, fungus diseases, plant poisoning, and other brood disorders, as well as from healthy larvae. June, '34] burnside : bacteria associated with European foulbrood 661 without having found or obtained B. alvei or 6". apis in culture, except on rare occasions when mixed infection was suspected. Lilcewise cul- tures prepared with honey and pollen from healthy colonies in which European foulbrood never existed have never yielded B. alvei. The writer's observations on this point differ from those of Maassen (P), who claims to have found B. alvei present in some cases in larvae dead of "virulent foulbrood" (American foulbrood). In a few cases the writer obtained B. alvei in cultures from combs infected with Amer- ican foulbrood, but a thorough inspection of the brood xomb and of the scales used in preparing the cultures generally revealed mixed infection and occasionally a scale of European foulbrood which resembled that of American foulbrood. It seems possible that Maassen may likewise have been dealing with cases of mixed infection. Transmission of European Foulbrood with Cultures. — When conditions are favorable, typical European foulbrood is readily trans- mitted by inoculation with bacteria taken from the digestive tract of sick or dead brood. On the other hand, typical European foulbrood has only rarely been produced by inoculation with cultures, although several investigators, in inoculation experiments with cultures of B. alvei (rods and spores), have obtained an atypical infection. In the writer's experiments an occasional larva or pupa was attacked by B. alvei when a water suspension of sporulating cultures recently isolated from infected brood was sprayed over developing brood. It appears that B. alvei in the usual sporogenic state may, under favorable circumstances, produce disease in larvae or pupae, but this disease is not typical European foul- brood. Likewise, attempts to produce European foulbrood by inoculation with pure cultures of 5". apis have usually been unsuccessful. Maassen (9) failed to demonstrate pathogenesis for 6". apis by feeding pure cultures, and White {12) states that "No disease results when the brood of bees is fed cultures of Streptococcus apis either by the direct or indirect method." In speaking of the coccoid form of B. alvei, Lochhead (d) states, "Our attempts to produce the disease in a colony of black bees through feeding cultures of the coccus have so far been inconclusive." On the other hand, Wharton {14), in inoculation experiments with cul- tures of a coccoid bacillus which Lochhead (5) says "appeared to be closely related to, if not identical with. Streptococcus apis," claims to have produced typical European foulbrood. Concerning this experiment Wharton says, "The writer has obtained infection in a healthy colony of black bees in four days, using as inoculum cultures of the organism de- rived from isolated colonies. The symptoms of the diseased larvae 662 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 27 accorded with those observed in naturally infected larvae and the micro- scopical picture was typical — B. alvei forms being also present, though only in small numbers." If Wharton's cultures were pure, as he as- sumes, to him belongs the credit of first producing typical European foulbrood by inoculation with pure cultures. The writer's inoculation experiments with 5". apis and with non-spore- forming rod cultures (resembling B. eurydice) isolated from sick or dead brood gave results that were largely negative or inconclusive. On one occasion typical European foulbrood was produced by inoculation with cultures of 6". apis freshly isolated from sick larvae. In isolating pure cultures plating was ordinarily done two or more times. Occasionally larvae inoculated with such cultures appeared to become infected and were removed by the bees, but the symptoms were not typical of European foulbrood and the infection disappeared promptly. In an experiment performed in 1933, bacteria from the digestive tract of a naturally infected larva were streaked on egg-yolk-agar plates. After 24 hours at 34° C. isolated colonies of S. apis were touched with a platinum loop and cultures were prepared on egg-yolk-agar slants. With the abundant growth obtained on these slants after 44 hours at 36° C, a colony of black bees was inoculated by spraying the bacteria, in water suspension, over two combs of young and hatching larvae. Two days later numerous coccoid bacteria were found within the digestive tract of some of the larvae. On the following day coccoid bacteria had greatly increased in number in many of the inoculated larvae and larvae were being removed rapidly by the bees. On the fourth day more than 90 per cent of the inoculated larvae had been removed. None of those remaining showed outward symptoms, but upon microscopical exami- nation coccoid bacteria morphologically identical with the bacteria in the inoculum were so abundant within the digestive tract that infection could be definitely ascertained. All the unsealed brood in the inoculated combs was finally removed by the bees and no dead larvae were found in the cells. A water suspension of bacteria from the artificially infected larvae was next sprayed over another comb of young brood in the same colony. After 3 days fully 25 per cent of the inoculated larvae in this comb were dead or dying from infection, of which the gross symptoms and the bacteriological picture were typical of European foulbrood. Pointed or lancet-shaped cells {B. pluton) were at first absent or present only in small numbers, but later they became numerous. The coccoid bacillus was reisolated, but out of about 100 cultures B. alvei was obtained in only one. With cultures prepared by rough transfer from those with June, '34] burnside : bacteria associated with European foulbrood 663 which infection was obtained three succeeding experiments gave negative results. The results of this experiment and the comparable experiment performed by Wharton (14) seem to point to retention of virulence by 5". apis during only about two generations on artificial culture media. It is recognized, however, that the purity of such recently isolated cultures may be questioned. Pleomorphism and Variability in Bacillus alvei. Several in- vestigators have observed variation in size and shape of individual cells in cultures of B. alvei. Maassen (P) says that cultures of B. alvei degenerate on the usual artificial medium and that nuclei or granules develop in the plasma while the ability to form spores disappears. Loch- head (5, 6), using a special nutrient agar, observed the origin of coccoid cells from rods of B. alvei, which he reported (6) to be indistinguishable morphologically from B. pluton. In the writer's experiments B. alvei, in the form in which it is usually isolated from dead brood, grew luxuriantly, spread rapidly over the agar, and formed spores promptly and abundantly on bouillon agar and on egg-yolk agar at 36° C. (Plate 7, A.) In repeated transfers at 36° C. on these agars no morphological or cultural changes were observed. In bouillon broth, potato broth, and milk, and in media containing sterile filtrate prepared from honeybee larvae, the luxuriance of growth and the tendency to form spores gradually decreased in repeated transfers. After about 10 generations in potato broth, cultures prepared by rough trans- fers to bouillon agar and egg-yolk agar grew slowly while "sporulation was incomplete and delayed or lacking. Growth either spread slowly or was confined to small colonies. (Plate?, S.) By planting and culturing from isolated colonies, strictly asporogenic cultures were obtained which in repeated transfers remained asporogenic. When cultured at room temperature the transformation in potato broth from a sporogenic to an asporogenic condition was more rapid. Bouillon broth seemed less effective in producing the change, and results with filtrates from honey- bee larvae were irregular. These asporogenic cultures of B. alvei varied in morphology and cul- tural characteristics (Plate 6, H, I, J), but in some cases the resemblance to cultures of asporogenic rods isolated by plating from sick larvae was marked. It seems probable, therefore, that B. alvei may exist in infected larvae in either sporogenic or asporogenic condition. Morphologically and culturally the characteristics of some of the cul- tures were indistinguishable from the characteristics given by White (11) for B. eurydice. (Plate 7, B; plate 6, H.) Concerning this form White (13) says: "In studying this species cultures were isolated which Plate 7 Types of Growth of Bacillus alvei A, Two spreading colonies of Bacillus alvei on a bouillon- agar plate, showing difiference in type of growth. B, Bacillus alvei on glucose-agar plate growing in small colonies after transformation from a sporogenic to an asporogenic condition. June, '34] burnside : bacteria associateu with European foulbrood 665 in some respects differed from it. Whether these are different species or belong to a group of which B. eurydice is a representative has not been definitely determined." Concerning methods of culture White further says : "Incubation must be carried out at room temperature. Growth of the species is always slow and never luxuriant." In view of the writer's observations it seems probable that the culture described by White as B. eurydice and cultures which "in some respects differed from it" may have been asporogenic variants of B. alvei. The variability of B. alvei in morphology and cultural characteristics appeared to depend upon the physiological condition of the organism as well as upon the culture medium. To retain viability of cultures frequent transfers were necessary. The description of the organism given below is of cultures produced as follows : Agar slant cultures of sporogenic B. alvei were prepared from isolated colonies. A water suspension of spores was boiled for 3 to 5 minutes, after which the organism was, cul- tured by transferring for 10 generations in potato broth. Cultures pre- pared from isolated colonies on agar plate by transfer to nutrient agar on which sporulation is ordinarily prompt were then asporogenic at room temperature. Glucose-agar plate. — Colonies slightly convex and rounded with uni- form outline, 1 to 2 mm in diameter, grayish by reflected light, bluish gray by transmitted light ; under a binocular appearing very light brown and finely granular. Morphology. — Variable; rods nonmotile and asporogenic, occurring singly, in pairs, or in chains, ends rounded ; protoplasm homogeneous or granular or broken ; smaller and more slender than sporogenic B. alvei in some cultures, of equal dimensions in others. Staining properties. — Stained readily with the usual dyes and Gram- negative ; gra:nules sometimes darkly staining and Gram-positive. Oxygen requirements. — Growth Occurring under anaerobic conditions but more luxuriant in the presence of air. Bouillon. — Medium slightly clouded after 48 hours, a slightly viscid sediment forming slowly at bottom of tubes. Sugars. — With the usual sugars acid but no gas produced ; both arms of tube clouded, but growth most luxuriant in open arm; litmus dis- charged. Brood filtrate. — In some cultures brood filtrate added to the medium increased growth, but in other cultures no effect observed ; growth also variable in water solution of filtrate. Milk.- — Slight growth with little or no change apparent in either litmus milk or plain milk. 666 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 27 Potato broth. — Growth slow, with sHght uniform clouding and slight sediment. Potato. — Feeble, grayish growth. Gelatine stab. — No liquefaction. In asporogenic cultures of B. alvei coccoid bodies were observed which morphologically resembled the coccoid bodies observed by Lochhead ((5), but attempts to isolate this form have thus far been unsuccessful. In recently formed asporogenic cultures the protoplasm (from bouillon or glucose agar) was usually homogeneous. After several transfers, especially on egg-yolk agar, the protoplasm often became granular or broken. At times the rods assumed a beaded appearance resembling chains of coccoid cells. One culture in brood-filtrate medium assumed a decided coccoid appearance with many forms indistinguishable mor- phologically from chains of coccoids observed in cultures of 5". apis. Rods were frequently observed in a state of dissociation, and in some cultures few rods remained undissociated after 4 or 5 days' incubation. Pleomorphism in Streptococcus apis. — On ordinary bouillon agar .y. apis, when freshly isolated, appears in diplococcoid form with occa- sional single cells and short chains. The cells are only rarely spherical, their length being usually aproximately lj4 times their thickness. In bouillon broth the tendency to grow in chains is accentuated, while on nutrient agar containing egg yolk the cells are smaller than in bouillon agar and appear singly or in pairs. The ends are sharply rounded and frequently pointed, many forms being morphologically indistinguishable from B. pluton. In some of the cultures on egg-yolk agar approximate- ly 50 per cent of the cells became more or less pointed after multiplication ceased. (Plate 6, E.) After prolonged cultivation further changes in morphology have been observed from time to time. Wharton {14) reported that his morphological studies suggest the identity of B. pluton and B. alvei and stated that "Cultures of B. pluton have been observed to change to B. alvei form, resembling biologically the B. alvei isolated from infected larvae." In a few instances the writer's cultures of 6". apis derived originally from isolated colonies have yielded rods (Plate 6, K) and eventually spores of B. alvei. This has been observed only in broth cultures prepared by transfer from old cultures on nutrient agar. After incubation for 7 to 12 days at 27° C, rods of B. alvei appeared in small numbers, but nothing was determined concerning their origin. Spores were produced in the original broth cultures and in transfers on nutrient agar. On other occasions rods that failed either to grow or to produce spores in transfers originated in broth cultures. Occasionally rods with length equal to about five times June, '34] burn side: bacteria associated with European toulbrood 667 their thickness, shorter rods, coccoid cells, and lancet-shaped cells were observed in the same chains in broth cultures of 5". apis. In cultures on bouillon agar to which 10 per cent honey was added, some of the cells were increased in size, many were distinctly rod-shaped, while others assumed lancet shapes indistinguishable from B ' alvei ( Plate 6, L ) . In broth cultures of 5'. apis containing both honey and unheated egg yolk, several variants were observed after 2 days, including large, irregu- lar, barrel-shaped, and spherical cells, occurring usually in pairs or in chains. Conclusions. — Several morphologically different bacteria forms are more or less constantly present in honeybee larvae sick or dead of European foulbrood. These forms are absent in larvae sick or dead of other causes. No evidence has yet been obtained which satisfactorily explains the etiology of European foulbrood or why these different bacterial forms are constantly associated with this disease. It has been found that Bacillus alvei is capable of morphological, cul- tural, and biological transformation and is also capable of stabilization, at least temporarily, as a sporogenic rod, an asporogenic rod resembling Bacterium eurydice, or a coccoid resembling Bacillus pluton. There seems to be insufficient reason for assuming that the lancet- shaped bacterial cell, B. pluton, found in late stages of infection in European foulbrood, is of different genus and species from the similar form Streptococcus apis, which is readily obtained in culture from sick larvae. The identity of Streptococcus apis and Bacillus pluton is suggested by morphological similarity, by the fact that the pointed or lancet shape is a variable character in both forms and appears to be only an expression of restricted growth or dormancy accentuated in infected larvae, and also by the usual, if not invariable, occurrence of Streptococcus apis in recently infected larvae, and by the fact that typical European foul- brood was produced in Wharton's and in the writer's experiments when young brood was inoculated with cultures of 6". apis prepared with isolated colonies. That Bacillus pluton and Streptococcus apis are variants, or stages in the life history, of Bacillus alvei is suggested by the occurrence of vari- ants resembling B. pluton in pure cultures of B. alvei and by the apparent origin on rare occasions of sporogenic B. alvei in cultures 5. apis. The transformation at room temperature of sporogenic B. alvei into an asporogenic nonmotile rod which morphologically, culturally, and bio- 668 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 27 logicall)' is closely allied to Bacterium eurvdice likewise suggests the identity of these forms. Regarding the etiology of European foulbrood and the variety of bac- terial forms present in sick and dead larvae much remains to be deter- mined. The writer is of the opinion that the evidence now available points more strongly to a pleomorphic organism as the etiological factor in this disease than to the secondary organism theory advanced by White. Literature Cited 1. BoRCHERT, A. 1926. Die seuchenhaften Krankheiten der Honigbiene. 98 p., illus. Berlin. 2. Cheshire, F. R., and Cheyne, W. W. 1885. The pathogenic history and his- tory under cultivation of a new bacillus (S. alve'i), the cause of a disease of the hive bee hitherto known as foul brood. Jour. Roy. Micros. Soc. (2) 5 pt. 4) : 581-601. 3. Hadley, p. 1927. Microbic dissociation. The instability of bacterial species with special reference to active dissociation and transmissible autolysis. Jour. Infect. Diseases 40: 1-312, illus. 4. Lehmann, K. B., and Newman, R. O. 1927. Bakteriologie, inbesondere bakteriologische Diagnostik. II Band. AUgememeine und spezielle Bakteriologie. 876 p. Munich. 5. LocHHEAD, A. G. 1928. The etiology of European foul-brood of bees. Science 67:159-160. 6. . 1928. Studies on the etiology of European foulbrood of bees. 4th Intern. Cong. Ent. Trans., v. 2, p. 1005-1009, illus. 7. Lohnis, F., and Smith, N. R. 1916. Life cycles of the bacteria. Jour. Agr. Research 6 : 675-702, illus. 8. . 1923. Studies upon the life cycles of the bacteria — Part II : Life history of Azotobacter. Jour. Agr. Research 23 : 401-432, illus. 9. Maassen, a. 1908. Zur atiologie der sogenannten Faulbrut der Honigbienen. Arbeiten K. Biol. Anst. Land u. Forstw. 6: 53-70, illus. 10. Mellon, R. P. 1926. Studies in microbic heredity. VI. The infective and toxonomic significance of a newly described ascospore stage for the fungi of blastomycosis. Jour. Bact. 11:229-252, illus. 11. White, G. F. 1912. The cause of European foulbrood. U. S. Dept. Agr. Bur. Ent. Circ. 157, IS p., illus. 12. . 1920. European foulbrood. U. S. Dept. Agr. Bui. 810, 39 p., illus. 13- . 1920. Some observations on European foulbrood. Amer Bee Jour. 60 : 225-227, 266-268, illus. 14. Wharton, D, R. A. 1928. Etiology of European foul-brood of bees. Science 66:451-452. Reprinted from the Journal of Economic Entomology, Vol. 14, February, 1921, No. i MIXED INFECTION IN THE BROOD DISEASES OF BEES By Arnold P. Sturtevant, Specialist in the Bacteriology of Bee Diseases, Bureau oj Entomology, United States Department of Agriculture The two principal brood diseases of bees, European foulbrood and American foulbrood, heretofore have not been found associated together commonly in the same colony. The generally accepted belief has been that it is indeed a rare occurrence to find both diseases under these conditions. Sacbrood, on the other hand, is much more Often found in greater or less quantity associated with either European foulbrood or American foulbrood, but seldom assuming dangerous proportions, either alone or in conjunction with the others. Statistics for the past few years, however, show that these cases of what may be called mixed infection are probably more common than was previously supposed and may account for some of the puzzling instances where colonies have not responded to treatment in the customary manner, thereby causing beekeepers to believe they have some new form of brood disease, or that the disease is showing some new unheard of characteristics. Cases of so-called mixed infections are not at all tmcommon among human diseases. Where this condition occurs, such as when a person affected with typhoid fever develops pneumonia at the same time, it is always the individual to whom the term mixed infection is applied. It is a somewhat different matter in the case of the brood diseases of bees. In the first place, so far as is known, the organisms causing these two diseases, Bacillus larvae of American foulbrood and Bacillus pluton of European foulbrood, have never been found together in the same individual larva. It is, therefore, the colony as whole which is to 128 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 14 be considered as the individual unit, as is the case in the majority of the manipulations of beekeeping practice. This fact makes the problem slightly different from a case of mixed infection as considered from the point of view of hiunan medicine. However, since different individuals are involved in the mixed infections there is no "a priori" reason for considering such cases as impossible. The first published report of an authentic instance where both Ameri- can and European foulbrood were found together in the same comb from a diseased colony was reported by McCray.i This report was concern- ing a sample (4982) received at the laborator)- for diagnosis May 4, 1916, from Stanislaus County, California. Previous to this case only one other such sample (2598 from Brown County, Wisconsin in 1911) had been received for diagnosis, showing the presence of both diseases, but no report concerning it was pubHshed. These two samples were the only known authentic cases on record either in the Bee-Culture Laboratory among practically 5000 samples received up to 1916, or in the beekeeping literature. These two cases were considered to be interesting in that they demonstrated that the presence of both diseases at the same time in a colony was possible, but not much importance was given the matter because of their rare occurrence. White^ states that "such a double infection has been encountered in the writer's experience very rarely. In such diagnoses, therefore, after European foulbrood had been found in the sample, American foulbrood is seldom looked for." This practice has been the custom generally as well when American foulbrood was found present in a sample, no further search for European foulbrood being made unless there were present strikingly prominent symptoms abnormal for American foulbrood. As a result the diagnostic records of the Office of Bee-Culture show but six cases of mixed infection up to December 31, 1918, among the approximately 6000 sample records. Developments during the year 1919, however, showed that mixed or double infection is more probable than had been previously supposed. These facts were particularly impressed upon the writer during the spring of 1919 while on a trip investigating the bee disease conditions in the State of CaUfomia. While in the field during a period of less than one month, and in three different counties of the State of Cahfomia, six cases were found showing both American foulbrood and European foul- brood in the same colonies. Each case was diagnosed posivitely at once in the field by means of microscopic examination of dead larvae showing characteristic symptoms of the two diseases and found to con- tain the specific causative organisms. It is interesting to note that three 'McCray, A. H. 1916. Report of the finding of American Foulbrood and European foulbrood in the same comb. Jour, of Eco. Ext. Vol. IX, p. 379. 2 White, G. P., 1920. European foulbrood . U. S. Dept. of Agric. Bui. 810. February, '21] sturtevant: mixed infections 129 of the six samples were found in Stanislaus County in the same locality as the sample reported by McCray in 1916. These cases were all found in regions where both diseases are exceedingly prevalent and of long standing. A few of the samples were fairly self evident from gross appearances, but the majority required a more minute examination. From that time on, particularly after returning to the laboratory in Washington, more careful examination was made, both gross and microscopic of all samples received because of suspicions aroused by the unusual prevalence of the obvious cases found in California. This was done in order to eliminate the danger of overlooking cases where one disease might be predominant over the other, whether both diseases were suspected or not, causing the less prominent to be overlooked. As a result, during the remainder of the year 1919 from June until December, twelve more such samples were received in the laboratory from various parts of the country, (18 in all for that year, total 24) all of which proved upon careful diagnosis to contain both American foul- brood and European foulbrood in the same sample of comb. Further- more, during the year 1920, up until November 15th, fourteen more such samples were received, making a total in all of 38. Tables 1 and 2 give the data from sample records. Table I. — Cases of Mixed Infection from Laboratory Records Apparent primary Date Lab. No .. State County invader from gross Remarks appearance 9-20-11 2598 Wisconsin Brown ? Diagnosed by G. F. White 5- 4-16 4982 California Stanislaus American fb. Diagnosed by A. H. McCray 6- 3-16 5061 California Stanislaus American fb. Diagnosed by A. H. McCray 5-16-17 5392 Missouri Jasper Probably Afb. 5- 9-18 5836 Mississippi Washington ? Apparently about equa 10- 9-18 6122 Wisconsin Barron ? More Efb than Afb 4-19-19 6437 California Santa Barbara Probably Efb. One cell Afb. 4-26-19 6441 California Sacramento American fb. From history of case 4-26-19 6442 California Sacramento American fb. 4-28-19 6445 California Stanislaus European fb. Few cells Afb. 4-30-19 6449 California Stanislaus American fb. Pew cells Efb. 5- 1-19 6452 California Stanislaus European fb. From history of case 5-20-19 6304 Missouri Lewis ? 6-11-19 6401 Ohio Ashtabula ? 6-27-19 6498 Iowa Johnson American fb. Efb early stages, also Sacbrood 8- 1-19 6629 Ohio Trumbull ? 8-15-19 6672 Connecticut Tolland Probably Efb. Afb slight amount 8-25-19 6698 Kansas Cherokee ? 8-29-19 6716 New York Cayuga American fb. Efb active Afb scales 9- 2-19 6721 Washington Pacific 7 9- 2-19 6722 Washington Pacific ? Efb more prominent 9-19-19 6768 California Santa Barbara 7 Afb 1st disease reported for county 9-26-19 6778 California Santa Barbara 7 10- 5-19 6834 California Santa Cruz ? 5-12-20 6985 California Butte European fb. Afb one or two cells 5-29-20 7023 Michigan Calhoun ? 5-29-20 7025 Michigan Calhoun ? 5-29-20 7026 Wisconsin Fond du Lac European fb. Few cells Afb 6-17-20 7119 Washington Lewis 5 6-17-20 7120 Washington Lewis ? Also Sacbrood 6-22-20 7143 New York Allegany European fb. Few cells Afb. 6-24-20 7158 Pennsylvania , Crawford ? 6-26-20 7172 New York Cayuga ? 6-26-20 7174 New York Cayuga ? 6-26-20 7177 Pennsylvania Crawford ? 7-21-20 7335 New York Seneca Probably Afb. 8- S-20 7386 Indiana Blackford ? 8- 5-20 7387 Indiana Blackford ? 130 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 14 Table II. — Samples of Mixed Infection by Years Samples of Total Samples Year mixed infection received 1911 1 1042 1916 2 374 1917 1 449 1918 2 429 1919 18 693 1920 14 698 1905-1920 38 7568 This marked apparent increase in cases of mixed infection carries the subject over from one of scientific interest to one of practical im- portance. As is shown in Table III, the 38 samples of mixed infection have come from 24 counties in thirteen states, most of these located in prominent beekeepiag regions. In eleven of these thirteen states both- European foulbrood and American foulbrood as shown by samples of disease received in the laboratory for diagnosis are prevalent and of long standing. There are only about three or four other states where both diseases have been found in quantity from which samples of mixed infection have not been received, while only from two states of the many where the diseases are only occasionally bad have such samples been received. Table III. — Samples of Mixed Infection by States and Counties State Counties Samples California 5 12 Connecticut Indiana Iowa Kansas Michigan Mississippi Missouri 2 2 New York 3 5 Ohio 2 2 Pennsylvania 1 2 Wisconsin 2 3 Washington 2 4 Statistics obtained from the sample records, however, are not entirely conclusive since a majority of the samples come to the laboratory unsolicited. If a careful survey could be made of the regions where the brood diseases are bad and widespread, probably many more such cases would come to light. February, '21] sturtevant; mixed infections 131 Table IV. — Distribution of Samples of Mixed Infection by Months April 5 May 9 June 10 July 1 August 6 September 5 October 1 November 1 These samples of mixed infection have been examined in eight out of the twelve months of the year, April to November inclusive, as shown in Table IV. Twenty -four of the total 38 samples, nearly 65 per cent., were examined during the months of April, May and June, the months during which European foulbrood is most prevalent.^ In contrast to the spring months, eleven samples of mixed infection were examined during August and September, and only one each in July, October and November, a total of fourteen. The question, however, of which diesase is most often the primary invader in a colony is difficult to answer, particularly without a history of the colony and locality. (Table I) . If only dried adhesive American foulbrood scales are found, accompanied by numerous coiled fresh moist melting larvae of European foulbrood, it is not difficult to say that American foulbrood was the primary invader, perhaps during the pre- vious season, as was the case of the sample reported by McCray. But often there is no such demarkation. Because the presence of American foulbrood depletes the strength of the colony this increases the probabil- ity of European foulbrood infection. Since the requirements of the treatment of the two diseases are so entirely different, the necessity for correct diagnosis becomes of im- portance, particularly in regions where both diseases have been prevalent for some time. The presence of both diseases in the same colonies or even in the same apiary is a complicating factor in the diagnosis and treatment. Furthermore there is danger from the possibility of con- tinued and confusing losses due to the ignorance of the presence of mixed infection in colonies under such circumstances and resulting therefrom, improper treatment which would only continue the losses. Several samples have been received for diagnosis which beekeepers have thought contained both diseases and which indeed seemed to have some of the characteristics of each. Upon careful examination, however, both gross and microscopic, these have mostly proven to be definitely not mixed infections. The recognition of cases of mixed infection in 'Phillips, E. p., 1918. The control of European foulbrood. U. S. Dept. of Agnc. Farmers' Bulletin 975, 16 pp. 132 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 14 colonies is often difficult because of the fact, as is particularly the case with European foulbrood, there are many irregularities and variations in symptoms that often add to the confusion of the beekeeper in making gross diagnosis hurriedly in the field. In order to more easily differen- tiate some of these confusing sjonptoms to assist in gross diagnosis, they may be divided into three classes. Occasionally in an unusually virulent case of American foulbrood or in one where the bees have deserted the brood because of its foul condition allowing what healthy brood there is to starve, larvae will be found which have died while still coiled in the cell, among the typical American foulbrood larvae.^ These coiled larvae often have much the same appearance as typical European foul- brood coiled larvae. However, the consistency is generally quite dif- ferent from European foulbrood, more like the typical slimy glue-like consistency of American foulbrood material. As a rule, however, the symptoms of American foulbrood are uniformly constant because of the fact that Bacillus larvae is almost always the only invader of the larvae causing death and a type of decomposition which prevents growth of other organisms. Several such cases were found in California. A second class of confusing sjanptoms are found in samples which come particularly from regions where European foulbrood has been allowed to run unchecked for a long time. Such samples were found in certain sections of California and have been received from various other sections of the country. These samples show along with more or less of the typically coiled European foulbrood larvae, large numbers of larvae which have died after extending and even being sealed in the cell, showing a consistency somewhat like that of American foulbrood but more lumpy or like an old partly rotten rubber band.^ Sometimes scales are found extended in the cells in such large n-umbers as to appear on casual examination like an old comb of American foulbrood. Close examination, however, shows the consistency, irregular shape and posi- tion with lack of adherence to the cell wall to be different from that in American foulbrood. This type was found to be quite prevalent in California. The third class is composed of cases of actual mixed infection where typical American foulbrood, ropy larvae or scales, are associated in the same comb with typical European foulbrood, coiled moist melting larvae, or possibly occasionally the abnormal rubbery irregular larvae mentioned above. The active stage of the two diseases often seems to be locaKzed more or less in different parts of the comb. This is probably due to *White, G. F. 1920. American foulbrood. U. S. Dept. of Agric. Bui. No. 809. ^Sturtevant, A. P. , 1920. A study of the behavior of colonies affected by European foulbrood of bees. U. S. Dept. of Agric. Bui. No. 804. February, '21] sturtevant: mixed infections 133 the fact that the queen would tend to desert that section of the comb containing the American foulbrood, particularly where this disease was the primary invader. In many cases one or the other of the diseases will be more prominent, at least in the active stages. This fact may be one of the causes for cases of mixed infection having been overlooked, the beekeeper seeing only the prominent outstanding symptoms. There- fore in cases where there is doubt or suspicion that both diseases may be present in the same colony, a positive laboratory diagnosis often appears to be desirable. As is well known, the shaking method of treatment in its essentials is so far the only successful way of treating American foulbrood.' The nature of Bacillus larvae has prevented success along any other line, because of its ability to form exceedingly resistant spores and especially to decompose the dead larva in such a way as to cause the mass contain- ing large numbers of these spores to adhere to the cell wall as if glued. It has been learned furthermore, often by sad experience, that the shaking treatment is practically never successful in the treatment of European foulbrood; in fact, often when used causes the disease to be spread all the more because of the weakening effect the shaking has on the colonies.' The requirements for the successful treatment of European foulbrood have been found to be fundamentally dependent upon ade- quately strengthening the colonies with young bees sufficiently to throw off the disease,' at the same time combined with the requeening of the diseased colonies with vigorous young Italian queens, permitting the bees themselves to remove the infected material. The apparent logical solution of the problem of the treatment for a known case of mixed infection, therefore, is to combine the treatments for both American foulbrood and European foulbrood as a single treatment. In other words, the one or more colonies known or strongly suspected to have mixed infection should be shaken as for American foulbrood, requeening them with vigorous young Italian queens and later strength- ening them by the addition of young bees or hatching brood from a healthy colony, or by uniting later. Strength of colony is the important- factor combined with the shaking and requeening with vigorous Italian stock. The problem of the control of mixed infections of American foulbrood and European foulbrood is primarily associated with the control of European foulbrood. In localities where both diseases are prevalent 'Phillips, E. P. 1920. The control of American foulbrood. U. S. Dept. of Agric, Farmers' Bulletin No. 1084. 'Phillips, E. F. 1918. The control of European foulbrood. U. S. Dept. of Agric, Farmers' Bulletin No. 975. 134 JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 14 and there is suspicion of both being present in the same apiary, and pos- sibly even some as mixed infection in the same colony, control of the two diseases will depend upon the elimination of European foulbrood first. This should be done by treating the entire apiary for European foulbrood, by strengthening and requeening all the colonies with young and vigorous Italian queens, which is after all only good beekeeping. After the elimination of European foulbrood it will be a simple matter to determine those colonies that have not responded to this treatment, as being American foulbrood. This method is possible because of the fact that American foulbrood seldom spreads with the rapidity of Euro- pean foulbrood, particularly if care is taken to prevent robbing and mixing up of combs. Those colonies which continue to show American foul- brood remaining may now be given the usual shaking treatment. K-228 f RELATION OF COMMERCIAL HONEY TO THE SPREAD OF AMERICAN FOULBROOD BY A. p. STURTEVANT (Contribution from Bureau of Entomology) Reprinted from JOURNAL OF AGRICULTURAL RESEARCH Vol. 45, No. S : : : Washington, D. C, September 1, 1932 (Pages 257-285) ISSUED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES AND UNIVERSITIES U. S. GOVERNMENT PRINTING OFFICE : 1932 JOINT COMMITTEE ON POLICY AND MANUSCRIPTS FOE THE UNITED STATES DEPAETMENT FOE THE ASSOCIATION OF lAND-GEANT OF AGEICUITUEE COIIEGES AND UNIVEESITIES H. G. KNIGHT, Chairman S. W. FLETCHER aief, Bureau „/ CKerai^ry a^ Soil, ^S^af^f/ZS^i.fS'^"''' ^"^ F. L. CAMPBELL S. B. DOTEN ETUomolosjist, Bureau of Eittomology Director, Nevada Agricultural Experiment Station JOHN W. ROBERTS C. G. WILLIAMS Senior Patkologiat, Bureau of Plard Director, Ohio Agricultural Experiment Iniuetry Station EDITOEIAL SUFEEVISION M. C. MERRILL Chief of Publicatiom, United States Departmem. of Agriculture Articles for publication in the Journal must bear the formal approval of the chief of the department bureau or of the director of the experiment station from which the paper emanates. Each manuscript must be accompanied by a state- ment that it has been read and approved by one or more persons (named) familiar with the subject. 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Address all correspondence regarding subscriptions and purchase of numbers and separates to the Superintendent of Documents, Government Printing GflBce, Washington, D. C. RELATION OF COMMERCIAL HONEY TO THE SPREAD OF AMERICAN FOULBROOD ' By A. P. Sttjrtevant ' Associate ApicuUurist, Division of Bee Culture, Bureau of Entomology, United States- Department of Agriculture INTRODUCTION The relation and importance of commercial honey to the spread of American foulbrood of bees has occupied the attention of the bee- keeping industry more or less prominently for many years. The- theoiy has been promulgated that honey which has not come from, disease-free apiaries is dangerous because of the possibility of its dis- seminating American foulbrood. A few States and at least one for- eign country require that honey intended for interstate shipment be accompanied by a certificate from the bee inspector of the State in' which the honey originated to the effect that such honey was produced^ in apiaries free from American foulbrood. It is a well-established fact that honey taken directly from the combs of the brood chambers of colonies affected by American foul- brood is capable of producing the disease if fed to healthy colonies. Since commercial beekeeping practice bans the extracting of honey from the brood nest, it is difficult to understand how heavily infected honey, in large quantities, could get on the market. Whether honey' from supers that have been on colonies affected with American foul- brood is of serious importance in transmitting the disease is still open to question. White {SO, p. S5y says: "The likelihood that the disease" will be transmitted by combs from diseased colonies, which contain honey but no brood, probably is frequently overestimated." On the" other hand, Millen {23) found that combs built from foundation and completely filled above an excluder with honey from colonies that had been destroyed by American foulbrood produced disease in all of 10 colonies made from package bees to which one comb each of the honey had been given. Corkins {8) expressed the belief, as a result-' of preliminary studies, that "Extracted honey produced above an excluder in a colony in the early stages of American foulbrood is" insignificant in the spread of this disease through commercial honey."' The conflicting nature of these observations emphasizes the need for further research before the certification of honey is required as a; means of alleviating the foulbrood situation. In both animal and plant disease bacteriology it is known that pathogenic microorganisms may vary considerably, even within indi- ' Eeceived for publication Feb. 1, 1932; issued September, 1932. ' For advice and assistance the writer Ls indebted to Profs. C. L. Corliins and O. H. Gilbert, of the Uni- versity of Wyoming; Prof. K. Q. Hiohmond, deputy State entomologist, apiary investigations, Colorado' Agricultural College; H. Bauohfuss, of Englewood, Colo.; N. L. Henthorne, of Greeley, Colo.; and C. H„ Banney, of Lander, Wyo. Appreciation is also expressed for the many courtesies extended by H. C. Hilton, supervisor of the Medicine Bow National Forest. ' Beference is made by number (italics) to Literature Cited, p. 284. Journal of Agricultural Eesearch, Vol. 45, No. 6 ■ Washington, D. C. Sept. 1, 1932 Key No. K-228 (257) 258 Journal oj Agricultural Research voi. 45, No. 5 vidual species, in virulence and in ability to produce disease. Fur- thermore, as stated by Zinsser {31, p. 188-189) — Whether or not infection occurs depends also upon the number of bacteria which gain entrance to the animal tissues. A small number of bacteria, even though of proper species and of sufficient virulence, may easily be overcome by the first onslaught of the defensive forces of the body. Bacteria, therefore, must be in sufficient number to overcome local defenses and to gain a definite foothold and carry on their life processes, before they can give rise to an infection. The more virulent the germ, other conditions being equal, the smaller the number necessary for the production of disease. The introduction of a single individual of the anthrax species, it is claimed, is often sufficient to cause fatal infection; while forms less well adapted to the parasitic mode of life will gain a foothold in the animal body only after the introduction of large numbers. In the case of American foulbrood the quantity of infectious mate- rial that honey must carry in order to produce disease in a colony has never been determined. White (SO, p. ^0, footnote 1) states, in con- nection with inoculating healthy colonies experimentally with Bacillus larvae: It was found that less than one scale is sufficient disease material to produce a considerable amount of disease in the colony. In some experiments one scale, therefore, might supply all the spores needed although the use of a somewhat greater quantity of material is advisable in most instances. While infected honey no doubt does become mixed with disease-free honey, it is probable in many cases that, because of the practice of using large settling and storage tanks, infected honey would be so diluted with spore-free honey as to make the spore content insufficient to produce infection even if fed to healthy bee larvae. Therefore, one object of these investigations was to determine the minimum number of spores of Bacillus larvae in honey necessary to produce American foulbrood in healthy colonies of bees as correlated with the infectivity or spore content of the average commercial honey. In order to obtain information relative to this subject, experiments were conducted in the apiary over a period of five years. In these experiments honey or sugar sirup with a known content of spores of Bacillus larvae was fed to healthy colonies and the minimum number of spores that would produce infection was determined. At the same time laboratory studies were carried on with cultures of spores of B. larvae, concerning certain growth phases of the organism, particularly the minimum number of spores that would produce vegetative growth on artificial culture media. Methods for demonstrating the presence or absence of spores of B. larvae in samples of commercial honeys were also investigated, and these honeys were studied in relation to their infectiousness as correlated with the spore-feeding experiments. These three phases of the investigation will be discussed in the order mentioned. MINIMUM NUMBER OF SPORES OF BACILLUS LARVAE NECES- SARY TO PRODUCE DISEASE IN HEALTHY COLONIES OF BEES methods of proceduee Location of Experiments These investigations were started during the summer of 1926 in a small experimental apiary located about half a mile from the bee culture laboratory of the Bm-eau of Entomology at Somerset, Md. The location at Somerset was undesirable, however, because of its Sept. 1, 1932 Commercial Honey and Spread of American Foutbrood 253 close proximity to the apiary connected with the laboratory and tO' other privately owned colonies of bees, necessitating extreme pre- cautions to prevent spread of the disease. In 1927 the experimental work was transferred to the Intermountain States bee culture field laboratory at Laramie, Wyo.* In Wyoming an ideal isolated location was foimd about 14 miles east of Laramie in the Medicine Bow Na- tional Forest, the nearest colonies of bees being at least 14 miles away and probably farther. Since this location is more than 8,000 feet above sea level, there is only a slight nectar flow from wild flowers, which assures the immediate use of any inoculated simp fed to colonies of bees. In fact, after the middle of the summer it was found neces- sary in most cases to feed the experimental colonies with uninocu- lated sugar sirup in order to prevent starvation. In 1927 and 1928 the colonies used for experimentation were located in two yards between a quarter and a half mile apart. The arrange- ment of the colonies in the two yards was such as to prevent drifting' as rnuch as possible. In 1929 and 1930, in order to limit still further the danger of transmission of disease because of drifting or robbing,. 20 colonies were stationed in pairs, so arranged as to minimize the danger from drifting, in 10 isolated locations at least a quarter of a. mile apart. Make-up of Colonies Five-frame nucleus hives were used for the spore-feeding experi- ments. The colonies were prepared either with two or three frames, of brood, honey, and adhering bees taken from healthy colonies, together with a young laying queen, or, as in 1927, 1928, and 1929, by placing a 2-pound package of bees containing a laying queen on foundation or on combs containing honey from healthy colonies and feeding them sugar sirup. During a good honey flow these small colonies were allowed to build up in the apiary connected with the laboratory until they consisted of three or four frames of brood before they were moved to the isolated locations. The bees making up the colonies used for the feeding experiments from 1927 to 193Q at Lara- mie, Wyo., were all from the same general strain. Material Used for Inoculation Spores of Bacillus larvae were obtained from American foulbrood scales in combs taken from diseased colonies located in the States of Maryland, Iowa, and Wyoming. The strain used at Somerset, Md., was obtained from a sample sent to that laboratory for diagnosis. Two different strains were used at Laramie during 1927, 1928, and 1929, one obtained from a diseased colony in the experimental apiary belonging to the University of W^yoming and one obtained from a bee- keeper at Lander, Wyo. In 1930 three other strains were used in the feeding experiments, one from Iowa and two from apiaries in Wyoming. Preparation of Spore Suspensions In preparing the spores for feeding to the healthy colonies, scales were removed from the combs by means of sterile forceps (the neces- sary precautions being taken against contamination) and placed in < This laboratory is maintained cooperatively by tbe University of Wyoming and the U. S. Department of Agriculture. 260 Journal oj Agricultural Research voi. 45, No. 5 a flask containing 50 c c of sterile water and glass beads. After the scales had softened in the water, the flask was shaken for one-halt hour to insure complete maceration of the scales. The suspension was then filtered through two thin layers of sterile absorbent cotton into another sterile flask in order to remove any lumps or debris. In preparing the stock suspensions of spores, at first 75 to 100 scales were taken by counting. Later it was found that the average American foulbrood scale weighs 0.0223 g. Therefore, the 100 scales for the stock suspensions were obtained by weight, the scales bemg •weighed in a sterile covered glass dish before they were deposited in the flask of sterile water. After the suspension had been filtered and tested for contamma- tion and was ready for use, the number of spores per cubic centimeter was determined by the following method: By means of a blood- diluting pipette giving a dilution of 1 to 20, the spore suspension was diluted with a weak solution of carbol fuchsin and a drop placed in the counting chamber of a Helber bacteria-counting cell 0.02 mm deep and ruled in squares of 0.0025 mm^ each.* With the use of two 15 X eyepieces in a binocular microscope and a 1.8-mm oil- immersion objective, the spores in 25 squares of the Helber chamber were counted. Then by means of the formula Total spores counted X dilution X 20,000 X 1,000 Number of squares counted the approximate number of spores per cubic centimeter in the sus- pension was determined. Later this method was checked by the method of Breed and Brew (2) for counting bacteria in milk. With the aid of a binocular micro- scope having two 15 X eyepieces and a 1.8 mm oil-immersion ob- jective, the area of a circle etched on an ocular micrometer disk was determined by means of a stage micrometer. One one-hundredth cubic centimeter of a 1 to 100 dilution of the stock suspension of spores was placed on a glass slide on which 1 cm^ had been ruled with a diamond pencil. This was mixed with a small loopful of carbol fuchsin stain and the whole spread over the 1 cm ^ of surface * and allowed to dry uniformly. The number of spores per cubic centimeter of the stock suspension was determined according to the formula Area 1 cm ^ total number of spores counted X dflution X 100. Area of circular field number of circular fields counted These two methods were found to check fairly closely within the limits of the precision of the methods used in counting. Further- more, by both methods it was found that in the majority of cases 100 scales in 50 c c of water give approximately 5,000,000,000 spores per cubic centimeter for each suspension made up in this way. Therefore, this number was used as a standard for making all dilutions. » Mm' and cm' are the abbreviations tor square millimeter and square centimeter, respectively, recently adopted by the Style Manual for United States Government printing. Sept, 1,1932 Commercial Honey and Spread qf American Foulbrood 261 After a considerable number of counts had been taken in making up several stock suspensions of spores, counting was eliminated and the spore content of the stock suspensions was standardized according to the method described by Gates {11, p. 114), as follows: "The opacity of a bacterial suspension is measured by the length of a col- umn of the suspension required to cause the disappearance of a wire loop." An instrument known as a suspensiometer was used for this purpose. The use of this method saved considerable time and labor without appreciably affecting the precision of the counts. One liter of a 50 per cent solution of sugar in water was used as the standard quantity of inoculated sirup fed to each experimental colony. A series of dilutions of the original stock suspension containing 5,000,- 000,000 spores was made by adding different quantities of the spore suspension to 1 liter of sugar sirup. In this way the approximate total number of spores in each liter of sugar sirup to be fed to colonies of bees was known. Method op Inoculating Colonies In 1926 at Somerset, Md., the sugar sirup containing the various dilutions of spores was fed to the colonies by means of galvanized-iron troughs that were hung inside the hives after two combs had been removed. In these troughs sterile excelsior was placed for the bees to walk on in order to prevent them from drowning. This method was found unsatisfactory, however. At Laramie, Wyo., the sugar sirup containing the spores was first placed in Boardman feeders, but owing to the danger of robbing at the entrance of the hives, the method finally used was to invert the jars in holes bored in the hive covers. In this way any leakage into the hives was cleaned up by the bees without danger of causing robbing. To prevent the jars from being broken or knocked over, box covers were placed over them and fastened to the hive covers. Each colony was usually inoculated only once with an individual dilution of spores. Duplicate colonies were inoculated with each dilution of spores. Uninoculated check colonies were placed among those that were inoculated. PRIMARY OBSERVATIONS Observations of the condition of the brood were made at least once a week, and sometimes oftener, after the colony was given the liter of inoculated sirup. In 1926 at Somerset, Md., as soon as diseased larvae appeared in a colony, the colony was killed and at once re- moved from the apiary. Because of the isolated location near Lara- mie, Wyo., the colonies were left until the end of the brood-rearing season, when fiaal observations were made. The results of the spore-feeding experiments are shown in Table 1. 262 Journal oj Agricultural Research Vol. 45, No. 5 Table 1. — Results of spore-feeding experiments " [Duplicate colonies of bees (A and B) were used in the first i years, and triplicate colonies (A, B, and C> in 1930] Extent of foulbrood in — 1926 1927 1928, repeat 192« 1929 1930, final Total number of spores fed During season Pinal During season Final During season Final A B A B A B A B A B A B A B A B A B c 5, 000, 000, 000 2, 600, 000, 000 1, 000, 000, 000 750, 000, 000 600, 000, 000 3,50,000,000 200, 000, 000 17.5, 000, 000 150,000,000 126, 000, 000 100, 000, 000 76 000 000 + + ?+ + + + + ^+ + + + + + + + + ?+ + + ?+ + ■>+ ?+ ?+ + + * + + + * + + + + * * * + _i_ + + + + + + + * + n + n + n * 60, 000, 000 25, 000, 000 n 10 000 000 5, 000, 000 2, 600, 000 1, 600, 000 600, 000 100, 000 Controls l+,12-0 l+,2-0 l+,2-0 1-0 l+,3-0 H-,3-0 8- -0 8 -0 2-0 ' +, Positive American foulbrood; ?+, probable American foulbrood, very slight and unconfirmed and disappearing by end of brood-rearing season; 0, no disease found during season; — *, disease cleaned out by end of brood rearing; — , no recurrence in second season. In 1926 a total of 200,000,000 spores fed to a colony was the smallest number that produced disease; in 1927, on the other hand, 75,000,000 was the smallest number. However, in the latter year the spores were obtained from another locality in which environ- mental conditions were quite different. In an effort to obtain check results, the feeding experiments were repeated in 1928. Through an error in maldng up the spore dilutions, which was not discovered until too late for rectification, no colony received less than 50,000,000 spores. This season one colony of the pair receiving an inoculation of 50,000,000 spores became infected. The feeding experiments were repeated again in 1929, with dilutions of spores from 75,000,000 down to 100,000 — ^considerably less than the minimum number in 1928. Again only one colony of the pair receiving a total of 50,000,000 became infected. As a result of two years' experiments this was foimd to be the apparent minimum number of spores of Bacillus larvae capable of producing infection when fed in 1 liter of sugar sirup. In 1930 spores from three different locaUties were fed in duplicate to six healthy colonies in dilutions of 50,000,000 and 25,000,000 without prod\icing disease. It is therefore apparent that a certain minimum number or mass of spores is required to start the initial action capable of producing American foulbrood in healthy larvae. Under the conditions of these expenments this minimum number was approximately 50,000,000 spores of inoculum per liter of sirup. SECONDARY OBSERVATIONS During the first tliree years of the experiments, or previous to 1929, at which time the experimental colonies were isolated in pairs, certain of the unmoculated control colonies developed disease, 1 out of 13 in Bept. 1, 1932 Commercial Honey and Spread of American Foulbrood 263 1926, 1 out of 3 in 1927, and 1 out of 4 in 1928. It was assumed that the disease was probably not spread by robbing, since no active rob- bing was observed at any time. In practically every case where a control colony became infected, it was so located in relation to the inoculated colonies that drifting of young nurse bees during play flights could account for the spread of the disease, in one or two cases quite definitely so. In 1929 all eight uninoculated colonies, although they were not located with the inoculated colonies but were within robbing range of all, remained free from disease. The prevention of drifting apparently eliminated the casual spread of disease. Occasionally a colony of bees affected with American foulbrood will try to clean out the diseased remains, often removing parts of the scales and sometimes actually tearing a comb down to the midrib in order to do this. White (SO, p. 34-35) states: There is considerable evidence to support the belief that occasionally in cases of light infection the disease may disappear unaided by treatment. * * * j^ should be emphasized that such a course for the disease, if it occurs at all, is unusual. Although American foulbrood spreads more or less rapidly within an infected colony, the fact remains that it frequently does not. Lineburg (16) in 1925 reported that in two colonies which were diseased in the spring the disease apparently disappeared later in the season. Three colonies were divided and used for maldng increase in June and July, but all remained free from disease, at least until the end of that season. Further observations were not reported. Cor- kins (8) in 1928 reported five colonies which were given combs con- taining scales of American foulbrood at the beginning of the honey flow of 1927 and developed no disease up to July 10, 1928. Two other colonies were observed to have cleaned out the disease and remained healthy for an entire season. However, during the several years of his experimental work on American foulbrood, the writer never observed a colony in which the disease was permanently cleaned out until 1927. In that year, of 16 colonies inoculated with various dilutions of spores, 4 colonies, 2 of which received more than the probable minimum dose causing infection, showed no disease during the season. The disease completely disappeared by the end of brood rearing ia 10 of the 12 other colonies that had showed either positive or probable disease some time during the summer. In 1928 package bees were placed on the combs of seven qf these colonies that had apparently cleaned out the disease during the previous summer and on two that had been inoculated with presumably a sufiicient number of spores but which had remained healthy. Three of the seven developed disease again the second season, while four remained healthy during the entire season. Neither of the two inoculated colonies that had remained free from disease in 1927 developed it in 1928. Of the 11 colonies inoculated in 1928 that developed disease, 4 cleaned up the disease by the end of the brood- rearing season and 2 inoculated colonies showed no disease. In 1929, 1 of the 2 colonies developing disease cleaned up by the end of the brood-rearing season, making a total of 15 cases in which the disease was cleaned up by the end of brood rearing. Two of the colonies inoculated with the minimum infectious dose or more showed no disease during that summer. It is possible that, in the high altitude of Laramie, and in similar places where the air is very dry, the scales of American foulbrood 131772—32 2 264 Journal oj Agricultural Research vu. 45, No. 5 become dried without adhering so tenaciously to the cell walls as they do in more humid climates at lower altitudes. These observa- tions iadicate the necessity of further work on the resistance of bees to the disease and variation in virulence of different strains of the organism. INOCULATION OF INDIVIDUAL BEE LARVAE WITH DEFINITE NUMBERS OF SPORES OF BACILLUS LARVAE In the light of the results of the foregoing experiments, in which colonies were inoculated with presumably a quantity of spores sufficient to produce infection but in which no disease developed, the question arises as to what became of the spores in the sugar sirup, some of which presumably were fed to healthy larvae. In those colonies developing disease that received a minimum number of spores, how many spores did each larva developing the disease receive? In order to obtain information on these points, a preliminary series of experiments was planned in which individual larvae were inoculated with known numbers of spores. Touraanoff (29) reports that he was unable to cause infection by giving individual larvae a drop of a rich emulsion of a culture of Bacillus larvae in salt solution. He found that many of the larvae so treated were removed from the cells by the bees, and those remaining failed to develop disease. He further found that larvae given only uninoculated salt solution were also removed in the same way. Therefore, in the present experiments sugar sirup was used instead of salt solution. In a comb from a healthy colony containing numer- ous coiled larvae, a drop of an uninoculated 50 per cent solution of sugar in water was placed in each cell containing a larva, as near the mouth parts of the larva as possible. The rim of each cell so treated was marked with a paint consisting of 1 part of liquid white shellac, 1 part of a paint pigment, and 4 parts of ethyl alcohol. The sugar sirup was slightly colored with water-soluble eosin in order to aid m determining the effect. Frequent observations showed that practically all larvae that were fed this colored sugar sirup developed normally and were sealed over, the pigment markings still being present on the edges of the cappings. In most of the cells a residue of colored sirup could be observed for several hours after the larvae had fed. A series of 5-frame nuclei was prepared, each containing one or two combs havmg a large number of unsealed larvae. A set of dilutions of spores was made from a stock suspension with a steriUzed 50 per cent sugar su-up m such a way that each 0.01 c c of the dilution would £?^*f 1^ a^ approximate Imown number of spores, as indicated in iable 2. btenhzed 2 c c Luer tuberculin hypodermic syringes Grad- uated m 0.01 c c the needles of which had been blunted, were "used in inoculating the cells containing coiled larvae. Fifty or more coiled larvae at least 4 days old were each given 0.01 c c of a dilution ot spores, each dilution being given to larvae in one comb in a separate colony, and the cells so inoculated were distinctively marked A few larvae that had just been sealed also were inoculated by puncturing the cappmg with the inoculating needle and depositing the 0.01 c c in the ceU Observations were taken at the end of 24 hours and at frequent intervals thereafter until the end of the brood-rearing Sept. 1,1032 Commercial Honey and Spread fff American Foulbrood 265 s < i 1 o a o 1 o 1 s- A OO lO O . 1 1 < 1 1 t < 1 t 1 1 1 1 ( 1^ i N Ml NNNNNNNN is CO ib"" != = ! i i i i i i i i i ! i i i i i i i i i : i 1" i i i i i i i i i : i i i i i i i i i i i i i rv ©OS CO ^ ^oocno o III i j i i i i 1 i 1 i ' i 1 1 1 I I 1 l'^ " Ml i i i i i i ! i i ! 1 i i i i i 1^ ^ ^oooo o 1 1 I 1 1 I 1 1 ! 1 1 1 1 ! 1 1 1 1 i I 1 1 1- i M i i M i i i 1 i i i i i M ! M i IS O H Larvae removed do Larvae not removed do One-half of larvae re- ||.§iiii , uaoioo o 1 1 ,1.1 e s '^"'^ " 1 1 1 ; : : 1 ! 1 1 1 : ; : : 1 : ; : ; : : o 0.2; (MCOQift Tjl iiil>ll>iiiiii I 2 1 1 1 ■s.i 1 I-- g^_oo lo O OO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1- i M M i 1 i i 1 i M i M i M i h 4^ P.C» i OO lo o OO ! 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 i 1- i M i i 1 i M i M i M i M M ■ CQ gj^OOMO O OO 111 1 1 I 1 1 1. 1- " i i i i M M i i M 1 M M M i ; 1^ i oooo o OO 1 I 1 I 1 I 1 1 1 ! 1 1 1 1 1 1 1 1 1 1- i i i i ! M i i M i M 1 1 i M ^ "o H §p §■§ II W-2 Larvae removed do Larvae not removed Three-fourths of larvae removed. One-half of larvae re- Larvae not removed do__ ||i|lil |— S" « '^^ i i i ; 1 i i i i : ; n i ; i i ; ; ! ^ I 1 i 1 : M 1 ; 1 1 1 : ; : I ; I 1 ! §11 cieooio ■* «Dw 1 [ 1 1 1 ; 1 1 1 1 ; ; 1 ; 1 1 1 ; ] 1 i a fl go III ill 1" e 1 1 1 1 O OOOOOOOOOOO < i 1 1 1^ 1 i 1 1 i 1 M M i i M 1 ^3 g ^__ I 1 1 1 O OOOOOOOOOOO 1 1 1 1 1 ! 1 1 i 1 1 l-ilM IliliiMili tM i i i 3 sssass^asas i i i i i i i i i i ■-■Six .IIS ^#11 s 1 i 1 1 1 1 1 1 1 1 1 1 looooooooooooo l^iill 1 MMMi i i i ; j i ; i isS3S2SS?S^SSSS3 P ,.g-l|.§ oao d'C fe||g§ I §|§|||||||||8gSSgSS""° Eo-O'o-O" O" o"o'"oo''i«OiQ'Ot-"lOTH' Soooo o o o lo o r- »o r, ^- ^ 266 Journal oj Agricultural Research ^'ol. 45, No. 5 In the first series of inoculations the number of spores fed each larva ranged from approximately 5,000 down to 1. None of the larvae inoculated developed disease. (Table 2.) Later a second series of inoculations was made. The same colonies were used be- cause of the limited number available^ but the larvae inoculated were in a different comb in each colony and a different color was used to mark the cells. In these inoculations the number of spores fed ranged from 5,000,000 down to 1,000 per larva. No disease devel- oped from this set of inoculations. It vv-as thought possible that the nurse bees might be removing most, if not all, of the inoculated sugar sirup before the larvae had had time to ingest a sufficient number of spores to bring about infection. Therefore, in a third series of experiments each inoculated comb was placed in a screen-wire queen-nucleus introducing cage, and this cage was put back in the colony for periods ranging from one-half to one hour before the unprotected comb was replaced in the colony, thus theoretically giving the larvae time to ingest some of the sugar sirup before the nurse bees had access to the inoculated cells. In these tests the larvae were kept from the bees so long that many of them, becoming hungry, were starting to crawl from the cells. The number of spores fed ranged from 50,000,000 down to 500,000 per larva. Twenty-four hours after the larvae were fed it was found that all re- ceiving 50,000,000 and 25,000,000 spores had been removed from the cells, while those receiving a smaller number of spores were either partly removed or remained in the cells, according to the strength of the dilution and the length of time that the larvae were kept away from the nurse bees. (Table 2.) Two days later another set of larvae was inoculated with the same dilutions as were previously used for these colonies but on the other ■side of the same combs. In this series the combs were kept away from_ the bees for periods ranging from 5 minutes for the heaviest dilution to 30 minutes for the weakest. Again all the larvae receiving the 50,000,000 and 25,000,000 spores were removed, while those receiving the 5,000,000, which were kept from the bees for half an hour, were partly removed, and those receiving 7,500,000 or 10,000,000 were not removed. Apparently there are two factors concerned in the removal of the larvae — the length of time they are kept away from the bees and the amount of foreign matter in the sirup, as indi- cated by the spore content, that is given to the larvae. The results of the last two series of inoculations showed that in the colonies in wliicli the larvae were not removed, or were not entirely removed, several larvae in the colony receiving 10,000,000 spores per larva developed disease, while those in the colonies receiving a smaller number remained healthy. (Table 2.) This work should be repeated with a different colony for each set of inoculations, although appar- ently the disease did not spread in the colonies used. Only one colony of the entire number developed disease, xilthough a certain degree of success was obtained, these results seem to bear out Toumanoff's {29) conclusion that the artificial infection of individual larvae is not brought about so easily as one had been in the habit of believing. Apparently, also, a considerable number of spores are necessary to establish an infection under these conditions. Sept. 1, 1932 Commercial Honey and Spread of American Foulhrood 267 MINIMUM NUMBER OF SPORES OF BACILLUS LARVAE PRODUCING. VEGETATIVE GROWTH ON ARTIFICIAL CULTURE MEDIA Bacteria are known to pass tlirough a definite cycle of growth, par- ticularly when cells from an old culture are transferred to fresh culture media. The growth stages have been described by Buchanan (3; IS, Ch. Tl, Henrici (12), and Winslow (IS, Ch. VI) somewhat as. follows: The initial stationary phase during which no growth takes place; the logarithmic phase when the organisms begin to divide, slowly at first but gradually accelerating; and so on through the com- plete cycle of growth. Henrici (IS, p. 21, 24) has observed that — Various factors, as temperature; the size, the age, and previous history of the? inoculum; and the composition and nutrient value of the medium, influence the- growth curves of bacteria. * * * Qf the various factors which influence the rate of growth and form of the growth curve, the initial number of cells introduced! into a unit volume of medium seems to be one of the most important. Robertson (25), in studies of cultures of certain protozoa, has shown that growth seems to be stimulated by the presence of other cells of the same type. This characteristic has been described at various times as mass action or communal activity. Early in 1929, in conjunction with the spore-feeding experiments.-. in the apiary, an investigation was started to determine whether there is a similar manifestation of mass action in the vegetative growth of spores of Bacillus larvae on artificial culture media. In a^ preliminary paper on this subject the writer (27, p. 4-56) made the following observations : Starting with a seeding of 5,000,000,000 spores^ of B. larvae on a suitable slanted solid culture medium, it was found at the end of 48 hours' incubation at 37° C. that growth had occurred, in the original and in a diluted seeding containing 60,000,000 spores, but not in one containing 50,000,000 spores. Growth occurred in a diluted seeding containing only 5,000,000 spores after six days' incubation, and in one containing 700,000 spores after 10 days' incu- bation. (Table 4, Group 1.) These observations indicated that a. certain initial mass of spores is necessary to start vegetative growth.. Furthermore, although the growth results were rather irregular owin^ to the comparatively small number of cultures made, they seemed to show that, within certain limits, the smaller the seeding the longer the incubation period necessary to obtain germination of the spores and vegetative growth. From this preliminary work it was assumed that the lower limits of dilution of the stock suspension that would give growth on longer incubation had not been reached. Ahrens (1) has observed, in cultural studies of scales treated with- formalin solution for different lengths of time, that growth may: occur in cultures from such scales after varying periods of incubation; up to 30 days, depending on the length of treatment and the per- centage of formalin in the solution. Burnside (7) states, in connec- tion with studies of disinfection of American foulbrood combs by- fumigation with formaldehyde gas, that "it is probable that if scales; had been washed and the incubation period increased, growth of Bacillus larvae would have been obtained in some instances whem negative results were recorded." Therefore, a single trial series of cultures was run (No. 7, Table 4)^ the total incubation period being 30 days. Results from this set of cultures showed that in some cases growth was obtained aftier 30i 268 Journal oj Agricultural Research voi. 45, No. s days' incubation where no growth was observed after 10 days' incu- bation. Work on this phase of the problem was continued during the summer and fall of 1930. Several sets of cultures were made in which Bacillus larvae from eight different localities were used in a series of seedings with a decreasing number of spores for each lot of the organism and all incubated for 30 days. (Table 4, Group 2.) methods op procedure Culture Media A culture medium was used similar to that employed by the writer in the preliminary experiments (^7) and also in earlier cultural work with Bacillus larvae (26} — -that is, a combination of the medium made of yeast-extract and egg-yolk suspension and the carrot-extract medium of Lochhead (18). The yeast-carrot extract medium was prepared as follows: (A) Dried yeast grams__ 10 Peptone do 10 Buffer (sodium glycerophosphate) do 2. 5 Water (distilled) cubic centimeters ._ 500 This solution was heated in flowing steam for one-half hour and, after a table- spoonful of siliceous earth had been added to assist in the filtration and clarifica- tion, it was filtered through filter paper on a perforated porcelain funnel with suction. (B) Two hundred grams of cleaned carrots was macerated in a meat grindei, added to 500 c c of distilled water, and allowed to stand for at least 30 minutes, preferably longer. The macerated carrot was removed by filtration through fine muslin, as much liquid as possible being squeezed from the mass. The filtrate was then clarified by the addition of siliceous earth and filtration in the same manner as the yeast-extract medium. (C) The final base medium was prepared by mixing 500 c c of A with 200 c c of B and adding 700 c c of a 3 per cent solution of washed agar. The reactidn of the medium was so adjusted that when 2 c c of sterile egg-yolk suspension, prepared as described in a previous paper (26), was added to 10 c c of the yeast-carrot extract base medium by means of the apparatus shown in Figure 1, and described previously (26), the pH value was 6.8. The medium was then sterilized in the Autoclave at 15 pounds' pressure (sea level) for 15 minutes. After it iad cooled to 45° C, 20 drops, or about 2 c c, of the sterile egg-yolk suspension was added to each tube of medium, mixed by shaking, and the medium was then allowed to solidify in a slanting position. The Lochhead yeast-extract medium was tried without the addition of egg-yolk suspension, but although it gave good growth with the heavier seedings of spores, the combination medium was found to give more uniform germination and heavier vegetative growth with the more dilute seedings. The addition of the carrot extract, while pos- sibly adding somewhat to the growth-producing qualities of the med- ium, served m these experiments as an indicator for vegetative growth because of the abiUty of Bacillus larvae to produce nitrite in the carrot- extract medmm without the addition of potassium nitrate (18). Pkeparation op Dilutions of Spores The stock suspensions of spores of Bacillus larvae were made up as described earlier m this paper. A series of primary dilutions, each one-tenth of the preceding dilution, was then made up in sterile 125 Sept. 1, 1932 Commercial Honey and Spread oj American Foulbrood 269 c c flasks bv adding 4 c c of a dilution to 36 c c of sterUe water. The series of dilutions containing gradually decreasing numbers of spores per cubic centimeter to be used in inoculating the culture medium were then prepared as indicated in Table 4. Sterile burettes were used in adding the proper proportions of spore suspension or spore- suspension dilutions to the proper quantities of sterile water in sterile test tubes, in order to make up the desired series of dilutions contain- ing approximately known numbers of spores. Inoculation op Culture Medium Swann has observed that in old cultures of anthrax a considerable percentage of spores are dead and therefore never germinate. Be- cause of the possibility that some of the spores in the stock suspensions of Bacillus larvae might not be viable, an effort was made to determine the approximate proportions of viable and dead spores in the stock suspen- sions. Since the determination of viable spores of B. larvae by means of plate cultures is difficult because of the opaqueness of the special culture medium that is required, an attempt was made to determine the percent- age of viable spores by the differential stain- ing method of Burke (4) as modified by Koser and MUls (IS). The procedure is as follows : A small quantity of the spore suspension is spread in a thin film on a slide and allowed to dry without heating. The slide, after immersion in a solution of carbol fuchsin at room temperature for two minutes, is washed in water and decolorized with absolute ace- tone for a few seconds, washed again, and immersed in Loefiler's alkaline methylene blue for two minutes, washed, dried, and examined. Very few solid-staining forms were observed in any of the suspensions ex- ^'<'™t^tinrotKii sXensS ^'' amined, possibly one or two spores in several fields. It was therefore assumed that the number of nonviable spores could be considered as negligible and probably within the limits of the precision of the measurements as indicated by this procedure. One cubic centimeter of each dilution was added to duplicate tubes of the slanted solid medium by means of sterile Ice pipettes, each cubic centimeter of inoculum containing an approximately known number of spores of Bacillus larvae. After inoculation the cultures were incubated at 37° C. In order to prevent the liquid in the tubes from drying out on long incubation, from time to time, as the water of condensation evaporated, 2 or 3 c c of sterile broth similar in com- position to that of the base medium, without the egg, was added to each tube by means of the apparatus shown in Figure 1. A total of 556 cultures was made during this series of experiments. 270 Journal of Agricultural Research voi. 45, No. 5 Method of Making Observations The culture tubes were incubated for 30 days at 37° C. Each tube was examined usually every 24 hours during this period. The pres- ence or absence of vegetative growth was noted at each observation, and ia cases of slight or doubtful growth the vegetative growth was checked both by microscopic examination of a stained smear and by testing for nitrite production in the culture medium by the sulphanUic acid and alpha-naphthylamine acetate test. After a large number of such observations had been made, it was found that vegetative ger- mination of spores of Bacillus larvae, almost too slight to be seen, would give a definite pink color on the addition of the reagents. Lochhead {17, p. 14) sta,tes: It was found, however, that ordinary nitrate-reducing species, such as B. cereus or Es. coli, which are able to form nitrites readily in nitrate media, were unable to produce nitrites in recognizable amount in the peptone-carrot media, though capable of doing so upon the addition of nitrates. Bacillus larvse under the same condition readily forms nitrites without the addition of nitrate to the medium. Despite this statement, a series of miscellaneous organisms was tested in standard nitrate broth, in carrot-extract broth, and on carrot-extract agar. Several organisms that commonly reduce nitrates and a few that do not were used . (Table 3.) Observations were made at short intervals during the first 24 hours. Most of these organisms gave positive nitrite tests within a few hours after inoculation in all the media used, but in the carrot-extract medium the nitrate had apparently disappeared in most cases after 24 hours' incubation, and in all cases after 48 hours. The same organisms on standard nitrate medium still gave positive tests after 48 hours' incubation. A positive nitrite test was obtained in cultures of Bacillus larvae that were incubated for 5 days and in one culture that was incubated for 4 days and then allowed to stand at room temperature for 16 days more before testing. Therefore, it appears probable — at least the results in Table 3 indicate — that in the case of many contaminating organisms having the power to reduce nitrite that might get into the culture tubes inoculated with spores of B. larvae the nitrite, if produced by the contaminating organism, would have disappeared after 48 hours' incubation, leaving contamination to be determined by gross appearence of the culture and microscopic examination. Nevertheless, in order to be sure that contaminating growth of any kind was not giving erroneous results with the nitrite test when this was used alone, any suspicious-looking growth in the culture tubes was examined under the microscope before it was tested with the reagents for nitrite production. Even though a positive mtrite test might be observed in some cases, the contaminations were recorded only as such. OBSERVATIONS AND EESULTS In no instance was positive growth obtained in cultures moculated with less than 50,000 spores, even after 30 days' incubation, and growth with 50,000 spores was obtained from only two of the eight lots of spores used, namely, Nos. 19 and 23. (Table 4.) In the other six strains the mimmum number of spores that produced positive growth ranged from 5,000,000 to 70,000. Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 271 si ■-< o S3 9 3 I 1 I I I I I I M + I I l+-H++i I 1 + ++-n+^+f; I +++SS+H-H I I I I I I I I I 1 + I I I I I I I I I M + 1 -H M I I l + l + l ii iitj++i I ci. I 'e +++ I + 1 + 1 I I + +++^+1+1 11+ +++++++ I +++++++ i ±1 i+±i I I 2 = &'S.§igl B o~ " k. S S ! S -Si's -St; I s «*i^ a p 3 £ u U V u t^ w ^ ses 272 Journal of Agricultural Research Vol. 45, No. 5 05 ,3 a < <.rtt ■^JfTi'-'* CO t=Q PO c3 + -x+++x+x77xTT d + IN + iX4-T++++++++++A 1 I++I M + + ++++4.-I- 1 "i 1 1 1 + 1 1 1 [ 1 1 -f Z + + ++++++ 1 1 + 1 ++ 1 r 1 ++ 1 + s + + ++++++++++X+ 1 1 1 1 1 I 1 o + + +++++++++^17 +!ji7 7 7 7 7 7 + + -r-r-r-\-x^+^ 1 1 1 1 X 1 1 i 1 + 1 1 lNM««tDSD(OOSDOQOQOOOOOOOOMO 0. g !? + + +++++++7 +7 +++7 7 7 +:ii7 o i + + + + +++++++++XX77777777 n(NiMcoeO"<*''*'^»o«;ooooooooo _(_ J 1 1 LJ L J L_l_« COmCQCOMPOCCCO + + ++++X 1 ++ 1 1 +XXXX (NcDOpOOOOOO 1— l_l_i-lMC*3(~HC0CQCCro 1 z + + +++++ N « o o o oo II + +++++ ,,,j ^,j 1..J vjvi 1 XX 1 XX 1 X 1 1 o PI z + + +++++ +X+++ 1 ++ 1 1 + + CO ■"^ +++++ 1 ++ 1 +X+ 1 1 1 o M (M t-t^coeOOO-S-Ot^O w 2 + + +++++ ++++5+^7^7 ++++ l+l 1 1 + 1 ++ 1 ++ 1 + 1 tig USOOOOOOOOOOOOOOOOOO ^; "^+++ 1 M + II II 1 1 1 1 1 M + + so o o ooooooooooooooooooo 1 B ^S + + +++++ 1 + 1 ++++ 1 1 1 1 + 1 1 li (O <£> «5 (D «3 O eO CO CD to eo *o i + + +1 I+++++I+ 1 13 M C^ClMNCSC>ltDtOC^cOQOOCDCOIou3■^coc4•-H c4'«raooodQdododo>?4-«coooac4-«cDoDO OOO-^TflO 00O-*N^^ H t-t ^.00 CO -^ W ^.00 CO -^ w ^, tcl *^+ n o o p^S + w b '■3 to S3 ll 9"- a-H o u £^ 274 Journal oj Agricultural Research voi. 45, No. 5 The length of the incubation period in relation to the decreasing number of spores used varied greatly with the different lots of spores, even with the duplicate inoculations of each lot. Table 5 gives the results of positive cultures obtained in relation to the period of incu- bation and the dilution of the spores. The coefficient of correlation {14, -p. 179) for the positive cultiu-es only, in relation to length of incubation and dilution of spores, was found to be 0.3558 ±0.0440. While this does not show a strong correlation, it indicates that with the smaller numbers of spores there is a tendency for growth to take place with longer periods of incubation. However, when the cases of positive growth were correlated with the dilution and incubation time on the basis of the percentage of positive cultures to negative cultures for each observation period of incubation time, an insignifi- cant negative correlation was obtained. Apparently there is a variable uncontrollable factor present, more obvious when spores are used from different lots of the organism, which makes it impossible to correlate the other factors closely. However, the data summarized in Table 6 indicate that, of the 120 cultvu-es made with seedings of between 5,000,000,000 and 9,000,000 spores per seeding, 98.33 per cent showed growth at the end of 10 days' incubation, while 100 per cent (120 cultures) showed growth after 30 days' incubation. This is 56.87 per cent of the 211 total cultures showing growth after 30 days. Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 275 3 o «3OU300":!»OiO-^(NiOTl'CO 1 in 1 1 " i -H n sD^o(o«(D(D^J40QOlOQOClOco.->^-T^^T^lO!OM<^lOi-^'-li-'I-n^^«l.He^o (N 1 1 1 o 1 1 1 > a g " i i s O OT j en 1 s i 1 ! 1 ! 1 i 1 i 1 i 1" O ^ 1 s^ 111! 1 1^ j 1 j 1 1(N^ l^w^N 1^ j 1 [ In 1 1 1 s 1 1 s s ! i ! 1 i i i i i i i i i i i !"" ; S i s ° S 1 CC 1 s o eg 1 a 1 1 i-H CO [ s 1 1 1 ::;:;;:;;;; I^H i-H " i: § 1 1 1 1 •-( 1 li-H CO r O) ° li oo lo 1 CO ' t^ 1 1 1 1 1 I 1 1 1 1 1 iH < rH 1 1 > 1 1 1 1 1 i 1 1 1 i CO i i i i : i i h i i i i i i :^ ! (N 05 . s ° K 1 ■* !l!lll.-iliii(Mii-ii^irt 1 ; ; p 1 1 1 1 1 1 1 m 1 s i ; 1 i : i ; : 1 : ; ; ; : i i 1 -" ; i i i i i i i i-^ i IN 00 ■ (N i i i i i i i i ! i i-""^ ! i " S : S i i i i i i i ;-" i i'" i i i i : i ; 1 ! 1 i 1 1 i'^ 1 CO i o Illlllllllllw^S^i-fmiii' '1 (D « 1 Ol 11 rM 1 1 1 1 1 1 1 CO 1 oo 1 i 1 i i- i 1 i i P-"^ i 1'=^ i 1 1 i H-^ r i " ii t^ 1 ! ■ 1 i O Percent- age nega- tive of total nf all cultures o inoocs s Percent- age posi- tive of total ot all cultures ^ s r-1 Percent- age nega- tive of total of all negative cultures o cooo >o «D(0 to Percent- age posi- tive of ' total of all positive cultures ^ to to 3;s o OK3 00 lO SoJg ill o o «cdo Total number of all cul- tures o s Total number of nega- tive cul- tures o MOCO S Total number of posi- tive cul- tures § t^ rH (M a o "ca 1 o s 4^ ffl S q > o o CO t- 2g coirio CO Num- ber of nega- tive cul- tures o ggg CO Num- ber of positive cul- tuies M r-;oOO CO ■* (=1 O 1 i T3 O Sb.> ^ ca g2 t>0>0 CM Percent- age posi- tive OOO o Num- ber of nega- tive cul- tures (M SsS CO Num- ber of positive cul- tures 00 «^o o 111 IP 6, 000, 000, 000- 9,000,000 8, 000, 000- 600, 000 400, 000-50, 000 40, 000-0 '3 o Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 277 Of the 171 cultures made with seedings between 8,000,000 and 500,000 spores per seeding, 48, or 28.07 per cent, showed growth at the end of 10 da>s' incubation, while 79, or 46.20 per cent, showed growth after 30 days' incubation. The latter number is 37.44 per cent of the 211 total cultures showing growth after 30 days' incubation. Of the 142 cultures made with seedings between 400,000 and 50,000' spores per seeding, only 4, or 2.82 per cent, showed growth at the end of 10 days' incubation, while 12, or 8.45 per cent, showed growth after 30 days' incubation. The latter figure is 5.69 per cent of the 211 cultures showing growth after 30 days' incubation. Of the 123 cultures made with seedings of 40,000 or fewer spores. per seeding, no growth was obtained after 30 days' incubation. Of the 556 cultures made with all seedings, 30.58 per cent showed growth at the end of 10 days' incubation and 69.42 per cent showed no growth. The 170 positive cultures after 10 days' incubation is- 80.57 per cent (not shown in Table 6) of the 211 total positive cultures obtained. In the interval between the 10 and 30 day incuba- tion periods, 19.43 per cent (not shown in Table 6) of the 211 total positive cultures, or another 7.37 per cent of all cultures made, showed growth, making a total of only 37.95 per cent of all cultures which showed growth at the end of 30 days' incubation, with 62.05 per cent still showing no growth. The initial growth phases as described by Buchanan (5; IS, Ch. V) are clearly more marked with spores than with simple vegetative organisms, since there is a varying length of time necessary for spores to germinate and start growing after implantation in a suitable medium. In the light of observations on other spore-forming organisms, it is probable that this factor, which seems to cause varia- tions in the germination time of Bacillus larvae even within a lot from a single source, is what has been termed "dormancy." Burke (5, p. 283) , working with Clostridium botulinum, foimd : The individual (unheated) spores in a given culture of CI. botuUnum vary greatlj' in the time required for germination under optimum growth conditions. The majority germinate relatively quickly, but a few lie dormant for a longer time. One hundred and forty-four days is the maximum period of dormancy recorded here * * *. Burke states : The primary factors which cause the spore to lie dormant for long periods of time under optimum growth conditions are believed to be inherent in the spore itself. It is thought that relative permeability of the spore wall is one of the factors. Environmental conditions may secondarily modify the period of dormancy. Burke, Sprague, and Barnes (6, p. 560) observed the same phe- nomenon with such non spore-bearing bacteria as Bacillus coli ( = Escherichia coli). They found that spores of B. subtilis remained dormant 39 days and those of B. megatherium 90 days, although a large majority developed in 4 or 5 days. They believe: Dormancy must be considered a factor in infection. It reduces the chances of infection by reducing the number of organisms that would otherwise start to grow at one time. Since the cells begin to multiply at different times, the body has an opportunity to initiate defensive reactions before all the cells develop. If dormant for a sufficient period, the organisms will be excluded from the body before development takes place. 278 Journal of Agricultural Research voi. 45, No. 6 Swann {28) has observed that there is a variation in the germination time of anthrax spores, depending on the age and condition of the spores. Morrison and Rettger {24, j). 339) recently stated — Because of the marked variability of germination, depending upon the stimuli supplied in the environment, the deduction is made that bacterial spores in the process of germination are vitally active bodies having requirements for meta- bolic function which are the same as or more exacting and specific than those of the vegetative cells. Experimental evidence is presented to show that the dormancy of aerobic bacterial spores is largely, if not entirely, determined by conditions in the environ- ment of the spores, and that these factors must be taken into consideration, perhaps specifically for each species, before so-called "inherent " or " normal " dor- mancy of bacterial spores can be established. This phase of the work with Bacillus larvae is being repeated with the organism obtained from a single source in an effort to determine the importance of this variable factor of dormancy. SPORES OF BACILLUS LARVAE IN COMMERCIAL HONEY A few instances have been reported in the bee journals, such as that by Merrill {22), in which American foulbrood has developed as a result of bees having access to cans of infected honey that have been carelessly thrown out. Without doubt in some cases honey has been allowed to get on the market from infected colonies through negligence of the beekeepers and without being diluted by mixing or blending with honey from disease-free apiaries. On the other hand, Fracker {10, p. 379-380) has shown, by a study of disease-inspection statistics for Wisconsin: 1. In Wisconsin the introduction of this disease into the State and into many individual localities is definitely known to have been in specific importations of bees and equipment. 2. Cases of infection in which the source appears to be infected honey in the •channels of trade are comparatively rare. 3. Even near such a large center as Milwaukee the infection percentage is greatest m locahties of active movement, such as greenhouse areas, and is relatively low within the city itself. 4. Towns and cities of from 3,000 to 40,000 which have been natural markets for infected honey from near-by counties, have remained for years free from disease either until the present or until infected bees and equipment were introduced. 5. No new centers of infection are known to have been started since the policv ot limiting movement of bees and equipment was begun in 1919 6 These observations appear to be confirmed by conditions in the South, in T^12l f^! t f""^ *^® "S-T-i °S ^'=*'^^ *^'slit of the bee tends to continue through the peak of honey distribution. Furthermore, F. L. Thomas, State entomologist of Texas, in an unpublished manuscript states: K J^^i;!^-""^*" ^V^ *^? estimates with reference to the quantity of honey that is brought into Texas ma year is 19 carloads. Most of this honey is produced in California, Colorado, New Mexico, Utah, and Wyoming * * * P™aucea in If 19 carloads of foulbrood-infected honey are distributed annually in this time tnipp^'?h-'!?'°°^^^"'-I° '"t^^"'^ ^^^* °"^ inspectors would have 1 hard time to keep this disease withm bounds. In fact, I would exDect to find thni thP Srat^ATrl'e sLfe^tthJj°"°^ TT"" '"^ thefrttemp'tfto^er^adl^^^^^^^^ wS ■?-!," t^ share of the honey which is imported is sold in west and north- west Texas where practically no bees are kept. The amount which is dbtributed in the beekeeping territory of the State is evidently less dangerous than is com- mon^^y supposed. The following facts, I think, will prove tStatement hafS ca^FeT?ntf'?or^''" I'- ^^^°f -t? ^"^^^^ ^1, 1926, the insTect on work has been carried into 100 counties. Fifty-six counties were found to be free Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 279 from contagious or infectious diseases of bees, but in the other 44 counties Ameri- can foulbrood lias been present. An average of 668 beekeepers have been visited each year and 38,661 colonies examined with the result that an average of 430 colonies, or 1.11 per cent, have been found to be diseased. American foulbrood is found now in only 23 counties, 21 of the 44 counties having been cleaned up. In 12 of the counties where disease occurs, only 30 colonies were found to be infected out of 7,642 examined — less than 0.4 of 1 per cent. Six counties had one diseased colony each. About 40 per cent of the beekeepers and 60 per cent of the colonies are rein- spected from year to year; the remainder, being free of disease and considered out of danger, are dropped and "new territory" is taken over and examined for presence of foulbrood. By "new territory" is meant beekeepers and their colonies visited and inspected for the first time. An average of 228 diseased colonies are discovered each year in "new territory." This is 1.6 per cent of the total number of colonies examined in this territory. The reinspection which has been made in the counties where disease has been present shows that there have been both gains and losses. But a net gain has resulted which has averaged 21 beekeepers and 368 colonies freed from American foulbrood and quarantine annually. From these facts it is easily seen that definite and really rapid progress in eradicating the disease is being made. Rarely do our inspectors find new out- breaks of disease that can not be traced to careless beekeeping methods, bees robbing infected and weakened colonies, or to the use of old and infected equip- ment. It is not my intention to imply that honey is not a carrier of American foul- brood. The above evidence simply indicates that the honey which has been imported into Texas has not been as dangerous a source of disease to bees as is sometimes thought. Practically no work has been reported on the microbiology of honey other than that in connection with the spoilage of honey through fermentation by yeasts {19, 21), and no work appears to have been done on the Bacillus larvae spore content of commercial honey. In 1925 the writer undertook to devise a method for demon- strating, at least qualitatively, the presence or absence of spores of B. larvae in honey and their significance in relation to the results of the spore-feeding experiments. Difficulties were encountered in obtaiaing cultures of B. larvae from honey. It was impossible to obtain vegetative growth of this organism, even when a considerable number of spores had previously been added to honey, because of the difficulty of eliminating contaminating organisms that developed rapidly in the honey, completely overgrowing any possible vegetative growth of B. larvae before it could get well started. Therefore, methods of concentratiag the spores from the honey and of identifying them by means of microscopic examination were attempted. Because spores of B. larvae have a characteristic appearance in stained smears {20, -p. 9), it was assumed that this method might give at least tentative 6V1(1gI1C6 METHODS OF,PE0CEDURE The first method attempted was the filtration of honey diluted with water through a membrane of ether-alcohol collodion or through filter paper impregnated with an acetic acid solution of collodion {9). Apparatus was devised in which both suction and pressure were tried in this filtering process. Stained smears were made of the sediment' retained on the surface of the filter. In several cases spores of Bacillus larvae were observed in stained smears of the sedi- ment filtered out of honey known to have a large spore content. However, with honey containing fewer spores it was found impossible to concentrate them on a small enough area of filter in sufficient 280 Journal of Agricultural Research voi. «, No, s numbers to recover and identify them under the microscope. Even with a comparatively large filtering surface, the process was so slow that the diluted honey would frequently start to ferment before it had all passed through the fUter. A filter of smaller area would become clogged, preventing the passage of a sufficient quantity of honey. Several unsuccessful attempts were made to recover spores of Bacil- lus larvae from honey by centrifuging samples diluted with an equal quantity of water. After considerable experimentation with honey of known spore content, it was found that it was necessary to dilute the honey to a much greater extent — 1 part to at least 9 of water — ia order to throw the spores down with the sediment. Apparently the specific gravity of these spores is so low that on centrifuging they remain in suspension in only slightly diluted honey. The procedure finally used for demonstrating the presence of spores of Bacillus larvae in honey is as follows: Five c c of warmed honey is thoroughly mixed with 45 c c of distilled water in a 50 c c cone- shaped centrifuge tube made of heat-resistant glass. Duplicate quantities of each sample of honey are made up for examination. The diluted honey is then centrifuged at 2,000 revolutions per minute for one-half hour. Because of the difficulty of obtaining a satisfac- tory stained smear from the sediment thrown down in the presence of the sugars of the honey solution, all but 2 c c of the solution in each centrifuge tube is drawn off by means of a 50 c c pipette. Another 45 c c of distilled water is added , the sediment is thoroughly shaken up in the water, and the tabes are centrifuged again for 20 minutes. After all but 2 c c or less of the wash water has been removed, 0.01 c c of the sediment is removed by means of a capillary pipette and smeared on a cover glass over a surface of 1 cm^, a small loopful of carbol fuchsin being mixed with the material before it is allowed to dry. After drying by gentle heat, the cover glass is mounted on a slide by means of a drop of distilled water and the smear is examined with an oil-immersion objective. Spores of B. larvae are identified by their size and shape in conjunction with their distinctive habit of breaking loose from the stained mass of the smear and of showing a delicate Brownian movement in the thin film of water between the two pieces of glass. In a few samples only one or two spores were seen in numerous fields examined or the spores did not have the typical appearance of spores of B. larvae. In such cases another test, in which twice as much honey was used, was made from the sample. OBSERVATIONS One hundred and ninety-one samples of honey were examined by this method. (Table 7.) Of tlfese, 187 were regular commercial samples purchased in the open market and 2 were from the experi- mental apiary at Laramie. The other two were miscellaneous samples, one of which was obtained from a brood comb from a dis- eased colony and the bther from a cappings melter which had been used with combs from an infected apiary. Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 281 Table 7. — Results of the examination of samples of honey for the presence of spores of Bacillus larvae Source Samples tested Samples showing positive presence of spores resem- bling Bacillus larvae Samples showing no evidence of spores ° Commercial samples from 30 States 187 2 2 15 172 2 2 Total 191 17 174 " 29 of these samples were doubtful on the first examination, but repeated examinations gave negative Tesults in each case. Of the 187 samples of commercial honey obtained from 30 different States or Territories, 15, or 8 per cent, showed the presence of a suf- ficient number of spores resembling spores of Bacillus larvae to be designated as positive. In 29 of the commercial samples, or 15.5 per cent, one or two doubtful spores were seen in each case, but on repeated examinations none of these samples could be considered positive. Two of the four miscellaneous samples from infected sources were also found to contain spores of B. larvae. Five of the samples showing the presence of spores of Bacillus larvae were fed to healthy 5-frame colonies during the summer of 1930. These samples consisted of from a pint to a quart of honey. No evi- dence of American foulbrood appeared in any of the five colonies during the entire brood-rearing season. In order to determine the approximate number of spores in the samples of honey in which the presence of Bacillus larvae was demon- strated, a series of dilutions of spores was prepared as described for the work with cultures. A stained smear was made of 0.01 c c of each dilution spread over a 1-cm^ surface of cover glass mounted with water and examined with the oil-immersion objective. By this means a definitely recognizable number of spores could be found down to the dilution of 2,000,000 spores per cubic centimeter, with a few single spores seen in occasional fields down to the dilution of 500,000 spores per cubic centimeter. (Table 8.) Then 1 c c of each dilution was added to 5 c c of distilled water in 15 c c centrifuge tubes and centrifuged at 2,000 revolutions per minute for 20 minutes. A stained smear made from 0.01 c c of each sediment showed a definitely recognizable number of spores down to the 5,000-spore dilution, with one or two doubtful spores in several fields from the 500-spore dilu- tion. The sample containing the 50,000-spore dilution, which would be comparable to the sugar sirup containing the minimum number of spores per cubic centimeter fed to colonies in the spore-feeding experi- ments that produced infection, showed a great many more spores in each field examined by this method than did the sample of commer- cial honey that showed the greatest number of spores. Therefore, until a better quantitative method is devised, it seems reasonable to believe, from the indications of the preliminary work on this problem, that, even though the presence of a few spores of B. larvae may be 282 Journal of Agricultural Research Vol. 45, No. 5 demonstrated in 5 c c quantities from a comparatively small per- centage of samples of commercial honey, the numbers are far below the minimum necessary to produce infection when such honey is used in healthy colonies of bees. Before definite conclusions can be drawn, it will be desirable to examine many more samples of coxn- mercial honey and to feed to healthy colonies samples of honey in which the presence of spores has been demonstrated. Table 8. — Microscopic examination of dilutions for spores of Bacillus larvae " Number of spores per cubic centi- meter in each dUution Direct exami- nation of 0.01 cubic centi- meter Exami- nation of sedi- ment after centri- fuging 1 cubic centi- meter Number of spores per cubic centi- meter in each dilution Direct exami- nation of 0.01 cubic centi- meter Exami- nation of sedi- ment after centrl- fuging 1 cubic centi- meter Number of spores per cubic centi- meter in each dilution Direct exami- nation of 0.01 cubic centi- meter Exami- nation of sedi- ment after centri- fuging 1 cubic centi- meter 5, 000, 000, 000 4, 000, 000, 000 3, 000, 000, 000 2, 000, 000, 000 1, 000, 000, 000 500, 000, 000 400, 000, 000 300, 000, 000 200, 000, 000 100. 000, 000 90, 000. 000 80, 000, 000 70, 000, 000 -j- -r + -t- -1- -1- + + + + + + + + + + + + H- -1- -t- + -t- -1- -t- -t- + -1- + + + + + + + + 10, 000, 000 9, 000, 000 8, 000, 000 7, 000, 000 6, 000, 000 5, 000, 000 4,000,000 3, 000, 000 2, 000, 000 1, 000, 000 900, 000 800, 000 700, 000 eoo, 000 SOO, 000 400, 000 300, 000 200, 000 -1- -+- -f -1- + + -f- + ±? — ^y- + + + + + + + -1- + -1- -f + + -i- + -t- -1- + 100, 000 90, 000 80, 000 70, 000 60, 000 60,000 40, 000 30, ( 00 20, 000 10, 000 5,000 4,000 3, f 00 2,000 1,000 500 50 6 "'"-'"" + + -1- + -t- + + 4- -i- -1- + 60, 000, 000 50, 000, 000 40, 000, 000 30, 000, 000 20, 000, 000 ± <• -j- indicates that spores were found; — indicates that spores were not found, by microscopic examina- tion; ± indicates that the result was doubtful; ±? indicates that the positive was more doubtful than the negative; — ? indicates that the absence of spores was not definite. SUMMARY AND CONCLUSIONS As a result of five years' study it has been found that, in order to produce American foulbrood infection in a healthy colony of bees, the sugar sirup used for inoculation must contain a certain initial number of spores of Bacillus larvae. Seventy-three colonies were inoculated during this time with numbers of spores ranging from approximately 5,000,000,000 to 100,000 per colony; 30 of these colonies receiving 50,000,000 spores or less. Of these 30 colonies, 2 out of 11 receiving 50,000,000 spores showed infection, but no colony receiving less than that number of spores developed disease. There- fore, the minimum infectious dose of B. larvae for a colony of bees seems to be approximately 50,000,000 spores in 1 liter of sugar sirup. PreUminary experiments in which individual bee larvae were given known numbers of spores of Bacillus larvae in 0.01 c c quantities of sugar sirup show that infection can be produced bv this method, but with considerable difficulty. From 50 to 100 larvae were inoculated with each dilution of spores, ranging in number from approxinaately 50,000,000 spores to, theoretically, 1 spore per larva. The minimum infectious dose was found to be 10,000,000 spores per larva fed in 0.01 c c of sugar sirup. These results indicate that the Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 283 minimum dose of spores of B. larvae that will produce American foul- brood infection must be large. The germination of spores of Bacillus larvae and vegetative growth on a suitable artificial culture medium resulting from the inoculation of 556 culture tubes with seedings varying from approximately 50,000,000,000 to 500 spores per culture also shows that a certain minimum initial number of spores in the inoculum is necessary in order to produce growth. This minimum number of spores produc- ing vegetative growth on a medium consisting of yeast-carrot extract, egg-yolk suspension, and agar was found to be approximately 50,000 in 1 c c of suspension inoculated. The production of nitrite in this medium by the vegetative growth of Bacillus larvae serves as a fairly delicate and reliable indicator of such growth. There was a tendency for the seedings containing the smaller num- bers of spores of Bacillus larvae to require a longer period of incubation than the larger seedings in order to produce vegetative growth. However, there was a considerable variation in the germination time of many of the seedings of spores, in one case a seeding of 9,000,000 spores requiring 27 days' incubation to produce growth and another of 70,000 spores requiring only 6 days. This variation, thought to be due to the variable character known as dormancy in bacterial spores, prevented more than a slight correlation. In the group of cultures comprising seedings between 5,000,000,000 and 9,000,000 spores, only 1.67 per cent required more than 10 days' incubation to produce vegetative growth, 100 per cent having shown growth after 30 days. In the group of cultures comprising seedings between 8,000,000 and 500,000 spores, 71.93 per cent required more than 10 days' incubation, while 53.81 per cent showed no growth at the end of 30 days' incubation. In the group of cultures comprising seedings between 400,000 and 50,000 spores, 97.18 per cent required more than 10 days' incubation, while 91.55 per cent of the group showed no growth at the end of 30 days. Below 50,000 spores no growth was obtained. In other words, below a seeding of 9,000,000 spores an increasing number of the smaller spore seedings required a longer period of incubation. About 80 per cent of all the positive cultures were obtained during the first 10 days of incubation, although this was approximately only 30 per cent of all the cultures made; at the end of 30 days' incubation only about 38 per cent of all the cul- tures had shown any growth. It was found possible to demonstrate the presence of spores of Bacillus larvae in 15 out of 187, or in 8 per cent, of the samples of commercial honey examined by means of the centrifuge and the microscope. "The preliminary results indicate that, even though spores of B. larvae may be demonstrated in a certain percentage of samples of commercial honey, in most instances they are probably present in such small numbers as to be less than the minimum number, 50,000,000 per liter, found to be capable of producing dis- ease, and therefore are ineffective in the spread of American foul- brood. 284 Journal of Agricultural Research voi. 45, No. s- LITERATURE CITED (1) Ahrbns, H. G. 1930. NEW FACTS ABOTTT FORMALIN TREATMENT. Amer. Bee Joup. 70: 61-62. (2) Breed, R. S., and Brew, J. D. 1916. COtTNTING BACTERIA BY MEANS OF THE MICROSCOPE. N. Y. State Agr. Expt. Sta. Tech. Bui. 49, 31 p., illus. (3) Buchanan, R. E. 1918. LIFE PHASES IN A BACTERIAL CULTURE. Joui. Infect. Diseases 23:109-125, illus. (4) Burke, G. S. 1923. studies on the thermal death time of spores op clostridium botulinum. 2. the differential staining of living and DEAD SPORES. Jour. Infect. Diseases 32 : [433]-438, illus. (5) 1923. STUDIES ON THE THERMAL DEATH TIME OF SPORES OP CLOSTRIDIUM BOTULINUM. 3. DORMANCY OR SLOW GERMINATION OF SPORES UNDER OPTIMUM GROWTH CONDITIONS. Jour. Infect. Diseases 33: [2741-284. (6) Burke, V., Spkague, A., and Barnes, La V. 1925. DORMANCY IN BACTERIA. Jour. Infect. Diseases 36: [565]-560. (7) BURNSIDE, C. E. 1931. DISINFECTION OF AMERICAN POULBKOOD COMBS BY FUMIGATION BY FORMALDEHYDE. Bee World 12:3-7, 16-19. (8) CORKINS, C. L. 1928. QUARTERLY REPORT. Wyo. Beeline 5 : 25-26. (9) Elford, W. J. 1928. ULTRAFILTRATION. (AN HISTORICAL SURVEY, WITH SOME REMARKS ON MEMBRANE PREPARATION TECHNIQUE). Jour. Rov. MicrOS. See. (3) 48:36-45, illus. (10) Fracker, S. B. 1925. are commercial honey shipments largely responsible foe the DISSEMINATION OP AMERICAN FOULBROOD? Jour. EcOn. Ent. 18:372-380. (11) Gates, F. L. 1920. a method op standardizing bacterial suspensions. jour. Expt. Med. 31:105-114, illus. (12) Henrici, a. T. 1928. morphologic variation and the rate op growth of bacteria. 194 p., illus., Springfield, 111., and Baltimore, Md. (Mono- graphs on Agricultural and Industrial Microbiology, v. 1.) (13) Jordan, E. O., AND Falk, I. S., editors. 1928. THE NEWER KNOWLEDGE OF BACTERIOLOGY AND IMMUNOLOGY. 1196 p., iUus. Chicago. (14) Kelley, T. L. 1923. STATISTICAL METHOD. 390 p., illus. New York. (15) KosER, S. A., AND Mills, J. H. 1925. DIFFERENTIAL STAINING OP LIVING AND DEAD BACTERIAL SPORES Jour. Bact. 10:25-36. (16) LiNEBURG, B. 1925. STRAIN OF IMMUNE BEES. Gleanings Bee Cult. 53 : 709-710. (17) LOCHHEAD, A. G. [1927.] FURTHER STUDIES OF BACILLUS LARV^, THE CAUSE OF AMERICAN FOULBROOD OP BEES. Canada Expt. Farms, Div. Bact. Rpt. (18) 1926:13-16. 1928. CULTURAL STUDIES OP BACILLUS LARV« (WHITE). Sci. Agr. 9r 80—89, illus. (19) AND Heron, D. A. 1929. MICROBIOLOGICAL STUDIES OF HONEY. I. HONEY FERMENTATION AND ITS CAUSE. II. INFECTION OF HONEY BY SUGAR-TOLER- (9m MnPo-, a'^'J?' ^^"^^w Canada Dept. Agr. Bui. (n. s.) 116, 47 p., illus. (20) McCray, a. H., AND White, G. F. ' > f < 1918. THE DIAGNOSIS OP BEE DISEASES BY LABORATORY METHODS. U. S. Dept. Agr. Bui. 671, 15 p., iUus. Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 285 (21) Marvin, G. E. 1928. the occurence and characteristics op certain yeasts found IN FERMENTED HONEY. Jour. Econ. Ent. 21:363-370, illus. (22) Merrill, J. H. 1927. AN INITIAL OUTBREAK OF FOULBROOD. Amer. Bee Jour. 67:414- 415. (23) MiLLEN, F. E. 1928. SPREADING FOULBROOD. Beekeeper 36: 134. (24) Morrison, E. W., and Rettger, L. F. 1930. bacterial spores. ii. a study of bacterial spore germination IN RELATION TO ENVIRONMENT. Jour. Bact. 20:313-342. (26) Robertson, T. B. 1923. THE CHEMICAL BASIS OP GROWTH AND SENESCENCE. 389 p., illus. Philadelphia and London. [Original not seen.] (26) Sturtevant, A. P. 1924. the development op American foulbrood in relation to the METABOLISM OF ITS CAUSATIVE ORGANISM. Jour. Agr. Research 28:129-168, illus. (27) 1930. PRELIMINARY REPORT CONCERNING FACTORS RELATED TO CERTAIN OF THE GROWTH PHASES OP BACILLUS LARVAE. Jour. EoOn. Ent. 23:453-459. (28) SwANN, M. B. R. 1924. ON THE GERMINATION PERIOD AND MORTALITY OP THE SPORES OP BACILLUS ANTHHACis. Jour. Path, and Bact. 27:130-134. (29) TOUMANOPF, K. 1929. NOTE SUR l'iNFECTION DBS LARVES d'aBEILLES PAR BACILLUS. LARV.E. Bul. Acad. V(St. France 2:45-49. (30) White, G. F. 1920. AMERICAN FOULBROOD. U. S. Dspt. Agr. Bul. 809, 46 p., illus. (31) Zinsser, H. 1927. a textbook of bacteriology; a treatise on the application op bacteriology and immunology to the etiology, diag- nosis, special therapy and prevention of inpectioos dis- eases, for students and practitioners op medicine and PUBLIC HEALTH . . . Rewritten, rev. and reset . . . Ed. 6, 1053. p., illus. New York and London. o K-269 QUANTITATIVE DEMONSTRATION OF THE PRESENCE OF SPORES OF BACILLUS LARVAE IN HONEY CONTAMINATED BY CONTACT WITH AMERICAN FOULBROOD BY A. P. STURTEVANT (Contribution from Bureau of Entomology and Plant Quarantine) Reprinted from JOURNAL OF AGRICULTURAL RESEARCH Vol. 52, No. 9 : : : : Washington, D. C, May 1, 1936 (Pages 597-704) ISSUED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES AND UNIVERSITIES U. S. GOVERNMENT PRINTING OFFICE : 1936 JOINT COMMITTEE ON POLICY AND MANUSCRIPTS TOR THE UiriTED STATES DEPARTMENT FOE THE ASSOCIATIOIT OF LASfD-GEANT OF AGRICTJITURE COIIEGES AND UNIVERSITIES H. G. KNIGHT, Chairman S. W. FLETCHER Chief, Bureau of Chemistry and Soils Director of Research, Pennsylvania Agri- cultural Experiment Station F.L.CAMPBELL j y p.TT ^lT?^,lf b^„";S^{''""'™''"""' ' director, Kansas AgricuUural Experiment and Plant Quarantine Station JOHN W. ROBERTS C. E. LADD Principal Pathologist, Bureau of Plant Director, New York {Cornell) Agricultural Industry Experiment Station EDITORIAI STTPEEVISION M. C. MERRILL Chief of Publications, United States Department of Agriculture Articles for publication in the Journal must bear the formal approval of the ■chief of the department bureau, or of the director of the e.xperiment station from which the paper emanates. Each manuscript must be accompanied by a state- ment that it has been read and approved by one or more persons (named) famiUar with the subject. The data as represented by tables, graphs, summaries, and conclusions must be approved from the statistical viewpoint by someone (named) competent to judge. All computations should be verified. Station rnanuscripts and correspondence concerning them should be addressed to S. W. Fletcher, Director of Research, Pennsylvania Agricultural Experiment Station, State College, Pa. Published on the 1st and 15th of each month. This volume will consist of 12 numbers and the contents and index. Subscription price: Entire Journal: Domestic, $3.25 a year (2 volumes) Foreign, $4.75 a year (2 volumes) Single numbers: Domestic, 15 cents Foreign, 20 cents Articles appearing in the Journal are printed separately and can be obtained by purchase at 5 cents a copy domestic; 8 cents foreign. If separates are desired in quantity, the}' should be ordered at the time the manuscript is sent to the printer. Address all correspondence regarding subscriptions and purchase of numbers and separates to the Superintendent of Documents, Gove rnment Printing Office, Washington, D. C. QUANTITATIVE DEMONSTRATION OF THE PRESENCE OF SPORES OF BACILLUS LARVAE IN HONEY CON- S^5!?i^^'^^° ^Y CONTACT WITH AMERICAN FOUL- BROOD ^ By A. P. Sttjbtevant ' Associate apiculturist, Division of Bee Culture, Bureau of Entomology and Plant Quarantine, United States Department of Agriculture INTRODUCTION In a previous paper ^ the writer showed that it is possible to demonstrate the presence of spores of Bacillus larvae, the cause of American foulbrood, in samples of commercial honey that have had contact with American foulbrood in the course of their production or prepa,ration for the market. Siace this work was reported, 25 additional samples, making a total of 212 samples of commercial honey, obtained on the open market from 28 States and 2 Territories have been examined by the same method, and spores of B. larvae have been found in 17, or 8 percent, of these samples.^ In most cases the spores were present in relatively small numbers. The method of examiaation used in the work thus far reported gave only a qualitative indication of the number of spores present, the observations being recorded as showing "the presence of a suflfi- cient number of spores resembling spores of B. larvae to be designated as positive."* This araounted to from one or two definite spores to a very few spores seen in numerous microscopic fields of each stained sediment examined. The primary object was to demonstrate only their presence or absence. It was assumed that iu most cases the number of spores found was considerably smaller than would be foimd in honey containing numbers comparable with the observed minimum infective dose of 50,000,000 per Hter. The only way of demonstrating the accuracy of this assumption has been to feed such "positive" samples of commercial honey to healthy colonies of bees. This was done with 15 of the 16 samples in which spores were demonstrated, and only 1 sample, or 6.7 per- cent, was found to contain sufficient infection to produce the disease in a healthy colony. These investigations indicate that the require- ment of certification of honey, as has been proposed and even placed in operation in certain States, is not a justifiable measure in the control of American foulbrood under the present conditions of inspection and control of disease in this country. To permit a more accurate, quantitative study of the infectivity of honey that has been in contact with American foulbrood, on the ' Beeeived for publication Jan. 27, 1936; issued June 1936. Tliis investigation was carried on at the Intermountain States laboratory of the Division of Bee Culture, which is maintained cooperatively by the University of Wyoming and the Bureau of Entomology and Plant Quarantine, XJ. S. Department of Agriculture. ' Acknowledgments are due to P. E. Hall, associate professor of commerce, University of Wyoming, for advice and assistance in the statistical analysis of the data. ' STOETEVANT, a. p. EELATION of COMMEBCIAL honey to the SPKEAD or AMEEICAN rOULBEOOD. Jour. Agr. Research 45: 257-285, illiu. 1932. * Stdetevant, a. p. honey or the inteemodntain eegion. Gleanings Bee Cult. 63: 463-468, illus. 1935. » Sttjetevant, a. p. See footnote 3. Journal of Agricultural Research, Vol. 62, no. 9 Washington, D. O. May 1, 1936 Key no. K:-269 57176—36 (697) 698 Journal oj Agricvltural Research voi. 62, no. 9 basis of its spore content^that is, a detailed study of the distribution of spores of B. larvae m. the honey from infected hives or apiaries, or in commercial honey obtained on the open market, or of the effect of mixing infected honey with disease-free honey in the course of production or blending and preparation for the market — a more detailed iavestigation has been made of the spore content of honey containing approximately known numbers of spores. This has been accomphshed by an improved and more accurate method of deter- mining the number of spores in such honey, and the accuracy of the results and method has been demonstrated by means of a statistical analysis of the data obtained. METHOD OF OBTAINING THE DATA PREPARATION OF SAMPLES OF HONEY A series of samples of honey containing approximately known numbers of spores per cubic centimeter were prepared in the manner described previously,' by adding to 100-cc quantities of spore-free honey the necessary quantities of various dilutions of a stock suspen- sion of spores of Bacillus larvae containing approximately 5,000,000,000 spores per cubic centimeter. Five samples of honey were prepared in this way containing approximately 1,000,000, 800,000, 500,000, 300,000, and 50,000 spores per cubic centimeter, respectively. These samples, each considered as a unit and not as a dilution of the 1,000,000-spore sample, were heated in a water bath to 120°-130° F., and then thoroughly mixed with a mechanical stirrer for 5 minutes. Duplicate 5-cc quantities of each sample were then placed in 50-cc conical centrifuge tubes, and 45 cc of distilled water of approximately the same temperature was added. When the honey and water were completely mixed, the samples were centrifuged at 2,000 revolutions per minute for 45 minutes. All but about 1 cc of the supernatant honey-water solution of each sample was then removed by means of a pipette and suction. Again approximately 45 cc of distilled water was added, and after thorough mixing the suspensions were centri- fuged for 30 minutes longer. The removal of the supernatant solu- tion was repeated until all but approximately 0.1 cc' of the water had been removed from each centrifuge tube, and each sample of sediment was completely suspended in this remaining quantity of water by blowing gently through a capUlary pipette dipped into the water. Duphcate 0.01-cc quantities of each suspension were then transferred with the capillary pipette (calibrated to deliver 0.01 cc) to microscope cover glasses. Circular cover glasses, size 12, no. 1 thickness, having an area of 1.13 cm 2, proved satisfactory for this P^Pj®^" u ^^^'^ ^^ *° ^ "^™) loopful of carbolfuchsm stam was added to the drop of suspension on the cover glass and thoroughly mixed with it. This stained liquid was then spread uniformly over a 1-cm area of the cover glass, a narrow ring at the outside edge being left uncovered. The smears were allowed to dry in the air and were then mounted on microscope slides either with water or pref- erably, with Canada balsam, for examination under the microscope, ihese stamed smears were not washed in water, as this might have caused some spores to be lost. • Stuetevant, a. p. See footnote 3. ' A mark was placed on the outside of the conical centrifuge tubes to indicate the 0.1-co volume. Uay 1, 1036 Spores /2 \ \ \\ /O A n 1 1 L. — _ wrRoc £Noas / 1 e 4 4 — -\ \ z N \ \ fi^T a ^rs / z 3 4 s 6 7 FiQ. 11.— Per cent composition of worker brood food (Table III) 144 Journal of Agricultural Research voi. xxvni, No. a 'f £ 6 7 a s /o /J Fio. 12.— Average chemical composition ol worker larvae at different ages (Table IV) Apr. 12, 1924 Development of American Foulbrood 145 Table IV. — Average chemical composition of worker larvce at (liferent ages, compiled from Straus (43) Weight of larvft Glycogen Fat Nitrogen Age Grams per larva Per cent of fresh substance Grams per larva Per cent of fresh substance Grams per larva Per cent of fresh substance Reducing sugar Days X Qrams 0.00030 .00340 .03000 .10010 . 12775 .14290 . 16140 .14300 .14200 .14500 .13000 2 0.00008 .0012 .0055 .0072 .0088 .0092 .0089 .0075 .0076 .0066 2.60 2.76 5.68 6.67 6.95 6.43 6.35 6.21 6.24 4.21 0.00004 .00005 .0031 .0047 = .0067 .0060 .0051 .0062 .0049 .0047 i.63 1.64 3.60 3.64 "3.98 3.71 3.53 3.66 3.60 3.26 0. 00009 .0006 .0016 . 0010 .0019 .0018 .0027 .0022 .0022 .0023 2.86 2.04 1.44 1.47 1.45 1.22 1.51 1.60 1.68 1.68 3 4 5 6 7 Trace. 8 Trace. 10 0.0002 11 o Calculated by interpolation and averaging. CHOICE OF REAGENT It was necessary to devise a special technic for the determination of the unassimi- lated reducing sugar in the larva by the application of procedures used in other analyses where small amounts of reducing sugars must be determined, such as in urine analysis. After studying the various methods of sugar analysis, a volumetric titration method seemed the most promising. For the purpose of determining quantitatively the unassimilated sugar in the bee larva at different ages, the modified copper sulphate solution of Benedict (5) was chosen, mainly because, as in urine analysis, it has proved more satisfactory than any other titration method for determining small amounts of reducing sugars quantitatively, and because this solution keeps indefinitely .without deteriorating. The potassium sulphocyanate in the solution produces, upon reductioil of the sugar, a white precipitate of cuprous sulphocyanate, which per- mits the end point of the reaction to be more accurately determined than with Fehling's solution. A trace of ferrocyanid is added to prevent precipitation of red cuprous oxid which, may be caused by certain impurities, which would interfere with the determination of the end point. The test solution is standardized to a known solution of dextrose so that 5 cc. equals 0.0102 grams of dextrose. CHOICE OF LAKVAE Since there is little likelihood of there being any appreciable amount of sugar elsewhere than in the intestine, analyses were made of entire larvse, because of the great difficulty attending the dissection of the intestines. Larvse for analysis were chosen from combs having large areas of brood of uniform size and age. In most cases 35 larvae as nearly of the same size as possible were carefully removed from the cells by means of a pair of fine forceps, care being taken to remove as little uningested food as possible. Any visible amount of adhering food was removed with filter paper and the 25 larvse were weighed. Several series were weighed for each age above the two-day age period through to about the fourth day after capping. DETERMINATION OF AGE OF LARVAE When choosing larva for the analysis, the approximate age was determined by comparison with drawings to scale by Nelson and Sturtevant (35) of larvse of known age at various age periods, 24 hours apart. Nelson and Sturtevant, as 5095— 24t 3 146 Journal of Agricultural ResearcJi voi. xxvm, No. 2 well as Straus (Table IV), also give weights for larvse of known age, but in order to eliminate the danger of variations due to the eflfect of different seasonal and environmental conditions, the average age of the larvse analyzed from various groups of 25 was determined by comparison with a series of weigh- ings of larvse of known age that "were made during this same period (35) . The various series of weights, with the corresponding determinations of reducing sugar, were arranged in age groups, 24 hours apart, as shown ip Table V. In some cases, such as the small two-day larvae, or the quiescent prepupffi, where the amount of unassimilated sugar is small, 50 larvae were taken for analysis, but usually 25 proved satisfactory. PREPAEATION OF MATERIAL FOE ANALYSIS Several difficulties were encountered in the preparation of material for sugar determination. At first, attempts to extract the sugar were made by macerating the larvae with distilled water and filtering through filter paper. This produced a cloudy opalescent liquid, indicating the presence of colloidal material, and this solution did not give the characteristic reaction with the Benedict reagent. Various clarification methods were tried. Precipitation with both neutral and basic lead acetate (10, p. 276) solutions proved unsatisfactory, something stiU remaining to interfere with the reaction. Mercuric nitrate solution, which is sometimes used to clarify liquids of animal origin such as blood, urine, and milk, was tried {10, p. 447). This method occasionally gave good results, mainly with the younger larvae, but often with older larvae and prepupae the colloidlike material still remained in the filtrate, interfering with the reaction. Furthermore, because of the numerous filtrations necessary to remove successive precipitates, it was feared that more or less sugar is lost by adsorption to those precipitates, even with careful washing. An attempt was made to clarify by filtration with suction through a celloidin membrane, and this gave a clear solution which reacted well with the test solution, but the method required too great time. The method finally adopted was by extraction with 50 per cent alcohol, similar to the method used in the extraction of sugars from grains and similar products (It). This method proved successful, since the alcohol causes precipitation of all solid matter, giving a clear filtrate which reacted properly with the Benedict's reagent. Since glycogen in water solution is colloidal in nature, and thereby difficult to remove by filtration from such a solution, it is doubtless the glycogen present in the larva which prevented clarification and interfered with the reaction. It is possible for this reason that Straus (43) failed to demonstrate reducing sugars. To determine this point, a small amount of glycogen was added to a known solu- tion of dextrose and tested with the copper sulphate solution, and the known reducing sugars could not now be demonstrated quantitatively. Since glycogen is insoluble in alcohol {10, p. 44S) the 50 per cent alcohol precipitates the glycogen and thereby removes materials interfering with the reaction in the filtrate. Even though there may stiU be a small loss of reducing sugar by adsorption or by some other means, the results obtained are of value for purposes of comparison. If any reducing sugar is lost by the method adopted, the amount is exceedingly small and may therefore be disregarded, since repeated washings failed to demon- strate its presence. TECHNIC ADOPTED After weighing, the larvae are renioved to a small porcelain mortar and mace- rated in 30 CO. of 50 per cent alcohol. This material is then washed carefully into a small flask and allowed to stand from two to three hours before filtering. The precipitate is washed with 60 per cent alcohol. The filtrate is then made up to 50 cc. with distilled water, and run into a burette. Five cc. of the stand. Apr. 12, 1S24 Development of American Foulhrood 147 ardized Benedict's solution are placed in a white porcelain casserole and di- luted with an equal amount of distilled water. To this are added about 5 grams of anhydrous sodium carbonate and a small amount of ground pumice. This solution is brought to a boil and the larval extract is run in slowly, drop by drop at the end, until the blue color disappears and a white precipitate forms. From the number of cc. of larval extract used, the milligrams of sugar per larva and the per cent of sugar per larva are calculated (Table V) . Table V. — Unassimilated sugar in intestinal content of larvx at different ages Larvse of known age, Sturtevant (SS) Larvffl analyzed for presence of unassimilated sugar {weights in grams) Ago Aver- Limits by weiglit tor age groups Weight Num- ber Aver- age Ex- Equiv- alent CuSO) Equiv- alent dex- trose Dex- trose Sugar in age Date of of weight tract number solu- per days weight sample lar- VEB otl larva used" of larva tion per larva larva Oram Qrarfi Wit Oram Gram Cc. Cc. Oram Oram P.d. 2 0.004745 .024626 Up to 0. 014685. 0. 014685 to 7-18 0.6233 50 0.01247 60 60 6 No re- action. 3 7-25 .4967 25 . 01987 60 25 5 No re- 0.059308. action. 7-25 1. 1072 25 > .04429 44 22 5 0.01020 0. 000463 1.13 8-2 ' 1. 0979 25 '.04392 23.6 23.5 6 . 01020 .000434 .98 5-9 • 2. 2906 50 .04581 90 46 10 . 02040 . 000463 .94 8-11 1. 1706 25 . 04682 42 21 6 . 01030 . 000490 1.04 Average 0. 093990 7-27 1.4009 25 .06604 20.25 10. 126 6 . 01020 .001007 1.79 .043222 .000476 .98 0. 059308 to 4 8-2 1. 6749 25 .06700 21 10.5 5 .01020 .00097 1.44 0. 120369. 8-11 ■> 1. 6817 25 . 06727 11 9.16 6 . 01030 . 00112 1.66 8-11 1. 9372 25 . 07749 12.76 .6.375 5 . 01030 .00161 2.07 8-11 1. 9916 25 .07966 18.1 9.05 5 . 01030 .00113 1.41 7-31 2.3044 26 ». 09218 6.8 3.4 6 .01020 .00300 3.25 8-2 2. 3566 26 ' .09426 8.6 4.3 5 . 01020 .00237 2.51 8-30 2. 3733 26 . 09493 6.68 3.29 6 . 01030 . 00313 3.29 7-25 2. 4901 26 . 09960 5.8 2.9 6 . 01020 . . 00351 3.52 8-2 2. 6748 26 . 10699 8.0 4.0 5 .01020 .00255 2.37 .8-17 2.6843 25 . 10737 4.76 2.38 5 .01030 .00431 4.01 8-18 2. 7781 26 . 11112 4.35 2.175 6 .01030 .00473 4.25 8-2 2. 7919 25 .11168 7.0 3.5 6 .01020 .00291 2.61 8-18 2.8205 25 .11282 4.35 2.175 5 . 01030 . 00473 4.19 6-1 2.8332 25 . 11333 12.7 6.35 8.9 . 018166 .00286 2.52 8-11 2.8972 25 .11689 6.8 3.4 5 . 01030 . 00303 2.61 8-4 2.9274 25 . 11710 7.1 3.65 5 .01020 .00287 2.45 8-2 2.9505 25 .11802 7.1 3.55 5 . 01020 .00287 2.43 8-31 2. 9749 26 .11900 5.2 2.6 6 .01030 .00396 3.32 8-2 2.9908 25 . 11963 6.0 3.0 5 .01020 . 00340 2.84 8-17 3.0038 25 .12015 4.5 2.25 5 . 01030 .00457 3.80 Average 0. 146748 8-4 3.0105 25 .12042 6.5 3.25 6 . 01020 .00314 2.61 . 10314 .00299 2.82 0. 120369 to 6 6-1 c 3. 0961 26 .12384 27.0 6.76 10 .02040 . 00317 2.67 0. 160876. 8-18 3. 1148 25 .12459 4.2 2.1 6 . 01030 . 00490 3.93 8-18 3.1953 25 . 12781 4.2 2.1 6 . 01030 .00490 3.83 8-11 3. 2141 25 .12856 6.4 3.2 5 . 01030 .00322 2.61 7-26 3.3153 25 . 13261 5.6 2.8 5 .01020 .00364 2.75 7-31 ' 3. 3278 25 .13311 5.0 2.5 5 .01020 .00408 3.06 8-11 3.3479 25 .13392 6.5 3.25 5 .01030 . 00317 2.36 8-10 3. 3602 25 .13441 6.15 3.075 6 . 01030 . 00334 2.45 7-31 3.3689 25 .13476 tl 2.05 5 . 01020 . 00497 3.69 8-11 3. 3706 25 .13482 6.1 3.05 6 .01030 .00337 2.49 7-27 3. 3721 25 .13488 4.6 2.25 5 .01020 . 00463 3.35 7-25 3. 4029 25 .13612 3.4 1.7 5 .01020 . 00600 4.41 7-25 3.4620 26 .13848 5.25 2.626 5 ,01020 .00388 2.80 8-17 3.4644 25 .13868 3.5 1.75 5 .01030 .00688 4.23 8-31 3. 4776 25 .13910 4.6 2.3 5 .01030 .00448 3.22 7-25 / 3. 6156 25 .14462 7.92 3.96 6.07 .01238 . 00312 2.15 8-17 3. 6394 25 .14558 3.6 1.8 6 .01030 .00572 3.92 8-17 / 3. 6971 25 .14788 4.2 2.1 5 .01030 . 00490 3.31 Average 8-18 / 3. 7164 25 .14866 5.2 2.6 5 .01030 .00396 2.66 . 13691 .00428 3.14 « Unless otherwise stated, total cc. of extract equals 50 ' Total extract, 25 cc. only. ' Total extract, 100 cc. <* Total extract, 30 cc. only. ' Just sealed, early. f Just sealed, still coiled. 148 Journal' of Agricultural Research voi. xxviii. No. 2 Table V. — Unassimilated sugar in intestinal content of larvx at different ages — Continued Larvae of knownage, Sturtevant (35^ Larvas analyzed for presence of unassimilated sugar (weights in grams) Age in days Aver- age weight Limits by weight for age groups Date Weight sample Num- ber of lar- vae Aver- age weight of 1 larva Ex- tract used Equiv- alent □umber of larva CuSO( solu- tion Equiv- alent dex- trose Dex- trose per larva Sugar per larva 6 Gram 0. 165005 Average 0. 141648 Average 0. 137165 Average 0. 133152 Gram. 0. 160876 to maximum and down to 0.148326. 19a 7-18 8-31 8-11 8-31 8-31 7-27 Grams ' 3. 8012 3.8038 3. 8925 « 3. 9783 4.1249 3. 7706 26 25 25 25 26 25 Gram 0. 16205 . 15215 . 15570 . 16913 . 16600 . 15082 cc. 18.05 6.4 4.35 6.0 7.0 6.65 4.51 2.7 2.175 3.0 3.5 3.33 «. 9.6 6 5 6 5 5 Gram 0. 19584 . 01030 .01030 . 01030 . 01030 .01020 Gram 0. 00434 .00381 .00473 .00343 .00294 .00306 P.ct. 2.85 2.50 3.03 2.15 1.78 2.03 .15581 . 00372 2.39 0. 148326 to 0. 139406. 7 8-18 8-10 8-10 8-4 8-18 3. 6980 3.6600 * 3. 5835 3. 5572 • 3. 4871 25 25 26 26 25 . 14792 . 14280 . 14334 . 14229 . 13948 7.3 8.9 11.5 7.4 45.0 3.66 4.45 5.75 3.7 2Z5 6 6 5 5 5 .01030 .01030 . 01030 .01030 . 01030 .00282 .00231 .00179 .00275 .00050 1.91 1.66 1.25 1.93 .36 . 14397 . 00203 1 40 0. 139406 to 0. 135158. S 7-26 7-18 3.4358 1 3. 4453 25 26 . 13743 ^ . 13781 50.0 50.0 25 25 5 6 No re- action. No re- action. . 13762 1 u fl 8-4 ' 3. 3232 25 .13293 50.0 26 6 No re- action. <: Total extract, 100 cc. « All sealed, coiled or with backs out. Feeding ended and spinning of cocoons started. * Cocoon partially spun, still some color in the intestine. i Cocoon not quite finished, still moving somewhat, no color in intestine. i Quiescent prepupae, intestines colorless, empty, histolysis started. • * First indication of change in external form. OBSERVATIONS Over 60 samples of 25 larvae each of various ages, containing over 1,600 indi- vidual larvae, were analyzed for the presence of reducing sugars. The largest number of analyses were made on larvae from 3i to 6J days of age during the active honey and poUen feeding period. At least five analyses were made of each of the other age periods which might show the presence of sugar. To obtain averages with a small probable error, the analyses are grouped by age periods of 24 hours each, as described earlier (Table V, fig. 13). All larva in the two- day group, as well as one sample of larvae nearly as heavy as the three-day aver- age larva, showed no reducing sugar. Larvae in the three-day group, averaging 0.043222 gm. in weight, gave 0.000475 gm. of reducing sugar per larva, or 0.98 per cent concentration. Larvae in the four-day group, averaging 0.10314 gm. in weight, gave 0.00299 gm. of reducing sugar per larva, or 2.82 per cent concentration. Larva; in the five-day group, comprising those just prior to seahng, with a few just sealed, averaging 0.13591 gm. in weight, gave 0.00428 gm. of reducing sugar per larva, or 3.14 per cent concentration. In the five-day group there were two samples which gave a concentration of over 4 per cent, the maximum being 4.41 per cent. The six-day group, comprised entirely of larva that had been sealed, had finished feeding and had started spinning, averaging 0.15581 gm. in weight, gave 0.00372 gm. of reducing sugar per larva, or 2.39 per cent concentration. This group contains larvae of maximum size (fig. 14). From Apr. 12, 1924 Development of American Foulhrood 149 this point on the gross weight decreases as preparation for metamorphosis begins. The seven-day group, comprising larvse which are still moving about in spinning, and most of which show only a slight remaining color in the intestines, indicating SO /■40 /20 \ N I /oo eo eo 40 20 \4 \ 1 // \ / / N ^ / i ^ *v^ // / ^ —— V 1 // 1 /C/VOW/V /tG£. : W£/0/fr OF i^/fM£ //V i sao^p. If i 1 i // ^i 1 1 / t I 1- i 1 1 / ^ 'I ^ \ 5 : i 3 4 £ 6 7 e 3 y^G£ /A/ O^I / X / --- '^--^ f/ ^ / "v. /v/Ty? OGe/^ ^- , / ^ s e 7 /o // Fig. 15. — Per cent composition of worker larvas at different ages (Tables IV and V) UTILIZATION OF GLYCOGEN According to Straus (Table IV, fig. 15) the greatest percentage of stored glycogen occurs just after sealing, when feeding has ceased. If an emulsion of the tissues of a larva of this age, or slightly older, at the age when prepupae usu- ally die of American foulbrood, is tested for the presence of glycogen with iodin solution,' the resulting deep reddish brown color shows that there are large amounts of glycogen present. If a prepupa which has ]'ust died from disease, sUmy in consistency, light brown in color, and which in the microscopic picture still shows the presence of vegetative rods, is tested with iodin solution, it will * Glycogen treated with iodin solution gives a color varying from brown to wine red, which disappears upon heating to 60° C, but returns again upon cooling. Soluble plant starch with iodin solution' gives the following reactions: Amylodeitrin, first dextrin of conversion, dark blue; erythrodextrin, second dex- trin of conversion, red; intermediate steps give various shades of purple or lavender. 152 Journal of Agricultural Besearch voi. xxviii, No. 2 be found that most of the glycogen has disappeared, although the iodin solu- tion gives a light yellowish brown color. The presence of a trace of reducing sugar also occasionally can be demonstrated with Benedict's solution in dis- eased material of this type where vegetative organisms are stiU actively present. In material which has decomposed completely, has reached the dark brown ropy stage (fig. 9), and contains only spores of Bacillus larvae, glycogen is found to be completelj' absent, nor can any reducing sugar be demonstrated, the sugars having been completely destroyed. This type of material stained with Sudan III or osmic acid {S^, p. 78) shows fat globules in practically the same condition and amount as in healthy larvae, so that fat is apparently not acted upon by Bacillus larvae even after drying down to the scale stage. Glycogen of the fat body of the healthy larva is hydrolyzed to dextrose to be used in metamorphosis, by the action of enzyms during the histolytic processes subsequent to sealing and prior to metamorphosis. This enzym action is demon- strated by the following e.xperiments: EXPERIMENTAL PBOCEDURE Several series of 50 healthy prepupae each that had reached the period of quiescence were macerated in 25 cubic centimeters of 50 per cent alcohol and incubated at 37° C. for from 3 to 24 hours. The extract was then filtered and diluted with an equal amount of water. A series .of test tubes were prepared, using for each tube 5 cubic centimeters of this extract and 5 cubic centimeters of 0.4 per cent glycogen in water, and also another series using 5 cubic centimeters each of a 0.1 per cent soluble starch. Both glycogen and starch were used, since it has been shown by Bradley and KeUersberger (S), as well as bj* experiments by the writer using commercial Taka-diastase, that diastase acts similarly on both glycogen and starch. These tubes were incubated for various periods and then tested with iodin solution for the presence of glycogen and starch (Table VI) . Hydrolysis of both glycogen and starch seems to be complete after incuba- tion for about five hours, and positively complete after incubation overnight, demonstrating the presence of diastase in the prepupae. In another experiment 50 prepupae were macerated in 50 cc. of water and in- cubated at 37° C. for 24 hours. Then sufiicient 95 per cent alcohol was added to precipitate any glycogen present, and the solution was filtered and tested with both the qualitative and the quantitative Benedict's solutions. In both cases definite traces of reducing sugar could be demonstrated, none having been present in the original solution before incubation, again demonstrating enzym activity of the larval tissues. This may have been due to action by bacterial contamina- tion, but if such had been the case the sugar would probably have been fermented and could not have been demonstrated. In a similar manner extracts with 50 per cent alcohol were made of ropy dis- eased material, enzym activity being demonstrated in the same manner as above. This, however, does not indicate whether the organism causing the disease has any diastatic power or whether the reaction was due to enzyms remaining in the decomposed tissues. Further extracts were made with 25 per cent and 50 per cent alcohol of several 48-hour vegetative cultures of Bacillus larvae grown on egg-yolk suspension medium. These extracts showed definite enzym activity with glycogen after a few hours' incubation, and more positive activity after incubation overnight (Table VI), while with starch marked hydrolysis was shown 'after only a few hours' incubation. Apr. 12, 1924 Development of American Foulbrood 153 Table VI. — Test for diasiaiic action with alcoholic extracf* Color with lodin after incubation of— Test material Ohour ihour 2i hours 6i hours 18 hours 5 CQ 1 3 CQ 1 5 M 3 CO 3 OQ 3 1 Extract of healthy pre- ++++ (brown) ++++ ++++ (blue) +++ ++ ++ ++ + + ++ + + ++ ± ± + Ertract of decomposed ropy remains. Extract of vegetative ++++ <* The following symbols are used: ++++ Deep color, brown or blue. +++ Slightly lighter brown than check or wine color. ++ Light coffee brown or lavender. + Trace faint brown or trace taint lavender. — No color or only iodln color, showing com. plete diastatic action. To further determine the production of diastase by Bacillus larvae, a series of Petri dishes were poured, using yeast-extract egg-yolk suspension agar, to which had been added respectively 0.25 per cent and 1 per cent of glycogen and 0.25 per cent and 1 per cent of starch, this being an adaptation from methods described by Vedder (45) and by AUen (i). After solidification of the media in the Petri dishes, smears were made upon the surface of the agar from 48-hour cultures of various previously isolated strains of Bacillus larvae. After several days the plates were examined, first by holding up to the light and then later by flooding with iodin solution, and comparing with control plates containing no starch or glycogen. In nearly all the plates good growth had occurred, causing clear areas to be pro- duced in the cloudy culture medium extending slightly beyond the edge of the area of growth. When flooded with iodin the halo around the culture growth, although not wide, was more prominently differentiated from the surrounding medium, showing in both glycogen and starch plates. These results, in con- junction with those of the extraction experiments, demonstrate that weak dia- static action is produced by Bacillus larvae. ACID PRODUCTION It has been shown that there is still an appreciable amount of sugar (reducing sugars in the food remaining in the intestines and dextrose available from gly- cogen) present in the larva after sealing and in the prepupa at the age when American foulbrood attacks, available for fermentation (Tables IV and V). In the various cultural investigations both by others and by the present writer, there is no evidence of carbon dioxid production. It would be expected, however, that at least some acid would be produced from the bacterial fermentation of these sugars, which is known to be present. To determine this more definitely than heretofore, a culture medium was devised for the qualitative determination of acid production, which gave good vigorous growth of Bacillus larvae. The method used Is an adaptation of the method of using agar slants for detect- ing acid formation, instead of liquid medium, described by Conn and Hucker {18) , in which the change in reaction can readily be seen. The regulation yeast- extract egg-yolk suspension agar was prepared for this purpose by adding to the yeast extract base before sterilization an indicator in the proper amount both to the plain medium and also to a portion to which was added 1 per cent of dextrose. 154 Journal of Agricultural Research voi. xxvin. No. 2 Brom thymol blue was first used, as it covers the range of the supposed optimum reaction for Bacillus larvx as described earlier. Baker (4) also has shown that brom thymol blue, used in about a 0.0024 per cent concentration in culture media, gives the most desirable color for comparison, without inhibiting acid fermentation. This concentration was obtained by using 12 cc. of a 0.2 per cent alcoholic solu- tion of the indicator per Uter. After marked acid production in the dextrose tubes was demonstrated with brom thymol blue, brom cresol purple was used as suggested by Conn and Hucker {18) in a 0.001 per cent concentration as a check on the end point. This concentration was obtained by using 8 cc. of a 0.2 per cent alcoholic solution of the indicator per liter. The yeast-extract base, both with and without dextrose, was adjusted so that after the addition of the egg- yolk suspension the final medium would have a primary reaction of approximately Ph=7.2, a definite blue grass green in the case of brom thymol blue and a marked purplish tinge with brom cresol purple, except in one series, where the primary reaction of the plain medium was Ph=7.6. These tubes after being slanted were inoculated as usual, both with vegetative cultures and with diseased material containing spores. The change in reaction was noted after different lengths of incubation, and the final reaction was determined by comparison with standard buffer tubes used in combination with tubes of plain egg-yolk suspension media slanted in the same manner. The approximate increase in hydrogen-ion con- centration was determined by this comparison (Table VII). Table VII. — Acid production by Bacillus larvae Brom thymol blue indicator Brom cresol purple indicator Culture No. Plain medium 1 per cent dextrose Plain medium 1 per cent dextrose Control Inocu- lated Control Inocu- lated Control Inocu- lated Control Inocu- lated 9693-1 Ph 7.6 7.2 7.6 6.8-7.0 7.2 7.6 7.6 7.6 Ph 6.8 6.6 •7.4 ±6.6 6.6-6.8 "7.4 "7.4 7.0-7.2 Ph 7.2 7.2 7.2 6.6 7.2 7.2 7.2 7.2 Ph 6.0 6.0 6.2 6.0 6.0-6.2 6.4 6.0 6.0 Ph Ph W Ph Ph S.8 9834-1 9834-2 (') (•) W 6.0 9863 9857 9867 i i 6.2 6.8 6.8 9869 9874 ' Doubtful growth. * Beyond end point, no growth. ' No change in color, good growth. '' Beyond end point, good growth. « No change in color, no growth. OBSERVATIONS Several interesting facts were observed from these experiments. Addition of buffer salts to the media delayed the approach to the final hydrogen-ion con- centration reaction somewhat, but eventuaUy practicaUy the same end point was reached. Also, in one series of media in which the plain medium was adjusted to about Ph=7.6, little if any growth occurred in these tubes except with two strains of Bacillus larvae, indicating that the alkaline limit for growth is about at this point. In cases where the initial reaction of the plain medium was Ph=7.2, the final reaction averaged Ph=6.6 to Ph=6.8 (Table VII). In the case of the medium to which 1 per cent dextrose had been added, the final reaction averaged about Ph=6.0 for brom thymol blue and from Ph=5.8 to Ph=6.0 for brom cresol purple (Table VII). WhUe, therefore, only a sUght change in reaction occurred in media without sugar, a marked production of Apr. 12, 1024 Development of American Foulbrood 155 acid was indicated in the tubes to which 1 per cent dextrose had been added. The maximum production of acid, however, required approximately 48 hours or more, the fermentation of the sugar apparently being relatively slow. As has been stated, however," the reaction of diseased material in various stages of decomposition and drying down is never found to reach a hydrogen-ion concen- tration of more than Ph =6.6, and usually averages Ph =6.8. PROTEIN DECOMPOSITION It is known that certain organisms have the ability to break down protein material under proper conditions, with the production of amino acids and alka- line decomposition substances, which latter tend to neutralize any acid produced from fermentation of sugar. If it can be shown that Bacillus larvae has this ability, it will explain the fact that the remains of larvje dead from American foul- brood do not show a greater acid reaction resulting from the fermentation of the sugar of the intestinal contents. A series of experiments was devised to demon- strate whether such is the case with Bacillus larvae. The prepupa at the age attacked by Americafi foulbrood contains nitrogenous substances as shown by the Kjeldahl nitrogen determination equivalent to 1.45 per cent nitrogen (4S) . The source of this nitrogen is mainly albuminous mate- rial, one of the constituents of the larval fat body. Its exact composition has not been determined, but without doubt it is complex in nature. There are certain color reaction tests by means of which the constitution of this nitrogenous material may be indicated. A delicate test for the presence of coagulable protein is that of Heller (3S, p. 1067). A suspension of healthy prepupse in water, treated by pouring about 4 cc. of concentrated nitric acid down the side of the inclined test tube, causes a white ring to form at the junction of the two liquids. Decomposed ropy material tested in this way gives no indication of such a ring, indicating that the complex protein has disappeared. One of the most characteristic reactions for complex protein is the biuret test (SS, p. 915). If some healthy prepupse are suspended in a few cubic centimeters of 10 per cent sodium hydroxid and are treated with a few drops of a 0.5 per cent copper sulphate solution, a distinct pinkish-violet color is formed, again indicating the presence of complex protein material. Decomposed ropy material tested in this way gives no indication of this color, again indicating the complete disappearance of the complex protein. There is also the xantho-proteic reaction (SS p. 916), which is given both by solid and by dissolved protein, and indicates the presence of the amino-acids, tryptophan, tyrosin, or phenylalanin in the protein molecule, or in solution. Tryptophan gives the reaction most intensely. Both healthy prepupse and ropy material, boiled with concentrated nitric acid, produce a lemon-yellow color which on cooling and neutralizing with sodium hydroxid changes to an orange, denoting a positive reaction. An even more delicate reaction for protein is that with Millon's solution (S2, p. 916). A few cubic centimeters of a suspension of healthy prepupae, treated with a few drops of Millon's reagent and boiled, cause a brick-red precipitate to form, leaving the liquid practically clear. A solution of decomposed ropy material, treated in the same way with Millon's reagent and boiled, causes a somewhat similar reddish precipitate, but the solution is also distinctly colored simDarly, indicating that the protein has been changed in some way, part at least being soluble in water. Tyrosin is the only amino acid in protein that gives this reaction. ]^ 5 6 Journal of Agricultural Research voi. xxviii, no. 2 Since tryptophan is probably one of the principal constituents of the protein molecule in the healthy prepupa as well as in solution in diseased material, certain tests were made to determine its presence, because this amino-acid is ' easily utilizable by bacteria and gives decomposition products indicating the nature of bacterial action. The following tests are specific tryptophan reactions: AdamUewicz reaction {SS, p. 917).— A suspension of healthy prepups or of diseased material in glacial acetic acid, treated by pouring concentrated sul- phuric acid down the side of the inclined tube, causes a violet ring to form at the junction of the two liquids, indicating the presence of tryptophan, either as part of the complex molecule or in solution. Rhodes reaction {41).—To a suspension of healthy prepup* or of diseased material in water, a few drops of a weak solution of dimethyaminobenzaldehyde is mixed and concentrated sulphuric acid poured down the side of the inclined tube. This produces a violet ring at the junction of the two liquids which, if shaken, produces a reddish violet coloration in the mixture. PROTEIN DECOMPOSITION PRODUCTS It is therefore evident that the composition of the nitrogenous material in the healthy prepupse is more or less complex but that certain amino-acids are avail- able for bacterial metabolism, or are produced as a result of bacterial action. In the decomposition of nitrogenous material, however, certain bacteria have the power of breaking down these amino-acids, such as tryptophan, to more simple compounds, some of them alkaline in nature, and often more or less foul smelling, or even to break them up into ammonia, the final product of nitrogenous decomposition. Indol is one of the products of such action of bacteria on material containing tryptophan. Its determination is largely used in the characterization Of various organisms (36). Two indol tests were used, Ehrlich's aldehyde test {19) and the vanillin test (19), using for both suspen- sions of diseased material as well as cultures. Test of suspensions of diseased material gave positive results for the presence of indol, both with the Ehrlich method and even more definitely with vanillin. For testing in pure culture a broth consisting of 2 per cent peptone, 10 per cent yeast extract, and a few cubic centimeters of egg-yolk suspension was inoculated, incubating at 37° C. for about one week. Growth took place in this broth sufliciently to give a slight positive pink color with the Ehrlich aldehyde test, increasing on standing, and a much more positive result with the vanillin test. AMMONIA PRODUCTION Test of a suspension of diseased material as well as some of the above culture broth with Nessler's reagent (SS, p. 1084) for presence of ammonia gave indica- tions, from the resulting slight production of characteristic yellowish color, that the decomposition had passed even to the ammonia stage. A more delicate qualitative test was devised, using the modification of the microchemical method of FoMn and McCaUura (3S, p. 1093) for the determination of urinary ammonia as described by Steel {42) . To 25 cc. of a suspension of diseased material, or to broth culture similar to the above, 1 gram of sodium hydroxid and 15 grams of sodium ohlorid are added and ammonia-free air bubbled through into 20 cc. of an approximately N/20 sulphuric acid, to which 10 drops of the indicator thymol blue are added. This showed the sulphuric acid solution to have a primary hydrogen-ion concentration of about Ph=2. After bubbling air through for an hour or more, in the case of the decomposed ropy material, sufficient ammonia had been carried over to neutralize part of the acid and change the hydrogen-ion concentration reaction from Ph=2 to Ph=2.8 or 3. Also one culture out of Apr. 12, 1924 Development of American Foulbvood 157 three showed a change from Ph=2 to Ph=2.8. Therefore apparently Bacillus larvae has the ability of producing at least small amounts of ammonia. It seems probable that the rather pungent volatile gluelike odor often associated with American foulbrood receives some of its characteristics from this ammonia as well as from certain of the protein digestion products. GELATINE LIQTJEPACTION The ability of putrefactive bacteria to liquefy gelatin is difficult to demonstrate with Bacillus larvae because of the cultural limitations. Maasen states i^8) that slow liqueflcation takes place, while White (SB) was unable to demonstrate any growth in gelatin. The writer inoculated a number of tubes of plain gelatin with several strains of Bacillus larvss, all of which showed slight growth, and one or two showed a slight softening of the gelatin about the culture growth. Tubes of gelatin to which some egg-yolk suspension was added showed this softening more markedly, but in no case was there sufficient liqueflcation to enable one to say that it was positive. Decomposed ropy material inoculated into plain gelatin, on the other hand, gives a marked liquefaction in a short time. This, however, probably is due not to enzyms produced by Bacillus larvae so much as to enzyms from the body tissues functioning in the histolysis previous to meta- morphosis. This series of experiments, however, demonstrates that sufficient alkaline decomposition products are formed by the action of Bacillus larvae in the prepupa to neutralize most of the acid formed by the fermentation of the sugar in the intestinal contents and the dextrose resulting from the hydrolysis of the stored glycogen. DISCUSSION PER CENT CONCENTRATION OP SUGAR In the data presented it may be seen that there is not an exact correlation be- tween the percentage of dextrose which inhibits the germination or prevents the growth of Bacillus larvae, and the percentage of unassimilated sugar in the larva as expressed. The reason for this is that the percentage of unassimilated sugar is calculated in relation to the entire weight of the larva, like the figures of Straus (45) on the percentage composition of the larva (fig. 15) . The percentage of dex- trose in the culture media gives the actual effective concentration of the sugar in the medium by weight. Since the unassimilated sugar is contained almost en- tirely in the intestine from which it is absorbed, the true concentration of sugar in the intestine should be determined in relation to the weight of the intestinal con- tent. Furthermore, as suggested by Maassen {^8), growth of Bacillus larvae occurs only inside the intestine until after the histolysis has begun, making pos- sible the invasion of the body tissues by the organisms. It is therefore in the in- testinal contents during the last part of the feeding period that the presence of sugar is primarily effective in inhibiting the growth of the organisms. The actual concentration of sugar in the intestine is, however, difficult to determine accu- rately, since the actual weight of food consumed by the larva for each 24 hours of the feeding period is unknown. Furthermore, the weight of the intestinal content is difficult to determine, because of the difficulty of dissecting the intestine free from the surrounding body tissues or of removing the contents intact. Several attempts were made, however, to remove intestines with as little adher- ing tissue as possible from larvae of different sizes during the last two days prior to seaUng, in order to obtain an approximately accurate figure for the relation be- tween the weight of the intestine and the weight of the larva. This in the several larvse dissected was found to be almost always about 1 to 5. Using this factor, 158 Journal of Agricultural Research voi. xxvm, No. 2 the percentage concentration in tlie intestine, at least during the progressive feed- ing period, should be approximately five times as great as the value calculated (in Table V) on the basis of the entire larval weight. The calculated percentages for the third and fourth days are now 4.90 and 14.10, respectively, and on the fifth day, just before sealing, the sugar concentration in the intestine should ap- proximate 15.70 per cent (fig. 16). There is, of course, the factor of dilution MPPA0X/M/^7£ PE/Z/OD ^pp/^x.f¥fuoP ^fip/?ox.P£fi^oz? /j/VKOx/flf>^r£/^£K/o/f arAf£y/t' ap cocoon SP/^- OPQi//£SC£^^C£ MOPPffQS/S,S//0*IWSi^£yy>t/^i?£S N/NO /INOMASTOCy^a m£Xr£flN/U.PO/lA^,£XT£A/D//fa r/ssu£s WG rpo/i^m££se. S 6 7 AG£/NOArS Fig. 16.-Correlation of time of death from American foulbrood to per cent concentration of reducing sugar m entire larva and to calculated per cent concentration in the intestine. Per cent sugar in the food and per cent glycogen in the larva are shown for comparison particularly toward the end of the feeding period, caused by the accumulation of undigested pollen shells, which may lower this figure somewhat. Still another approximate check may be calculated from the molecular weights of dextrose, glycogen, and fat, and from the percentage composition of the constitu- ents of the larva (Tables IV and V), in order to obtain the percentage of sugar which was present in the intestine at any one time previous to assimilation neces- Apr. 12, 1924 Development of American Foulhrood 159 sary for the formation of the stored glycogen and fat, on the basis of the relation of their carbon atoms. Molec- For- ulor inula weight Equivalent sugar per cent Dextrose (unassimilated) CoHi20e = 180 *3. 14 Glycogen CJi,o05 = 162 ^^°~^^^ X5. 57+5. 57= 6.13 loU Fat (oleic acid)... ..CsHmOj =282 ^^sxiSO^^ '^'^' ^^'^^' ^^'^ ^' ^^ Total 14.65 Since more sugar is used for energy in the production of a molecule of glycogen or of a molecule of fat (SS, p. 77) than is indicated by the actual relation of the carbon atoms, this figure should be somewhat higher, thereby more nearly corresponding with the figure calculated from the weight ratio between in- testine and larva. The average concentration of sugar in the food of a larva of the age during which inhibition of bacterial growth takes place is 13.48 per cent (.40) (Table III, fig. 11). The percentage composition of the larva at the different age periods (Table IV, fig. 15) in relation to food composition indicates, however, that the food probably is not assimilated as rapidly as it is ingested by the larva. If this is the case, an increase in the unassimilated sugar in the intestine would occur, as is indicated by the results obtained and the calculated percentage figures. The percentage of glycogen and fat increases slightly by the third day, as a result of the change in the composition of the food and the resulting increase in nursing {S6) . This is accompanied by the appearance of unassimilated sugar in the larva (Table V, fig. 15). There is then a marked increase by the fourth day, when apparently the limit for assimilation is reached, as shown by the constant percentage of glycogen in the larva as a whole between the fourth and fifth days in spite of the increase in weight, until after feeding ceases, when theie is another increase in storage during the next 24 hours (sixth day) . The latter is the result of the consumption of the food remaining in the cell after capping. The amount of unassimilated sugar increases continually, however, until feeding ceases, soon after the larva is sealed in the cell. This fact probably accounts for the slight difference between the percentage of sugar in the food and the calculated per- centage in the intestine, but the correspondence is so striking as to substantiate the assumption. Even though these calculations are only approximately accurate, it is known that some such condition must exist, since observations which have been made on the nursing habits of the honeybee (^6), considered in relation to the figures for unassimilated sugar and food composition, give adequate foundation to the conclusion that there is considerably more than enough sugar in the intestine at the time infection occurs, or soon after, to inhibit the growth of the organism causing American foulbrood. THE ACTIVE FEEDING STAGE IN THE LIFE HISTORY OF THE LARVA The feeding stage of the honeybee larva has been divided into two parts, de- scribed by Lineburg {S6) as the mass feeding period and the progressive feeding period. These two periods are characterized by a difference in the manner of feed- ing and in the amount of time spent by the nurse bees in the process, as well as by the change in chemical composition and character of the food {40) (Table III) , and by the chemical composition of the larvae themselves {4$) .(Table IV). It has been determined, however, that this change in composition of food occurs ' From Table V. ' From Table IV, Straus. 160 Journal of Agricultural Research voi. xxvm, No. 2 much earlier than stated by Von Planta (35, 26). The young larvae receive a food rich in nitrogenous material and relatively low in sugar and in which pollen grains are absent during about the first two and a half days of larval Ufe. A large part of this, which is several times in excess of the weight of the larva during the first 24 hours or more {SB) (Table VIII), seems to be placed in the ceU with the newly hatched larva at one time soon after hatching, which justifies the assumption of mass feeding. During this period, assimilation must be very rapid, because the greatest relative growth occurs during the first two to three days (Table VIII) and also because no unassimilated sugar can be demonstrated in the larva during this period, even though the food contains about 5 per cent reducing sugar (Table V) . The high nitrogenous content of the food apparently serves for rapid cell building, while the sugar is largely consumed in producing energy for this rapid growth; little storage of glycogen or fat occurs during this period. The nature and composition, as well as the biochemical reactions of this early food, as described by Koehler {26) , suggest that it is a glandular secre- tion rather than a regurgitation of predigested honey and poUen from the ven- triculus. The chances of larvae of this age during mass feeding receiving infective material are, therefore, slight. Table VIII. — Ratio of weight in milligrams of unconsumed food in cell to weight of larva at different approximate ages {from Sturtevant {SB)) Approximate age (days) Number of ob- serva- tions Average weight of food per cell Average weight of larvae Average ratio, food to larva Egg _ Several. 33 131 24 36 25 65 17 MilU- grams None. 3.96 3.23 9.10 11.79 6.06 7.76 8.76 MiUi- grams OO.IO 1.02 1.36 7.20 17.48 25.22 63.46 116. 16 1.1 .._ 1.4 _ 2 37 2.1 _ .._ 2.6 _ _. 3.0 _ 20 35 12 4.4 " From Nelson {SS), Soon after the second day, a change in the composition of the food of the larva occurs, accompanied by a change in the method of feeding it by the nurse bees {26) . The larva is now fed at approximately the rate at which the food is ingested by it, the demand for food rapidly increasing, accompanied by the great increase in actual body weight, until the time of sealing. The food now contains many entire pollen grains and has a much higher sugar content, nearly 14 per cent, and a relatively lower nitrogenous content (Table III). The principal ingredi- ents are now honey or nectar and pollen. It is well known that honey which is gathered while disease is present in the hive usually carries infection. There is, therefore, a much greater opportunity for infection to occur when the larvae are being given a food containing unmodified honey as one of its chief ingredients. Furthermore, the constant care of the larvae during the period of progressive feeding and the large number of nurse bees which visit the cell still further increase the chances of infection being introduced during the period of progressive feeding. It is the young bees in the colony which act as nurses, and these bees are also the ones which clean the hive, so that they are more apt to have infected material on their mouth parts and elsewhere than are old field workers. There can, therefore, be little doubt that it is almost exclusively during the period of pro- gressive feeding that infection normally occurs. Under normal feeding condi- tions the disease organisms can not develop in the larval intestine until after Apr. 12, 1924 Development of American Foulbrood 161 feeding has ceased and the sugar-containing food has largely been assimilated, because, with the rate at which the larva is given food containing about 14 per cent sugar, the sugar concentration in the intestine must rapidly increase beyond the 3 to 4 per cent concentration which inhibits growth, so as to prevent the development of the disease in the larva. From Tables IX and X it may be seen that from the second day on, increasing numbers of visits are made to the larva with an increasing amount of time spent in nursing (Table X), so that on the last day of feeding a nursing visit, averaging six seconds in duration, is made approximately every 30 seconds, or an average total during the last 24 hours of 2,855 visits, or 36 per cent of the average 7,858 visits made during the entire feeding period (Table IX). The rapid consumption of this great amount of food, which is supplied in almost a steady stream, is indicated by the fact that the amount of food in the cell with the larva decreases to less than 10 per cent of the weight of the larva just prior to sealing. Even though the exact amount of food consumed may not be known, the actual concentration of sugar in the intestine during this period must rapidly rise, because of the high concen- tration of sugar in the food and the volume at which it apparently is fed to the larva, to several times the concentration necessary to inhibit the growth of Bacillus larvae. There is, as mentioned above, a diluting factor due to the accumulation of undigestible material until the connection is made with the end intestine, but, as shown by the microscopical examination of the intestinal contents, this, because it is largely insoluble, is probably of relatively slight importance in its effect upon the actual sugar concentration in the intestine during the period when inhibition occurs. Table IX. — Relation of nursing to increase in weight of larva "■ Average weight of larvaB Average daily increase Average daily ratio of increase to pre- ceding weight During active feeding period Age (days) Number of visits in 24 hours Average increase in weight per visit per 24 hours Gram 0.000100 .000650 .00474S .024626 .09399a .148748 . l.WOOfl Qram Oram 1 . 0.000560 .004096 .019881 .069364 .052768 .008257 5.5 6.3 4.19 2.82 .66 .06 921. e- 833.8 1163.5 2083,7 2865 5 0.000000597 2 .00000492 3 -- .00001709 4... .00003329 5 .00001848 6 " From Sturtevant (SJ) and Linehurg (2S). Table X. — Time spent in feeding " Age (days) Average time spent in nursing per visit per 10 minute period Average number of visits per 10 minute period Average number of seconds per visit Average frequency of visits Average number of seconds between visits 1 _ Seconds 2a 73 9.93 11.42 41.00 118.08 6.40 6.79 8.08 14.47 19.83 Seconds 3.2 ■ 1.0 1.4 2.8 5.9 Seconds 93.7 103.6 74.3 4L5 30.3 Seconds 90.5 2 102 6 3 72.9 4 38.7 5 24.4 " Calculated from Lineburg {IS). 162 Journal of Agricultural Research voi. xxvm, no. 2 Since it is shown that the concentration of reducing sugars in the larval intes- tine is usually sufficient to inhibit the growth of Bacillus larvae and thus to prevent the manifestation of American foulbrood until after sealing, it is now necessary to explain the rare cases in which advanced stages of the disease are seen in younger coiled larvse. Such cases are exceedingly rare, except in colonies where almost every cell in the brood combs is filled with a dried scale, and where the bees have deserted the brood-nest because of this diseased material. There can be no doubt that in these cases the earlier manifestation of the disease is due to the fact that in such colonies the progressive feeding of the larvse is ser- iously reduced by the fact that the colony has already been depleted in numbers of adult bees. Since there must be a decrease in progressive feeding in such cases, the concentration of reducing sugars in the intestine of the larva is obviously reduced, causing a condition to exist in these intestinal tracts which- no longer inhibits the germination and growth of the causative organism. Such rare cases of young larvse dead of American foulbrood do not, therefore, disprove the theory regarding the time of the development of the disease which has been here set forth, but rather serve as further substantiation of it. THE COCOON-SPINNING STAGE Sealing usually takes place on about the fifth day, at which time apparently the intestine contains a maximum amount of unassimilated sugar. After sealing occurs and feeding ceases, a different set of factors influence the concentration of sugar in the intestinal contents, so that there is a rapid steady decrease from this time on. The storage of glycogen and fat, however, continues for a short while from the assimilation of reducing sugar. Soon the movements of cocoon spinning and the histolysis of tissues make necessary the utilization of energy stored in form of glycogen and fat, so that the percentage of these substances begins to decrease as the larva loses weight. The emptying of the intestine of fecal material during this period also tends to decrease the sugar in the intes- tine, so that by the time the cocoon is finished, some time between the seventh and eighth days, the intestine is empty. The larva has straightened out and become quiescent by the eighth day, and all remaining sugar has now been assimilated. It is during this period that, as the concentration of sugar decreases, a point is reached where the growth of the organism can proceed. This appar- ently occurs when the sugar concentration in the intestine has decreased to about 3 to 4 per cent or less, probably not until some time between the sixth and seventh days. In the cultural experunents it was found that with 2 per cent or less dextrose, vigorous vegetative growth occurs. This vigorous growth requires, however, from 24 to 48 hours to develop, depending somewhat on the amount of initial inoculum. It is probable, as suggested by observations of Herman and Rettger (6), that this sugar in the food furnishes the energy for vegetative growth, while the soluble nitrogenous constituents of the intestinal contents furnish material for ceU metabolism, until the time when the organisms have increased in number sufficiently to cause death and are able to invade the tissues of the larva, causing their subsequent decomposition. THE QUIESCENT STAGE As also is known from long observations of symptoms, the death of the larva and invasion of the body tissues do not in the majority of cases take place until after the larva has at least reached the age of 8 days and has become quiescent. This fact explains the characteristic uniformity of position and appearance of the majority of the larvse dead from American foulbrood (figs. 7, 8, and 9). There are occasional cases in which death is still further delayed for some reason Apr. 12, 1924 D&velopment of American Foulbvood 163 until external transformation in form has begun, so that after death the pupa tongue is seen extended and often attached to the upper side wall of the cell in a characteristic manner (fig. 17.) It is possible that in such cases the initial inoculum was smaller than the average, thereby retarding maximum growth of the organism, as was noted in the cultural experiments, and delaying the production of sufficient toxin to kill until this stage of development had been reached. While the biochemical relations of the bee larva to the disease are seemingly quite adequate to explain the delay in the time when American, foulbrood is manifest, there is one other consideration which should be mentioned. The abUity of the larva to resist the invasion of the bacteria is a subject on which virtually nothing is known, yet there must be some such ability, as is suggested by the fact that a slight initial inoculum in a colony may not cause the disease to be manifest. At the time when the biochemical conditions are most favorable for the germination and growth of the invading organisms, the larva itself has reached that stage of its development ^X^'^V when its internal structure is materially modified by .-^'^BH^fc^. histolysis in advance of pupation, and it must follow almost necessarily that its power of resistance is reduced. The extent to which this factor is involved is, for the time being, purely a matter of speculation. The variation in the content of reducing sugar of the healthy honeybee larva and the inhibition of the germi- nation and growth of Bacillus larvae by a concentration of over 3 or 4 per cent yield an interesting fact concerning the other serious disease of the brood of bees. European ^'°- i^-Decomposed, dried *^ down remains of pupa foulbrood makes its attack on the bee larva at an earlier ^^^^ {^qj^ American foul- stage in its development, while the content of reducing brood, showing character- sugars is still high. It must, therefore, be concluded that istic tongue attachment to Bacillus pluton has the ability to grow and rapidly to ^^^^' ''*" "' ™" '^^^"^ produce toxic substances sufficient to kill the larva in a medium of much higher reducing-sugar content than has Bacillus larvae, probably as high as 15 per cent. THE EFFECTS OF BACTERIAL METABOLISM IN THE LAEVA There is little unanimity of opinion concerning the effect of dextrose (glucose) upon nitrogen metabolism by various bacteria. Kendall and Walker (2^) con- cluded that the presence of glucose in the medium delays the production of proteolytic enzym, indicating the "protein-sparing" action of carbohydrates. Fischer {SS) believed that proteolytic enzym is inactivated by glucose, indicated by inhibition of indol formation. Berman and Rettger (6) state that the presence of a carbohydrate in a culture medium may inhibit protein metabolism, depending on the nature of the medium and on the type of the organism as related to hydro- gen-ion concentration. DeBord (SO) believes that some bacteria destroy glucose without marked increase in the hydrogen-ion concentration and that the rate of production of amino nitrogen or ammonia nitrogen, which may be affected by the presence of carbohydrates, indicates different types of metabolism of bacteria. The results of the present investigation, although more or less incomplete on this subject, seem to indicate that Bacillus larvae has the abiUty to decompose nitrogenous material in the presence of carbohydrate, since there must be dextrose available until the stored glycogen of the fat body is entirely hydrolyzed. Many organisms are unable to attack complex protein unless there is some other source of food present, because, according to Berman and Rettger (6), to be 164 Journal of Agricultural Research voi. xxvm, No. 2 utilizable by bacteria sufficient growth is necessary by which to produce the enzym capable of splitting the complex protein molecule into its simpler amino- acid forms. In the healthy larva at the age when Bacillus larvae starts growth in the intestine, Mie remaining sugar in the intestinal contents and other material of nitrogenous nature in the food is sufficient, as stated earlier, to produce the energy for the initial growth of the organism. By the time the invasion of the tissues by the organisms occurs sufficient proteolytic enzym has been produced to attack the body proteins. Furthermore, the process of histolysis itself, as stated in relation to gelatin liquefaction by ropy material, has probably broken up sufficient of the body proteins to serve as food for bacterial metabolism. Since Bacillus larvae belongs to the spore-forming group of organisms, its processes of metabolism may be similar to those of Bacillus subtilis, as described by Berman and Rettger (6), as follows: "The ability of Bacillus subtilis to break down protein in the presence of fermentable sugar, and in the absence of an added buflfer, may be explained as follows. This organism attacks glucose slowly, and for this reason it is able to produce its proteolytic enzym before the hydrogen- ion concentration reaches a point unfavorable to further growth. When the enzym is thus formed the products of the nitrogen metaboUsm neutralize the acid, at least in a measure, and the metabolism therefore continues uninter- ruptedly." The fact that the production of indol and even of ammonia can be demon- strated, although they may be produced slowly and in small amounts, indicates that even though considerable acid may be produced by the fermentation of the carbohydrate in the food and the hydrolysis of the glycogen. Bacillus larvae has putrefactive functions which bring about the formation of sufficient alkaline protein-digestion products from the larval tissues to neutralize this acid produc- tion, thereby maintaining the hydrogen-ion concentration at approximately Ph=6.8. SUMMARY AND CONCLUSIONS 1. It has been shown by the work of others that the glycogen and fat content of the bee larva increases in a definite manner for a time and then decreases. In the present work it is shown that the per cent and amount of reducing sugar likewise increase after the third day of larval feeding, but decrease rapidly im- mediately after feeding has ceased, until by the eighth day no reducing sugar remains in the larva. 2: The presence of reducing sugar can be determined only after the progressive feeding of the larva begins. 3. The best method for extracting reducing sugars from the bee larva was found to be by the use of 50 per cent alcohol and the removal by filtration of the insoluble materials. By this means the interference of glycogen with the action of the reducing solutions is prevented. 4. The constitution of the protein molecule of the normal tissue content of the healthy bee larva is complex, containing, however, among other amino-acids, tryptophan. 5. The best medium for the growth of Bacillus larvae so far devised is a yeast- extract agar medium to which sterile egg-yolk suspension has been added. The optimum reaction for cultural growth is Ph=6.8. 6. Reducing sugars in the culture medium of more than 3 or 4 per cent usually inhibit germination of spores and growth of Bacillus larvae. A few spores may germinate at higher concentrations, but the resulting vegetative forms fail to increase in number and show granular disintegration due to autolysis. Less than 2 per cent of reducing sugars seems to stimulate growth of the vegetative forms. Apr. 12,1924 Development of American Foulbrood 165 7. The food of the older honeybee larva contains a high percentage of reducing sugar, which is derived from the honey or nectar used in its production. The concentration of reducing sugar in the larval intestine is more than sufficient to inhibit the growth of Bacillus larvae until after feeding has ceased. After feed- ing ceases, the remaining reducing sugar is rapidly assimilated, so that by the seventh day the concentration of sugar has been reduced sufficiently for the active growth of Bacillus larvae to occur. 8. The incubation period of Bacillus larvae is 24 to 48 hours, so that growth sufficient to kill the larva does not occur until it has completed the spinning of its cocoon and has extended quiescent in the cell, on or after the eighth day, by which time all reducing sugar has disappeared from the larva. 9. The delayed death of the larva in American foulbrood is, therefore, corre- lated with the inhibiting effect of unassimilated reducing sugar in the intestine upon the germination and growth of Bacillus larvae. 10. Bacillus larvae has the ability to produce considerable acid, but the hydro- gen-ion concentration of the decomposing material is not thereby increased, because of the neutralizing effect of protein decomposition products. The hydrogen-ion concentration of the diseased larva throughout its decay varies only slightly from Ph = 6.8. * 11. Bacillus larvae not only utiUzes reducing sugar for its initial growth, but also completely hydrolyzes the glycogen of the larval body tissues in the process of decomposition. 12. Bacillus larvae has the ability to decompose nitrogenous materials, with the formation of amino-acids, indol, and ammonia, but the hydrogen-ion con- centration is not decreased by this action, because of the concomitant pro- duction of acids from carbohydrates. 13. Bacillus larvae apparently has no action on fat. 14. The biochemical data herein presented for the first time explain the remarkable characteristics of American foulbrood, which were left entirely unexplained from observations on etiology alone. LITERATURE CITED (1) Allen, P. W. 1918. A SIMPLE METHOD FOR THE CLASSIFICATION OF BACTERIA AS TO DIASTASE PRODUCTION. Jour. Bact. 3: 15-17, illus. (2) Ayers, S. H., and Rupp, P. 1919. EXTRACTS OF PURE DRY YEAST FOR CULTURE MEDIA. Jour. BaCt. 5: 89-98. (3) MuDGB, C. S., and Rupp, P. 1920. THE USE OF WASHED AGAR IN CULTURE MEDIA. Jour. BaCt. 5: 589-596. (4) Baker, H. R. 1922. substitution of brom-thymol-blue for litmus in routine LABORATORY WORK. Jour. Bact. 7: 301-305. (5) Benedict, S. R. 1911. A METHOD FOR THE ESTIMATION OF REDUCING SUGARS. JoUr. Biol. Chem. 9: 57-59. (6) Bbrman, N., and Rbttgbr, L. F. 1918. THE INFLUENCE OF CARBOHYDRATE ON THE NITROGEN METABOLISM OF BACTERIA. Jour. Bact. 3: 389-402. (7) Bishop, G. H. 1922. cell metabolism in the insect fat-body. i. cytologicai changes accompanying growth and histolysis of the pat- BODT OF APIS MELLiFicA. Jour. Morph. 36: 567-601. 166 J ourinMl of Agricultural Research voi. xxvin, No. 2 (8) Bhadlet, H. C, and Kellersbbbger, E. 1913. THE PROBLEM OP ENZYME SYNTHESIS. II. DIASTASE AND GLYCOGEN OP ANIMAL TISSUES. Jour. Biol. Chcm. 13: 419-423. (9) Browne, C. A. 1908. 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HEMMUNG DEE INDOLBILDUNG BEl BACTEEIUM COLI IN KULTUBBN MIT zucKBRzusATz. Biochem. Ztsehr. 70: 105-118. Apr. 12, 1924 Development of American Foulbrood 167 (24) Kendall, A. I., and Walker, A. W. 1915. OBSERVATIONS ON THE PROTEOLYTIC ENZYME OP BACILLUS PROTEUS. Jour. Infect. Diseases 17: 442-463. (25) KOEHLBR, A. 1922. NBUE UNTBRSUCHUNQEN tjBER DEN PUTTERSAPT DBR BIBNEN. Verhandl. Deut. Zool. Gesell. 27: 105-107. 26) LlNEBURQ, B. 1924. THE FEEDING OP HONEYBEE LARVAE. U. S. Dept. Agr. Bul. 1222: 25-37, illus. (27) Maassen, A. 1906. paulbrutseuchb der bibnen. Mitt. K. Biol. Anst. Land- u. Forstw. 2: 28-29. (28) 1908. ZUR ATIOLOQIB DER SOQENANNTBN PAULBRUT DEE HONIQBIENEN. Arb. K. Biol. Anst. Land- u. Forstw. 6: 63-70, illus. (29) 1913. WEITERE MITTEILUNQEN tjBER DIB SBUCHBNHAPTEN BRUTKRANK- HBITEN DER BIBNEN, INSBBSONDERB TJbER DIB PAULBRUT. Mitt. K. Biol. Anst. Land- u. Forstw. 14: 48-68. (30) McCray, a. H. 1917. SPORE-PORMING BACTERIA OP THE APIARY. Jour. Agr. Research 8: 399-420, illus. (31) and White, G. F. 1918. the diagnosis op bee diseases by laboratory methods. U. S. Dept. Agr. Bul. 671, 15 p., illus. (32) Mathews, A. P. 1920. physiological chemistry. Ed. 3, 1154 p., illus. New York. (33)lMooRE, V. A., and White. G. F. 1903. A preliminary investigation into the cause op the inpectious^ bee disease prevailing in the state op new YORK. N. Y. state Dept. Agr. Ann. Rpt. Com. (1902) 10: 255-260, iUus. (34) Myers, V. C, and Fine, M. S. 1913. essentials op pathological chemistry, including description OP THE chemical METHODS EMPLOYED IN MEDICAL DIAGNOSIS. 137 p. New York. (35) Nelson, J. A., and Sturtevant, A. P. 1924. the rate OP growth op the honeybee larva. U. S. Dept. Agr. Bul. 1222: 1-24, illus. (36) Norton, J. F., and Sawyer, M. V. 1921. iNDOL PRODUCTION BY BACTERIA. Jour. Bact. 6: 471-478. (37) Phillips, E. F. 1906. THE BROOD DISEASES OP BEES. U. S. Dept. Agr., Bur. Ent. Circ. 79,. 5 p. (38) 1918. THE CONTROL OP EUROPEAN pouLBROOD. U. 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EXPLANATION POR SOME OP THE CON- FUSING SYMPTOMS IN THE GROSS DIAGNOSIS OP BEE DISEASES. Gleanings Bee Cult. 50: 298-302, illus. (47) 1923. LES MALADIES DU COUVAIN D'aBEILLES TELLES QU'oN LES CONNAIT Aux etats-unis. Cong. Internat. d'Apioulture Marseille (1922) 6: 168-177. (48) Vedder, E. B. 1915. STARCH AGAR, A USEFUL CULTURE MEDIUM. Jour. Infect. Diseases 16: 385-388. (49) White, G. F. 1904. the further investigation op the diseases affecting the APIARIES IN THE STATE OP NEW YORK. N. Y. State Dept. Agr. Ann. Rpt. Com. (1903) 11: 103-114. (50) (51) (52) 1906. THE BACTERIA OP THE APIARY, WITH SPECIAL REFERENCE TO BEE DISEASES. U. S. Dept. Agr., Bur. Ent. Tech. Ser. 14, 50 p. 1907. THE CAUSE OP AMERICAN FOUL BROOD. U. S. Dept. AgT., Bur. Ent. Circ. 94, 4 p. 1912. THE CAUSE OP EUROPEAN POULBROOD. TJ. S. Dept. Agr., Bur. Ent. Circ. 157, 15 p., illus. 1917. SACBROOD. U. S. Dept. Agr. Bul. 431, 54 p., illus. 1919. UNHEATED EGG YOLK MEDIA. Science 49: 362. 1920. AMERICAN POULBROOD. U. S. Dept. Agr. Bul. 809, 46 p., illus. (53) - (54) - (55) - (56) - 1920. EUROPEAN POULBROOD. U. S. Dept. Agr. Bul. 810, 39 p., illus. (57) Zander, E. 1921. handbuch der bienbnkundb in EINZELDARSTELLUNGEN. IV. DAS lbben dbr bibne. Aufl. 2, 195 p., illus. Stuttgart. O > 3 1 °3 U12J 11 s n ■Rfe 1 h 3 °1 11 a p I"* bo a -1 i^ 1 a 1 s l|^ 11 3 cd S CI rst com ing op growth cultures M o CD Is tn z 'A 'A u u '^ f^ 'A o O o f^ C.f. Ce. n.e. C.C lO.R 80 20 4 4 u » 1 4 4 n s 80 ?0 4 4 u » 1 4 2 2 1 80 ?o 5 6 u 5 6 1 4 Third. 1 80 so 6 b u 4 1 J 5 2 3 fl. Do. Half-normal sodium 1 80 20 1 6 6 6 6 B .0 oleate. 1 75 •m R 6 « u 6 ft fi n 1 80 m 1 U » u 8 1 9 M 8 Second 1 80 90 1 6 5 5 5 2 3 First. 2 80 20 2 U « 8 1 y 2- 7 Sixth. >2 80 20 1 6 6 u 6 .6 4 2 FiKt. 1 80 20 1 9 9 9 1 8 2 80 20 2 9 U u 9 9 '2 7 Third. 1 80 20 1 2 2 u 2 2 d,i 1 Second. 1 SO 20 1 4 4 u 3 1 4 "2 1 1 First. 1 80 ?fl 1 7 '7 7 7 4 a Second. 10 per cent solution of hard toilet soap. 2 2 2 76 75 SO 25 26 20 2 4 4 6 4 4 «6 c 4 4 6 4 4 '6 3 1 1 B First. Sebond. 2 80 20 -2 fr '6 u ,6 '6 e n s 90 10 4 4 4 4 1 3 First. 1 00 10 4 4 4 4 M 3 Fourth. las 85 16 4 4 »2 2 First. 4 2 2 First. ' Few spares germinated. ' Cottonseed^U soap. • One showed few spores germinated. •Used entire brood combs in tank. ■ Sealed cells with cappings perforated compared with open cells. In the case of the solutions of hard toilet soap in 20 per cent formalin, 39 open and 39 sealed cells were cultured. Cultures from 38 open cells showed no growth, and the culture from 1 was contam- inated. From the sealed cells, 22 cultures showed no growth^ 1 was contaminated, 13 showed good growths of Bacillus larvae, and in 3 only a few spores had germinated; in all, there were 16 posi- tive cultures, showing that 41 per cent of the sealed cells were not sterilized. The variation in the quantity of soap added to the solution seemed to have little effect on the variable penetration and sterilization of the scales in sealed cells, but the hard-soap solutions appeared somewhat less efficient than the solutions of soft soap. Three short series (see bottom of Table 1) were tried, using smaller quantities of formalin, but these solutions proved inefficient as germicides in open as well as in sealed ceUs. 21137°— 26 2 10 Department Circular 284, U. S. Dept. of Agriculture UQUIDS OF LOW SURFACE TENSION OTHER THAN ETHYL ALCOHOL The results of tests with these liquids are presented in Table 2. Acetone gave indications of being a rather more efficient carrier than those previously used, both in the proportions of 80 parts acetone to 20 parts formalin, and even when diluted with water in the pro- portions of 50 parts acetone to 30 parts water and 20 parts forma- lin. Two series, one of 4 open and one of 4 sealed consecutive combs, and one series of 9 sealed combs, showed no cells giving growth of BaGilhts larvae, while each of three series of sealed combs showed only 1 cell giving growth. Two short series were tried with the use of smaller proportions of formalin, 15 parts and 10 parts, respectively, per 100 parts. These proportions proved to be inefficient as germicides in both open and sealed cells. In the light of later work, it seems possible that if more cells from each comb had been examined a few' more positive cultures from sealed cells might have been found; but iu comparison with other solu- tions tested under the same procedure of cultural examination, ace- tone gave about the best results as a carrier. The comparatively high cost of acetone eliminates it as a substance that can be used economically in apiary practice. The ketone solution previously mentioned was mixed with forma- lin in the proportions of 80 parts ketone to 20 parts formalin. This solution, as may be seen from the table, sterilized the open cells, but was unsatisfactory in its action on scales in sealed ceUs. Table 2. — Cultural results of various tests with samples of comt) treated for ^8 hours in liquids of low surface tension, other tha/n ethyl alcohol Composition of solution 'p. .£ ill si" Ago Open cells First comb in series contain- ing open cells showing growth of B. larvae in cul- tures Sealed cells .§ 5 ** fen"! O M^ Liquid used "3 3 1 1 .9 ■a a S 1 1 o P o a bo a gfeb S 1 o i i IB S 1 ho H s B ca a g 3 o C.c. (80 86 80 60 50 40 85 .90 (80 ]80 (50 \90 C.c. I 30 30 40 30 30 c.c. 20 20 20 20 20 20 15 10 20 20 20 20 20 4 10 10 9 9 4 3 3 5 6 6 7 7 4 10 10 9 9 4 3 3 5 5 6 7 7 ■ 1 »1 4 10 10 9 9 3 2 2 5 4 3 7 7 1 1 2 1 10 10 9 9 4 3 3 5 6 5 7 7 1 1 14 s 3 1 1 ■4 9 9 9 B. 4 2 2 1 1 3 6 7 0. rO 1 1 Tenth. Do. Second.. Third... Second. Third. First. Methyl-ethyl ketone _ _ - Do. Fifth. ' Few spores germinated. ' Three showed few spores germinated. ' Two showed few spores germinated. Iso-propyl alcohol, another liquid manufactured on a commer- cial scale resembling ethyl alcohol in its physical properties, but too The Sterilization of American Foulbrood Combs 11 expensive for practical use, was also tested. Only four series, each of seven cells, were tried with this carrier for purposes of oora- parison, two of open and two of sealed cells, the proportions used being 50 parts of iso-propyl alcohol, 30 parts or water, and 20 parts of formalin. Of all the 28 cells cultured only one sealed cell was found to contain viable Bacillus larvae. This solution is there- fore in about the same class as that of acetone. MISCELLANEOUS SOLUTIONS The results of experiments with these solutions are presented in Table 3. The use of iodine solutions proved entirely impracticable for sterilizing infected combs. In dilutions of 1 to 50,000 in water and even 1 to 500, with an immersion of 48 hours, no germicidal action on the spores from diseased material in either Open or sealed cells could be demonstrated. A solution of 1 to 20 iodine killed all the spores, both in open and sealed cells, but the comb was attacked to such an extent as to make it too soft for further use. The probable reason for the lack of germicidal action on the part of iodine is that this substance combines readily with the fatty-acid constituents Table 3.-^Gultural results of various tests with samples of comb treated for 1 hours in various solutions Composition of solution g-9 — ' 3 §■32 la Open cells O-t-a CO Vi 1^3 SboS S.9l ■a ten lie Sealed cells P o I. bOJ3 II m T! 9" s43 d lodtne, 1-50,000 water iodine, 1-600 water bdine, 1-20 water lieiiatnred alcohol (50 per cent), 93 , c. «., hydrocbloric scid, 6 c. c, formalin, 2 c. c. Ijenatured alcohol, 90 c. c, hydro- ; diloric acid, Sec. tormaun, 6 c. a. -B^anatored alcohol (50 per cent), '93 c. c, hydrochloric acid, 5 c. c, formalin, 2 c. c. ;;^niatared alcohol, 90 c. c, hydro- ehlorlc add, 6 c. c, formalin, See. 'Jtoetle add, 10 c. c, water, 70 c. c, formalin, 20 c. c Do — Acetic acid, 6 c. c, water, 76 c. c, formalin, 20 c. c. Do... Denatured alcohol, 80 c. c, formalin, 20 e. c, glycerin, 2 c. c. Do.... 1 per cent solution of gelatine, 80 ■e. 0., formaUn, 20 c. c. Do 10 per cent ketone solution added to 9S per cent alcohol,80 c. c, forma- Un, 20 c. C: Hmirs 48 4S 48 First., ...do.. Third... First... First. Do. Do. Do. Do. Do. Fourth. Do Do. Second. First. Third. Second. Do. Do. ' One showed Savr spares germinated. ' Few spores germinated. 12 Department Circular 28i. U. JS. Dept. of Agriculture of the wax, as well as with the fatty residue in the diseased remains of the brood, thereby nullifying the action of the iodine as a germi- cide. ' The use of denatured alcohol in various dilutions contaimng 5 per cent concentrated hydrochloric acid, and a smaU quantity of iocmalin to prevent deleterious action of the acid (10) on the supporting wires of the frame, gave unsatisfactory results with im- mersions of 24 and of 48 hours. Each of the sealed cells tested gave a good growth of Bacillus larvae; in two cases growth was obtained from open cells, although some spores apparently were killed. This type of solution was not tested further. Varying quantities of acetic acid added to a 20 per cent solution of formaliu in water contributed nothing to the germicidal or pene- trating action of the solution so far as sealed cells were concerned, as several such cells in each series tested gave each a good growth of Bacilliis larvae. These results are very similar to those obtained with plain water-formalin solution, which will be considered later. Several other miscellaneous solutions were tried whose composition is indicated in the table, all containing 20 per cent of formalin, but varying in the composition of the carrier. AH proved imsatisf actory when used with sealed cells, and will not be discussed further. SOLUTIONS WITH DILUTED ALCOHOL A few dilutions of denatured alcohol, as well as dilutions of alcohol-formalin solution, were made, varying the alcohol content from about 30 per cent to over 60 per cent, as indicated in Table 4, the 20 per cent formalin, however, being kept constant in the mix- tures with denatured alcohol. Tests were made of combs treated for 24 hours and for 48 hours. In every case no growth was ob- tained from open cells, and, as would be expected, there were fewer positive cultures from sealed cells in combs treated 48 hours than from those treated 24 hours, although several sealed cells showed no growth, even in the 24-hour series. In each of the 48-hour series there was at least one sealed cell from which was obtained a growth of Bacillm larvae, but, with the few observations made, no signifi- cant differences could be found between the few dilutions tested of alcohol or alcohol-formalin solution. Further work is necessary to demonstrate whether results comparable with those of Kronig and Paul can be obtained. For purposes of comparison, as a preliminary test, three series of six samples each from diseased combs were treated with a solution containing no alcohol, composed of formalin 20 parts and water 80 parts (Table 9). No significant difference could be seen between these results and those obtained with the various alcoholic solutions. No open cells treated with this solution gave cultures showing growm, but 8 sealed cells, 1 or more in each series, gave cultures Stowing growth of Bacilhis larvae, the growths in 3 of the 8 cultures being noted as " few spores germinated. rne Sterilization of American Foulbrood Combs 13 Table 4. — Cultural results of various tests with samples of comT) treated for Si hours and for 4S hours in various dilutions of alcohol-formalin and commer- . oial ^Icohol-foirnmli/n solutions Composition of solutions 3 .a 3 8l Open cells 11 O . Scaled calls g Commercial alcohol-formalin solution, CO c. c, 20 per cent formalin In water, CO c. c Do. Commercial alcohol-foiteaUn solution, 40 c c, 20 per cent formalin in water, 60 o. c. Do. Denatured alcobol (62.5 per cent), 80 c. c, formalin, 20 c. c Do. Denatured alcohol (50 per cent), 80 c. c, formalin, 20 c. c Do Denatured alcohol (40 per cent), 80 c. c, formalin, 20 c. c Do Denatured alcohol (50 per cent), 75 c. c, formalin, 20 c. c, glycerin, 5 c c Do.. Hu. 24 48 24 48 24 48 24 48 24 48 12 FIrsf. Sixth. Third. Seventh. Third. Fourth. Third. Fourth. Fburth. Sixth. , First. Fourth., 1 One showed few spores germinated. • Few spores gerrainatedi COMMERCIAL ALCOHOL-FORMALIN SOLUTION Ten series of combs were treated with the Gommercial alcohQl- formalin solution' at various times. From these combs 61 open and 61 sealed sells were cultured. It was intended to use these series as controls for comparison with other solutions, since published reports of the success obtained with this commercial solution in apiary practice indicated that negative cultural results would be obtained. As may be seen in Table 7, however, in the case of every one of the 10 preliminary 48-hQur series 1 or more sealed cells, 22 in all, or 36 per cent, were found to give a growth of BacUhis larvae. In 3 cases of the 22 the growths, were noted as " few spores germinated." No growth was obtained from the 61 open cells cultured. At first it w£|is thought that the solution might have deteriorated; fresh solution was obtained and tested, with practically the same results. A chemical analysis of the actual iormaldehyde content of the fresh solution, and a similar analysis of solution through which sonie 500 combs had been passed, showed ah actual increase with use in the percentage of formaldehy(ie. This increase was without doubt due to the more rapid evaporation of the alcohol than of the formaldehyde cspntent. These results seem to indicate that there must be a variation in the permeability of ca,ppings, the decrease in permeability slowing up or even pre- venting the pehetratioA of disinfectants to the scal^, and spores. 14 Department Circular 28^^, U. S. Dept. of Agriculture PERMEABILITY OF CAPPINGS A. simple experiment was undertaken in an effort to learn whether the permeability of the cappings is variable. Such a variation might account for the fact that sealed cells are not always sterilized, and for the variation in the number of cultures of Bacillus larvae from sealed cells in different combs. If cappings are carefully removed from a piece of comb and examined under the microscope, it is seen that their structure is apparently very variable. The cappings are composed of criss- crossing cocoon fibers, pollen grains, and granules of wax, and conse- quently vary in structure. Cappings from brood cells of different ages are found to vary greatly in thickness. Freshly sealed cappings are much thicker and more opaque than cappings from the cells of more nearly matured pupae, the latter often being gnawed by adult worker bees. A simple piece of apparatus was devised to test the variability in porosity of cappings. A piece of glass tubing slightly smaller in outside diameter than the inside diameter of worker cells was drawn to a fine capillary tube and broken off, making a capillary opening on the end of a 6-inch tube. Numerous pappings were then removed from various samples of comb, both diseased and healthy, with a sharp scalpel, and cut so as to leave on the capping a rim of cell wall about one-sixteenth to one-eighth-inch wide, care being taken not to rupture the capping. These cappings were then sealed on the larger end of the glass tube with liquefied beeswax. The end of the tube covered by the capping was then submerged 3 centimeters below the surface of the chosen disinfectant, so as to have a uniform upward pressure on all the cappings successively tested. The rise of the liquid in the tube was measured at the end of a five-minute period unless the liquid was able to pass rapidly through the cap- ping and to rise to the level of the outer liquid in less than that time. The capillary opening in the upper end of the tube somewhat retarded the exit of air and, unless the capping was cracked, or slightly perforated, the contained liquid would not reach the level of the outside liquid in five minutes. When the rise was more rapid the apparent reason was recorded. A considerable number of cap- pings were tested with fresh and used alcohol-formalin solution, and with fresh and used water-formalin solution (Table 5), As will be seen, there was not much difference in the results between the fresh and used solutions. It was clearly indicated, however, that there was a great variation in the rapidity with which the solutions passed throu^ the various cappings. Twenty cappings were tested in the alcohol-formalin solution. In 5 cases no solution passed through the cappings within the five- minute period ; in 6 cases the liquid in the tube rose 1 centimeter or less; in 2 cases it rose between 1 centimeter and 2 centimeters; in 4 cases there was a rise of between 2 centimeters and 3 centimeters ; and in 3 cases the liquid rose to the 3 centimeter mark in less than five minutes. Twenty cappings were likewise tested in the water-for- malin solution. In 9 cases no solution passed through the cappings in periods varying from 5 to 10 minutes, and, in 1 case, even 60 minutes ; in 4 cases there was a rise of liquid in the tube of 1 centi- meter or less, one of these after 10 minutes ; there were 3 cases of a rise The Sterilization of American Foulbrood Combs 15 of between 1 centimeter and 2 centimeters, 1 of these being attained after 5 minutes, 1 after 15, and 1 not imtil 60 minutes had elapsed ; there were 3 cases of a rise of between 2 centimeters and 3 centi- meters, 2 of them after 5 minutes, and 1 after 15 minutes had elapsed ; in 1 case the liquid rose to the 3-centimeter mark in 3 minutes. When no liquid passed through within the test period, ropy, diseased material was found smeared on the inside of the capping, or the cap- ping appeared unusually thick. As was to be expected, the alcohol solutions passed through more readily in most cases than did water solutions. One capping, not recorded in the table, was submerged in water- formalin solution for three days with no perceptible passage of liquid into the tube. Table 5. — Tests of the penneaHUty of brood cappings to disinfectants* Description o{ cappiags Immersion Rise In tube Minuta Ctntimeten 6 2.2 6 .9 3 3.0 6 .2 2 3.0 5 S 1.1 6 S 2.8 6 6 2.1 S .g 6 1.2 6 .1 5 3 3.0 5 «0 6 2.8 5 1.0 6 .2 5 0.9 10 15 1.7 5 10 15 2.« 5 2.8 10 10 10 10 0.4 5 L5 10 6 .2.1 60 1.1 3 3.0 60 10 5 .7 5 .8 Fresh commercial alcohol-formalin solation: Dark brown from diseased comb, medium thick Dark brown from diseased comb, fairly thick Dark brown from diseased comb, quite thin Dark brown from diseased comb, tnick Dark brown from cliseaaed comb, slightly cracked Dark brown from diseased comb, thick Dark brown from diseased comb, medium Dark brown from diseased comb, thick.. Dark brown from diseased comb, thin Dark brown from diseased comb, thick Used commercial alcohol-formaMn solution: Light brown from diseased comb, dried, medium thick., . Dark brown, old, from diseased comb, dried, thick Brown, old, from diseased comb, mediuin thin Scale smeared over Inside, thick Dark brown from diseased comb, thick .... Dark brown from diseased comb, thin Dark brown from diseased comb, some scale in capping. Dark brown from diseased comb, dry, medium thick Light brown from diseased comb, good condition Dark brown from diseased comb, thick Fresh water-formalin solution: Dark brown from diseased comb, dried, thick Dark brown from diseased comb, thick — Dark brown from diseased comb, thin Dark brown from diseased comb, normal Dark brown from diseased comb, dried, thin Do - - Dark brown from diseased comb, slightly cracked Dark brown from diseased comb, tbick Do - Do - - Dsed water-formalin solution: Dark brown from diseased comb, dried, thick _.. Dark brown from diseased comb, dried, cracked Dark brown from diseased comb, thin Dark brown from diseased comb, very thin ..- Dark brown from diseased comb, thin — Dark brpwn from diseased comb, small hole -- Dark brown from diseased comb, dried, thin Dark brown from diseased comb) thin » --- Do -. Dark brown from diseased comb, (packed ' Tests made at a uniform depth of 3 centimeters below surface of solution. • Trace. To observe what actually takes place in a sealed cell when sub- merged in a disinfectant solution, some artificial cells were made with pieces of glass tubing of the same diameter as those used in the experiment just described. Pieces of tube about three-quarters of an inch long were sealed at one end and sterilized in a hot-air steril- izer, cotton plugs closing the open ends. Scales containing virulent spores of BogUIus larvae removed from diseased combs were then 16 Department Circular 28i, U. S. Dept. of Agriculture with aseptic precautions placed in a number of these glass cells, and cappings were sealed on the open ends as in the previous experiment. The sealed glass cells were then submerged for 48 hours in alcohol- formalin and water- formalin solutions, after which they were al- lowed to dry for a few days. Observations made at the end of 48 hours showed that no perceptible quantity of liquid had entered the cells. Enough moisture had been absorbed through the cappings, however, possibly in the form of vapor or of an indistinguish- able film, to cause the dried scales to become slimy or almost ropy, like diseased remains before they have dried down. After drying, cultures were made in the usual manner {Table 6). Three scales out of 20 so treated, 1 in alcohol-formalin solution and 2 in water- formalin solution, apparently were completely sterilized, and from several, most of which had been treated in the alcohol-formalin solu- tion, cultures were made which showed only a comparatively few germinated spores and having a slight growth. This seemed to in- dicate that not much actual disinfectant gains access to some at least of the sealed cells. Table 6. — Cultural results of various tests vnth spores of Bacillus larvae inclosed i/n artificial glass cells, capped, and treated for 48 hours w formalin solutions Solution tested Cells cultured Cells showing B. lanae CeUs showing no growth Cells contami- nated Bemarks Fresh alcohol-formalin. 6 6 5 5 2 16 '3 >S "3 2 1 1 Scarcely perceptible growth. Frflflh wHti^r-forniaiin Do. TTsw^l wn.t-'^r-forTnaliTi.. 2 Do. Control, not treated - 1 All showing few spores germinated; one very few. « Two showing many spores germinated. > One showing very few spores germinated. VACUXJM TREATMENT A method of forcing disinfectant solution into the cells was de- vised to demonstrate whether alcohol fills all spaces in a submerged comb. Pieces of comb of the same size as those used in previous experiments and containing numerous sealed ceUs were cut from infected brood combs and submerged in 180 cubic centimeters of disinfectant solution in graduated cylinders. One of these was allowed to soak for 48 hours. The cylinder containing the sub- merged comb was then subjected to a vacuum of 28 inches, which caused the air still remaining in many of the open cells to rush out in considerable amount and the air in sealed cells to bubble through and in some cases to burst the cappings. When the pressure was allowed to become normal the free liquid in the cylinder had de- creased by from 25 to 30 cubic centimeters in displacing the air in the cells. Similar results were obtained by applying the vacuum as soon as the combs were immersed. When combs so treated were subjected to a vacuum again after 48 hours' immersion they were found still to release a few air bubbles from sealed cells. These ex- periments made it evident that even in open cells of combs immersed for 48 hours at ordinary atmospheric pressure a considerable por- The Sterilization of American Foulbrood Combs 17 tion of the cell space is often filled with air. Of course the solution, particularly an sucoholic solution when used with open cells, forms a film on the surface of the cell around the bubble, as has been de- scribed by Demuth (9), and thus comes in contact with the larval remains. In the case of capped cells, however, under normal atmos- pheric pressure this bubble can not get out, and only a small quan- tity of liquid can gain access, in some cases probably not enough to form this moist film. It was thought at first that this vacuum treatment might be a satisfactory method of disinfecting foulbrood combs, but aside from the cost of apparatus it was found that after this procedure it was practically impossible to remove the disinfectant from the sealed cells, even by means of centrifugal force, particularly from those whose cappings had not been broken, untU after removal of the cap- pings. Liquid in the perfectly capped cells evaporated slowly, and a deposit or solid paraformaldehyde would probably remain in them. This residue has been found objectionable if not positively detri- mental to the bees when combs with such a residue are given to a colony. Cultures made from a few combs so treated and allowed to dry for a long time gave completely satisfactory results, no growth being obtained from any cells, either open or sealed. For combs from which all cappings have been completely removed this method of filling all the cells with liquid, thus assuring actual contact of all cell surfaces and cell contents with the water disinfectant, shoud be satisfactory, provided a simple and inexpensive vacuum apparatus can be devised. PERFORATION OF CAPPINGS It seemed evident that because of the greater impermeability of many of the cappings the diseased material in some of the sealed cells of the immersed combs was prevented from coining in contact with sufficient disinfectant to kill the spores of Bacillus larvae in the infected remains. A preliminary method of perforating brood cap- pings was tried. By means of a blunt needle holes variable in size and intended to resemble perforations in cappings made by the bees, were made in aU cappings, through whidi the solution might be able to enter the cells more readily. Series of samples of combs vidth cappings perforated in this manner were treated in both alco- hol-formalin and water-formalin solutions, some for 24 and others for 48 hours, as well as in two lots of soap solution for 48 hours. In the 24-hour tests with alcohol-formalin solution, of the 20 scales cul- tured (Table 8) 5 gave positive growths of B. larvae, whereas in the case of the water-formalin solution only 1 scale from a perforated cell, of 20 cultured (Table 10), showed a few germinated spores. In the 48-houE tests with alcohol-formalin solution only 1 scale showed growth out of 20 cultured from perforated cells (Table 7). In the 48-hour tests with water-formalin solution 2 scales, of the 20 cultured from perforated cells, showed a few germinated spores (Table 9). In tests with soap-formalin solution and perforated cap- pings (Table 1) the contents of aU such cells were apparently ster- ilized as far as this particular experiment was carried. These results indicate that in the case of both solutions the action was aided by perforating the cappings, but in a few instances the sterUiztng action 18 Department Circular 28i, U. S. Dept. of Agriculture was apparently still incomplete, probably because a trapped air bubble had prevented sufficient solution froni passing through the opening. Table 7.- -Cultural results of various tests with samples of comb treated for 48 hours in commercial alcohol-formalin solution 3 q3 CI ial "at" Open cells ■a o o 35 ll o " 5 o 3 "■3 Sal Sealed cells > boo H OQ'O 1.-. * ■S3 II o |5 I-. ■3 o s-si .aal Preliminary tests.. Open (uncapped) cells compared with sealed cells; used solution. Open (uncapped) cells compared with sealed cells; fresh solution. Fresh solution; control for samples washed after treatment. Open cells, washed in water after treatment Cells with perforated cappings compared with open cells. 4 3 3 7 7 8 6 7 7 10 10 10 ID 10 10 10 10 First., First Second. SV8t.,- ftfth. ThiEd. .. FireT Thifd- Secoqd. First. Ninth. Third. Do. First. Tenth. I One showed few spores germinated. • Few spores germinated. • Three showed few spores germinated. ' Eight showed few spores germinated. Table 8. — Cultural results of various tests loith samples of comb treated for i hours vn, commercial alcoJiol-formaUn solution Preliminary tests Uncapped cells only; used solution Uncapped cells only; fresh solution Fresh solution; control for samples washed after' treatment; imcapped cells only. Open cells, washed in water after treatment.. Cells with perforated cappings compared with open cells. One showed tew siwres germinated. ' Seven ^owed few spores germinated. Table 10. — Cultural results of various tests with samples of comi treated for '. hours in water-formalm solution •a m Demtjth, G. S. 1923. American fonlbrood ousted. In Gleanings Bee Cult, vol. 51, No. 11, pp. 7i2-7n. (9) 1924. Sterilizing diseased combs. In Gleanings Bee Cult, vol. 52, No, 4, p. 211. (10) Gbiffin, R. C. 1920. The solubility of metals in adds containing formaldehyde. In Jour. -Indus, and Engin. Chem., vol. 12, No. 12, pp. 1159-1160. (11) Hdtzelman, J. C. 1922. Can the combs be saved? New treatment for American fonl- brood by immersion in disinfectant solution. In Gleanings Bee Cult, vol. 50, No. 12, pp. 764-766. (12) - 1924. Honeycomb sterilizer. [U. S. Patent No. 1511762. Patented Oct 14, 1924.] U. S. Patent OfBce, OfC. Gaz., vol. 327, p. 387. (13) Jasvis, G. L. 1925. Apiary experience in treating combs. In Beekeeper, vol. 33, No. 3, pp. 27, 38. (14) JoNiB, Dan H. 1924. Control of American foulbrood. Summary of some laboratory tests with disinfectants in this disease. In Gleanings Bee Cult, vol. 52, No. 6, pp. 364-365 and 396. (15) 1925. Chemical treatment of foulbrood. In Beekeeper, vol. 33, No. 1, pp. 4-5 and 10. (16) Kino, Geobge E. 1925. Disinfecting diseased combs. In Gleanings Bee Cult., vol. 53, No. 7, pp. 438-440. (17) Kkonio, B., and Padi., Th. 1897. Die chemischen Grundlagen der Lehre von der Giftwlrkung und Desinfectlon. In Ztschr. Hyg. u. lufectionskrank., vol. 25, pp. 1-112. 27 28 Department Circular 28^, U. S. Dept. of Agriculture (18) MAA88EN, A., and Boecheet, A. 1920. tJber die Bekampfung der antsteckenden Bienenkrankheiten und liber Entseucliungsversuclie mit Formaldehyd in der Form des Autanverfalirens. In Mitt. Biol. Relchsanstalt Land- u. Forstw., Heft 18, pp. 151-156. (19) MooEE, William. 1921. Spreading and adherence of arsenical sprays. Univ. Minn. Agr. Exp. Sta. Tech. Bui. 2, 50 pp. (20) EnjEAL, S., and Rideal, E. K. 1921. Chemical disinfection and sterilization. London. 313 pp. (21) Stuetevant, a. p. 1924. The development of American foulbrood in relation to the meta- bolism of its causative organism. In Jour. Agr. Research, vol. 28, No. 2, pp. 129-168. (22) United States Tkeasubt Department, Btiebau of Internal Retvbnue. 1920. Regulation No. 61. Laws and regulations relative to the produc- tion, tax payment, etc., of industrial alcohol and to the manu- facture; sale, and use of denatured alcohol. 107 pp. (23) Vansell, G. H. 1925. Promising experiments in sterilizing foulbi'ood combs. In Amer. Bee Jour., vol. 65, No. 2, p. 55. (24) Vincens, F. 1925. Desinfection des rayons contamin^es par la " loque pernicieuse " k I'aide d'une solution aquense de formol. In La France Apic, vol. 31, No. 8, pp. 174^175. (25) White, G. F. 1904. The further investigation of the diseases affecting the apiaries in the State of New Tork. In N. Y. State Dept Agr. Ann. Rpt. Oomr. 1903, pp. 103-114. (26) (27) 1906. The bacteria of the apiary, with special reference to bee diseases. U. S. Dept. Agr. Bur. Ent., Tech. Ser. 14, 50 pp. 1920. American foulbrood. U. S. Dept. Agr. Bui. 809. ORGANIZATION OF THE UNITED STATES DEPARTMENT OP AGRICULTURE NoTember 17, 1026 Secretary of Agriculture W. M. Jabdinb. Assistant Secretary R. W. Dunlap. Director of Scientific Work A. F. Woods. Director of Regulatory Work WAiiTBB G. CAMPBELii. Director of Extension Work C. W. Wakbubton. Director of Information Nelson Antbim Cbawfobd. Director of Personnel and Business Admin- istration W. W. Stockbbbqeb. Solicitor R. W. Williams. Weather Bureau Chables F. Maevin, Chief. Bureau of Agricultural Economics Lloyd S. Tennt, Acting Chief. Bureau of Animal Industry John R. Mohlke, Chief. Bureau of Plant Industry 1 William A. Tatloe, Chief. Forest Service W. B. Gbeelet, Chief. Bureau of Chemistry C. A. Beowne, Chief. Bureau of Soils Milton Whitney, Chief. Bureau of Entomology L. O. Howaed, Chief. Bureau of Biological Survey E. W. Nelson, Chief. Bureau of Public Roads Thomas H. MacDonald, Chief. Bureau of Home Economics Louise Stanley, Chief. Bureau of Dairy Industry C. W. Labson, Chief. Office of Experiment Stations E. W. Allen, Chief. Office of Cooperative Extension Work C. B. Smith, Chief. Library Claeibbl R. Barnett, Librarian Federal Horticultural Board C. L. Mablatt, Chairman. Insecticide and Fungicide Board J. K. Haywood, Chairman. Packers and Stockyards Administration John T. Caine III, in Charge. Grain Futures Administration J. W. T. Duvel, in Charge. This circular is a contribution from Bureau of Entomology L. O. Howard, Chief. Bee-Culture Investigations J. I. Hambleton, in Charge. 29 ADDITIONAL COPIES 07 THIS PUBLICATION MAY BE PEOCORED fBOH THE SDPEBINIENDENT OF DOCUMENTa 60VEENMENT PErNTING OWICE WASHINGTON, D. C. AT 5 CENTS PEE COPY U. S. DEPARTMENT OF AGRICULTURE FARMERS' BULLETIN No. 1713 AMERICAN FOULBROOD is a disease of the - brood of bees which causes serious losses to bee- keepers. Its occurrence is practically world-wide, it attacks all races of bees, and strong colonies are as liable to infection as weak ones. It is important that the beekeeper recognize the symptoms of the disease and be familiar with the manner in which it is spread, in order that he may take precautions to keep it out of his apiary and to prevent it from spreading from one colony to another in case any of his colonies become infected. He should also know how to treat the disease in the most effective way. Such information is given in this bulletin. It is impossible to manipulate colo- nies so that they cannot contract the disease, but much worry and financial loss can be avoided by dealing promptly and effectively with the disease as soon as it appears. Samples of brood suspected of being diseased are diagnosed free of charge by the Division of Bee Culture, Bureau of Entomology, United States Department of Agriculture, Washington, D.C. This bulletin supersedes Farmers' Bulletin 1084, entitled ■■ Control of American Foulbrood." Washington, D.C. Issued October 1933 THE TREATMENT OF AMERICAN FOULBROOD By Jas. I. HAMBiLHTON, senior apicuUurht, in cfiarge, Division of Bee C'ulture, Bureau of Entomology CONTENTS Page Cause of the disease. _ 1 Spread of the disease 2 Symptoms... 3 Otlier brood diseases sometimes mistaken for American foulbrood 3 Treatment... , 9 Burning diseased colonies '---. 9 Disinfecting empty hives after burning . . 10 Treatment— Continued ^**® Shaking not recommended 11 Objections to the use of disinfecting solu- tions 11 Disinfecting super combs _ 11 Treatment by State inspectors 12 Handling and disposing of honey from affected colonies 13 AMERICAN FOULBROOD is a disease of the brood of bees XTL which causes series losses to beekeepers. Not only does it take a heavy toll in the actual destruction of colonies and increase the cost of operating an apiary, but, what is perhaps of equal import- ance, the presence of disease in an apiary, or in the vicinity of one, causes such mental anxiety to some beekeepers that it unquestionably prevents them from succeeding in the bee industry. The disease is practically world-wide in its distribution and is of common occurrence in the United States. All races of bees are sus- ceptible. There have been statements in the beekeeping literature that lead one to believe that the brood of certain strains of bees is immune, but there is no evidence upon which to base this assump- tion. There may be various strains of the disease, differing in viru- lence, and individual colonies may react differently. Not enough variation has yet been detected, however, to warrant giving such suspected cases special treatment. Worker larvae are particularly susceptible to the disease, although queen and drone larvae are occa- sionally affected. Adult bees are immune to it. CAUSE OF THE DISEASE American foulbrood is caused by a species of spore-bearing_ bac- teria known as Bacillus larvae. The living honeybee larva is its only known host and the disease is transmitted primarily by means of the spores. The bees that work within the hive become contami- nated with these spores in attempting to remove the diseased brood, and carry them from one cell to another. Once the disease has spread generally throughout the brood nest, the bees cease trying to remove the dead brood, and it accumulates until the colony dies owing to the absence of emerging bees. 4939° — 33 1 2 FAHMEBS' BULLETIN 1713 In performing such tasks as feeding the larvae, building the cells, ripening the nectar, and transferring it from one part of the hive to another, the bees may contaminate not only honey that is in the brood nest, but also that in the supers above the queen excluder. This does not mean, however, that all the honey in the brood nest or supers necessarily becomes contaminated. Spores may come in contact with larvae of any age, but the larvae rarely die until they have developed to the point where they lie lengthwise in the cells or the cells are being sealed preparatory to transformation to pupae. During the early coiled stages the sugar content of the larva is usually high and, since the germ of American foulbrood will not grow in highly concentrated sugar or honey, it is only after much of the body sugar has been utilized that the spores can develop. Underfed larvae have a low sugar content, and in contact with such larvae the spores are able to germinate and to kill the larvae while they are still coiled. The spores of American foulbrood are invisible to the naked eye, and they are extremely resistant to sunlight, drying, heat, and commonly used chemical disinfectants. The maximum time that the spores retain their virulence has not been determined, but they are faiown to remain alive for years in honey and brood combs. SPREAD OF THE DISEASE It has always been considered that the disease is spread from colony to colony most commonly by the robbing of hives containing disease-weakened colonies or bees that have died of American foul- brood, by bees from healthy colonies. Robbing* unquestionably scatters American foulbrood. A disease-weakened colony does not defend itself well; therefore it is possible for robber bees from healthy colonies to help themselves to contaminated honey. On the other hand, the beekeeper himself often unwittingly spreads the disease within an apiary when he moves combs of brood and honey from one hive to another, or unites weak colonies, which may be diseased, with strong, healthy ones. These are probably the most common means of spread. The disease may also be carried from one colony to another when bees enter the wrong hive, a practice gen- erally referred to as drifting. The dissemination of the disease beyond the range of flight of the bee can be accounted for by the transportation of infected material, including honey, into a disease-free area, where it is later made accessible to healthy bees. Experimental evidence indicates that the commercial shipment of honey is not such an important means of spread as many persons suppose. The sale of used, contaminated equipment is, however, one of the principal avenues through which this disease is spread from one locality to another. A beekeeper who does not know anything about American foul- brood, or how to check its spread in his apiary, will not be able to maintain his colonies with profit if they become weakened by the disease. Finding this to be the case, he may sell his empty hives, combs, and other accessories, perhaps to another beekeeper who knows no more about the bee diseases than he does. Purchasers of THE TREATMENT OF AMERICAN POULBROOD 6 used beekeeping equipment should make sure that it is free from disease material. Some States wisely restrict the sale of used bee- keeping equipment to that which is known to be uncontaminated. SYMPTOMS In the apiary American foulbrood can be detected only by the presence of brood remains. The spores of the disease organism can be recovered and identified only by bacteriological technic. The disease may be recognized by the sunken and perforated cap- pings and the isolated sealed cells in the midst of recently emerged brood. The dead larvae have a melted-down appearance and are usually extended lengthwise in the cells (fig. 1). Occasionally the bees die while in the coiled stage, and in this condition the brood may resemble that dead of European f oul%rood. Dead larvae are slightly yellowish or dirty white in color at first, but become chocolate brown or black upon further decay. Shortly after death of the larvae, and until the contents of the cells become too thick, the brood remains can be drawn out with a toothpick into fine silklike threads, and are quite ropy and gluelike. Upon drying, the brood remains, called scales (fig. 1, F, G) , become tough or brittle and adhere so tightly to the floor and base of the cells that the bees cannot remove them. The scales are very thin and in old, dark brood combs are easily over- looked. Pupae that die of the disease undergo similar changes in color and consistency and in the final formation of a scale (fig. 2). Occasion- ally the tongue of a dead pupa adheres to the roof of the cell. This is a significant, but not an infallible, symptom. Bees remove many of the cappings from cells containing dead brood, and this makes it appear that the larvae or pupae died before being sealed. American foulbrood has a characteristic odor, which is pronounced when the disease is in an advanced stage. Although adult bees are not attacked, loss of brood causes an infected colony to become gradually weaker, and usually to die during the second year of the disease. The constancy and the uniformity of the symptoms characterize this disease more than does any one symptom. Isolated sealed, sunken, or perforated cells in the midst of healthy emerging brood should be examined whenever disease is suspected. It is not diffi- cult to make a reliable diagnosis in the apiary except perhaps when only 1 or 2 recently dead larvae or pupae are present. In such cases a portion of the comb containing the suspected brood should be sent to a competent inspector. In the meantime the entrance of the suspected hive should be contracted and the colony left undis- turbed until the nature of the trouble has been determined. OTHER BROOD DISEASES SOMETIMES MISTAKEN FOR AMERICAN FOULBROOD Many colonies have been destroyed or treated in the erroneous belief that they were infected with American foulbrood. On the other hand, the disease has been spread when American foulbrood FABMEES' BULLETIN 1713 Figure 1. — Stages in the aecomposition of larvae (prepupae) dead o£ American foul- brood : A, Healthy larva at the age when most of the brood dies of American foulbrooQ. B, C, D, B, Progressive stages i» the decomjposition of dead larvae. These stages can usually be detected only by removing the cappings. P, Scale of American foulbrooQ. Except in new combs the scale is difficult to see by looking straight into the cell. The comb should be held so that the line of sight falls on the long floor of the cells. This can be done by grasping the comb by the top bar and holding it 8 or 10 Inches below the eyes and tipping the bottom bar slightly away from you. O, Longitudinal view of an American foulbrood scale. THE TREATMENT OF AMERICAN FOULBROOD has been mistaken for some of the less serious brood diseases, which require different treatment. It is therefore of the utmost importance that a correct diagnosis be made before corrective measures are applied. For this reason brief descriptions are here given of the FiGDBB 2. — Stages In the decomposition of pupae dead of American foulbrood : A, B, C, Heads of pupae showing progressive stages of melting down and decay. In B and O • the tongues show prominently. D, Scale of American foulbrood formed from the dry- ing down of a diseased pupa. E, Scale of American foulbrood formed from' the drying down of a diseased pupa, with a vestige of the tongue adhering to the roof of the cell. two other common brood disease of the apiary, European foulbrood and sacbrood. European foulbrood usually kills the larvae in the coiled stages (fig. 3). The dead larvae are slightly yellowish white in color. The brood remains are watery, pastelike, or granular, the appearance varying according to the age at which the larvae die. The scales PAEMEKS' BULLETIN 1713 Figure 3. — Coiled and unsealed larvae sick or dead of European foulbrood : A, Healthy coiled larva at the earliest stage at which larvae die of European foulbrood ; B, scale formed by a dried-down larva ; O, one of several positions assumed by a sick larva prior to death ; D, B, longitudinal views of scales formed from larvae that had assumed a nearly lengthwise position at the time of death, quite difteient from the scale shown in B. do not adhere tightly to the cells and are removed by the bees in a strong colony. Occasionally larvae dead of European foulbrood become brown and ropy and present other symptoms similar to those THE TKBATMENT OF AMERICAN POULBROOD dead of American foulbrood (fig. 4). In such cases a correct diag- nosis can usually be made only after a microscopic examination. f ^ FiGnBB 4. — Larvae (prepupae) which may or may not be in sealed cells and which are lying lengthwise at the time of death from European foulbrood. Stages similar in appearance to those illustrated here are encountered in Ansricani foulbrood. A. Sunken and perforated capping of a cell containing a larvq,, dead of European foulbrood ; B, larva lying lengthwise in the cell and recently dead of European foulbrood ; O, same as B except in a more advanced stage of decomposition ; D, scale formed by dried-down larva dead of European foulbrood ; E, the remains of a larva dead of European foul- brood, part of which has been removed by the bees. Sacbrood kills the larvae while they are extended in the cell (fig. 5), and the cappings become sunken and perforated, as in American foulbrood. The dead larvae are yellowish at first, but become 8 FAEMEKS' BULLETIN 1713 Figure 5. — Appearance of larvae (prepupae) dead of sacbrood: A, B, Stages In the course of the disease ; O, the erect head end of a dead larva showing through an opening that the bees have made In the capping ; D, E, two views showing the scale of sacbrooo ; F, the head portion of this larva has been gnawed away by the bees. Note how the head remains erect in all stages. THE TREATMENT OF AMERICAN FOULBROOD 9 brown or black as decay advances. The heads remain erect during the process of decay. The larval skin becomes tough and saclike, thus giving the disease its name, and the material inside is watery and granular. The brood remains do not adhere to the cells ; there- fore, the bees are able to remove them and the disease does little damage. TREATMENT BURNING DISEASED COLONIES It is now commonly recognized that the safest, and in the end the most economical, means of stamping out American foulbrood is to burn the diseased colonies. While this procedure may seem wasteful to those who believe that less drastic measures afford ample protection, it is the only method that leaves no op- portunity for the disease to recur, thus relieving the mind of the bee- keeper. Diseased colonies should be burned as soon as possible aft- er the infection is discovered. Before this is done, how- ever, the bees must be killed. A table- spoonful of calcium cyanide, an ex- tremely poisonous chemical which must be handled with great care, spread on a sheet of paper or cardboard and slipped into the entrance of the hive (fig. 6), which should be left open, will kill the bees in a few minutes. As as an extra precaution additional cyanide may be thrown into the top of the hive, since occasionally the bees fall onto the poison placed in the entrance so rapidly as to prevent the fumes from penetrating all parts of the hive. AH field bees that try to gain entrance to the hive will also be killed. Gasoline is sometimes used to kill the bees. In such cases the entrance to the hive is closed, a pint or more of gasoline is then poured over the top frames, and the hive is closed tightly. After the bees have been killed, the contents of the hive should be burned with the least possible delay in order to avoid trouble Figure 6- -Killing the bees of a diseased colony calcium cyanide. with 10 FARMERS' BULLETIN 1713 from robber bees, as both calcium cyanide and gasoline act as repel- lents for only a short time. Before the bees are killed, a pit 18 inches or more deep, and wide enough to hold all the material to be burned, should be dug in a place not likely to be plowed or otherwise disturbed. A hot fire should then be kindled, with plenty of scrap material and with cross mem- bers stout enough to support the weight of the frames and placed so as to permit plenty of ventilation underneath. A brisk hot fire is necessary thoroughly to burn the brood and honey. The hives containing the dead bees should be carried intact close to the pit and the bees and frames fed to the fire as fast as circum- stances permit. The bottom board, hive bodies, inner covers, and tops are not burned. By placing the hives on pieces of burlap or stout paper it will be easy to gather up and burn the bits of comb honey or dead bees which may be dropped during the operation. After everything has been completely burned, the topsoil surrounding the fire should be raked into the pit to prevent bees from healthy colonies from having access to any dead bees or honey. The pit should then be filled. If the killing of the bees and the burning are done at night, the danger of interference from robber bees will be lessened. It is essential, of course, to have everything well planned and all necessary material at hand. No beekeeper should wait for an inspector to discover and burn his infected colonies, but should, himself, periodically inspect all colonies and promptly destroy every diseased one. It should be understood that the burning of all diseased colonies in an apiary gets rid only of the colonies in which American foul- brood in an active form is plainly manifest. If there has been any equalization of the brood, if supers or combs have been transferred from one colony to another, or if diseased colonies have been robbed out, it is highly probable that the disease will show up later in other colonies. So even where burning is done carefully and thoroughly, it is usually at least 3 or 4 years before the disease can be stamped out of an apiary. DISINFECTING EMPTY HIVES AFTER BURNING After the burning, the hive bodies, bottom boards, inner covers, and taps should be taken into the honey house, thoroughly scraped to remove all propolis and wax, and then scrubbed, both inside and out, with a hot soap or lye solution and a stiff brush. The scrapings should be burned and the wash water disposed of in such a manner that it is not accessible to the bees. Washing with soap and water is also the best way to remove spores from the hands, clothing, tools, and extracting equipment. Disin- fectants strong enough to kill the spores are injurious to the hands. If it is not feasible to wash the hive bodies, they may be stacked 7 or 8 high to form a chimney, the inside walls sprinkled with kero- sene, and ignited. A little ventilation and fuel at the bottom of the stack will produce a hotter fire. Gasoline can also be used for this operation, but extreme precaution is necessary. As soon as the in- side is scorched, the fire should be smothered by placing a board THE TREATMENT OF AMERICAN POULBROOD 11 over the top super. The outside of the hive bodies should then be thoroughly washed to remove all traces of honev. A gasoline blow- torch is a handy tool for scorching, but its use is rather slow. SHAKING NOT RECOMMENDED For many years the Department of Agriculture recommended the treating of infected colonies by the shaking method, whereby the bees in a diseased colony are shaken from the old combs into a clean hive on clean frames. This procedure reduces the losses due to the disease, and a careful operator, who thoroughly understands the dis- ease, may be able to maintain his apiaries in this way. The disease is rarely eradicated by this method, however, and it usually has to be adopted as a routine manipulation. Treated colonies have to be nursed along, and the very act of shaking, if not done with meticu- lous care, is apt to spread the disease. Moreover, there is always a doubt as to whether the shaking is successful. A treated colony or a colony on disinfected combs cannot be pronounced clean for 2 years. Now, after many years during which colonies have been shaken to get rid of the disease and at the same time save as much as possible in the way of bees and equipment, the disease situation in the United States has not materially improved. OBJECTIONS TO THE USE OF DISINFECTING SOLUTIONS Disinfecting solutions are of only limited value in the treatment of American foulbrood. In the first place, their use for treating brood combs does not obviate the shaking treatment, as the bees must be removed before the combs are disinfected. Moreover, the careless handling of combs during the disinfecting operation may result in failure. When disease reappears after a colony has been shaken and later placed on treated combs, it is impossible to tell whether the method of shaking was at fault, the disinfection inadequate, or the colony reinfected. Although there are several disinfectants which, when properly used, will kill the spores of American foulbrood without destroying the comb, none has yet been found to sterilize the spores in sealed honey without destroying the comb and making the honey poisonous to bees and brood. Individual sealed cells are easily overlooked and it is probable that many, if not most, of the cells of honey in the brood chamber of a diseased colony are contaminated. Another disadvantage in the use of disinfectants is that bees are loath to accept treated combs, and as a result the size of the honey crop is reduced. DISINFECTING SUPER COMBS Disinfectants can be used effectively in treating super combs that have never contained brood. Super combs are ordinarily used on whatever colony needs them and are not set aside for designated colonies. After all the diseased colonies in an apiary have been disposed of, it is often not possible to know wJiether any of the general suppljjr of super combs have become contaminated by being used on colonies with foulbrood. Therefore, it is safest to disinfect all the super combs in an outfit in which there has been appreciable 12 FAEMEES' BULLETIN 1713 amount of American foulbrood. This is the only use recommended for disinfecting solutions in connection with the treatment of this disease. Super combs can be disinfected with a 20 percent formalin-water solution — that is, 20 parts of formalin^ to 80 parts of water, liquid measure. The combs to be disinfected should be free of honey. They should be kept inmiersed in the solution at a temperature of not less than 70° F. for at least 24 hours. At lower temperatures sterilization proceeds much more slowly. In order that the solu- tion may come in contact with all parts of the cells, after being placed in the solution the combs should be agitated to dispel as many air bubbles as possible. This can also be accomplished by pouring the solution into the tank so that it rises slowly enough to fill each cell completely. The 20 percent formalin-water solution may be used repeatedly without much deterioration in strength. It is advisable, however, to add formalin occasionally to maintain full strength of the solution. Formalin is unpleasant to work with, although not dangerous. It is well for the operator to protect his hands with rubber gloves. Formalin-alcohol solution, formaldehyde gas, and chlorine also kill the spores of American foulbrood without necessarily destroying the combs. Formalin-alcohol solution, a patented article, is slightly less effective than the 20 percent formalin-water solution and is more expensive. Formaldehyde in gaseous form cannot be recom- mended for treating American foulbrood combs. The use of chlo- rine, although still in the experimental stage, has produced disap- pointing results and, moreover, it is extremely dangerous to handle. TREATMENT BY STATE INSPECTORS Under most conditions inspectors are justified in burning every diseased colony immediately, because such a colony constitutes a menace to all healthy colonies in the vicinity. The maintenance of such a serious nuisance as a colony containing American foulbrood should not be tolerated. The best interests of the industry demand the prompt disposal of all such colonies. When a State inspection force is applying- the area-clean-up method for the first time, however, and when the incidence of the disease is high, the use of the shaking treatment is sometimes justi- fiable. The advisability of using it depends not only upon the amount of disease in a particular apiary or area, but upon the char- acter of the beekeeping, the kind of equipment employed, and the facilities and experience of the beekeepers for doing the job. Where shaking is used in the first steps of an eradication program, the State inspectors should give every consideration to the protec- tion of healthy colonies. The establishment of temporary yards where diseased colonies can be treated without endangering healthy colonies is strongly recommended in this connection. These tem- porary yards are not to be confused with hospital yards, which were recommended at one time and with which many beekeepers are » Formalin or formaldehyde solution is an aqueous solution containing from 37 to 40 percent of formaldehyde gas. THE TEEATMEIjrT OF AMERICAN FOULBKOOI) 13 familiar. Temporary yards are used only to shake and reestablish the bees in a place where healthy colonies are not endangered. The contents of the hives other than "bees should be burned with the least possible delay. HANDLING AND DISPOSING OF HONEY FROM AFFECTED COLONIES The honey from a diseased colony, if it constitutes a super or more, may be saved and marketed. The handling of this honey, however, requires special attention. At no time should it be accessible to the bees. Therefore, since no honey house is, strictly speaking, bee- tight, the honey should be bottled or canned as soon as possible, every vestige of honey washed from the outside of containers and from the extracting equipment and honey house, and the empty combs burned. No attempt should be made to recover honey from diseased colonies unless there is a distinct economic saving. Since it is often impossible to ascertain the source of honey pur- chased on the open market, such honey should not be fed to colonies of bees if it can be avoided. If such honey has to be used, it should first be diluted with an equal volume of water and boiled for an hour in a closed vessel. Boiled honey, however, should not be fed for winter stores. ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE WHEN THIS PUBLICIATON WAS LAST PRINTED Secretary of Agriculture Henkt A. Wallace. Assistwnt Secretary— Rexfoed A. Tugwell. Director of Scientific Work A. F. Woods. Director of Extension Work C. W. Waebukton. Director of Personnel and Business Adminis- W. W. Stockbebgeb. tration. Director of Infonnation M. S. Eisenhowee. Solicitor Seth Thomas. Bureau of Agricultural Economics Nils A. Olsen, Chief. Bureau of Agricultural Engineering S. H. McCeoey, Chief. Bureau of Animal Industry John R. Mohlek, Chief. Bureau of Biological Survey Paul G. Redington, Chief. Bureau of Chemistry and. Soils H. G. Knight, Chief. Office of Cooperative Extension Work C. B. Smith, Chief. Bureau of Dairy Industry O. B. Reed, Chief. Bureau of Entomology C. L. Maelatt, Chief. Office of Experiment Stations Jambs T. Jaedine, Chief. Food and Drug Administratimi Waltee G. Campbell, Chief. Forest Service R Y. Stuaet, Chief. G-rain Futures Adininistration J. W. T. Duvel, Chief. Bureau of Home Economics Louise Stanley, Chief. Library Claeibel R. Baenbtt, LibrariOrn. Bureau of Plant Industry William A. Taylor, Chief. Bureau of Plant Quarantine Lee A, Strong, Chief. Bureau of Public Roads Thomas H. MacDonald, Chief. Weather Bureau Charles F. Marvin, Chief. Agricultwal Adfustment Admiiiistratlon George N. P.ebjk, Administrator. Chas. J. Brand, Coadministrator. 14 U. 5. GOVERNMENT PRINTING OFFICE: 1933 For sale by the Superintendent of Documents, Washington, D.C. - - Price 5 cents ;/ CIRCULAR No. 392 JULY 1936 UNITED STATES DEPARTMENT OF AGRICULTURE WASHINGTON, D. C. DIAGNOSING BEE DISEASES IN THE APIARY By C. E. BuRNSiDE, asslstamt apiculturint, and A. P. Sttjrthvant, assooiate apicultwnst, Divislcm of Bee CiiUiirc, Bureau of Ento-mology and Plant, Quarantine CONTENTS Foreword Importance of bee diseases and their recogni- tion Brood diseases - What to observe whenloolcing for brood diseases American foulbrood European foulbrood- Parafoulbrood- Sacbrood Infection with two or more brood diseases- Fungous diseases of brood. Diseases of adult bees What to observe whenlooliing for diseases of adult bees Page 1 Page Diseases of adult bees — Continued. Nosema disease 25 Acarine disease --- 27 Septicemia 29 Amoeba disease — 30 Fungous diseases of adult bees - 31 "Paralysis"- 32 Sending samples for laboratory examination. 33 How to prepare samples of brood__ 33 How to send samples of adult bees 34 How to send samples of treated comb — S4 How to address samples 34 FOREWORD Bees, like all other living creatures, are subject to diseases, and their manner of living in crowded hives makes it almost inevitable that any contagious disorder will spread within the hive or to other colonies unless it is detected and the appropriate treatment given. Other publications of the Department furnish information on the methods of treatment. This circular tells where to look and what to notice in the examination of colonies for possible or suspected disease. More than one disease may be present in a colony, therefore the beekeeper should not discontinue the search on findingthe symptoms of one disease. Especially is it important that American foulbrood be detected if it is present in the apiary. If the nature of the disease is not apparent, samples of brood comb or the adult bees should be sent to the State apiary inspector or the Bee Culture Laboratory of the Bureau of Entomology and Plant Quarantine, National Agricultural Eesearch Center, BeltsviUe, Md., as directed on page 34. IMPORTANCE OF BEE DISEASES AND THEIR RECOGNITION Bee diseases are found throughout the United States wherever bees are kept. These diseases cause large annual losses in bees, honey, and equipment and very materially add to the cost of honey 51309°— 36 1 1 2 CIRCULAK 3 9 2, U. S. DEPARTMENT OF AGRICULTURE production. Unless bee diseases are recognized and controlled, indi- vidual colonies or even those of entire apiaries may be seriously weakened or destroyed. It is important that beekeepers recognize bee diseases in their early stages so that they can apply proper methods of treatment, since practically all the diseases are more or less contagious and can spread from diseased to healthy colonies. Some of the diseases cause only slight losses, and can, to a certain extent, be disregarded. Oth- ers, however, are serious, and prompt treatment is required to pre- vent their spread. Consequently it is necessary that the beekeeper be able to recognize even the less serious diseases so as not to con- fuse them with the serious ones. The symptoms of sacbrood and European foulbrood, for instance, are often confused with those of American foulbrood. Furthermore, American foulbrood may be mistaken for European foulbrood, and if the usual treatment for the latter is applied, the disease not only will not be arrested but is likely to spread to healthy colonies. In recent years new bee diseases have been discovered. One of these, parafoulbrood, a serious brood disease, at present appears to exist only in limited sections of the South. It is highly desirable to prevent the further spread of these newly discovered diseases and, consequently, beekeepers should learn to differentiate them from the other more widely distributed diseases. There are also a number of abnormal conditions of bees that at times cause heavy losses and can easily be confused with some of the diseases. It has recently been found that nectar or pollen, or both, from certain plants may cause the poisoning of brood and adult bees. Then, too, the symptoms of poisoning or other abnormal con- ditions of bees such as chilling, starvation, or the presence of brood of infertile queens or laying workers, can easily be confused with the sA'mptoms of some of the diseases. No attempt has been made in this circular to describe methods of treatment, since these are available in other publications of the Department of Agriculture and in State bulletins and other bee- keeping literature. Abnormal conditions of bees that are often difficult to distinguish from diseases are not discussed in detail, since it is planned to describe them in another publication. BROOD DISEASES WHAT TO OBSERVE WHEN LOOKING FOR BROOD DISEASES To identify the brood diseases, any dead brood found in the cells should be examined carefully. The appearance of the combs may indicate which brood disease is present, but final diagnosis should always depend upon the symptoms shown by the dead brood. Dead brood m open cells can be seen clearly if a comb is held so inclined that the direct light of the sun falls on the lower side and bottom of the cells (fig. 1). If there is no dead brood in the open cells, any sunken, discolored, or punctured cappings should be removed and these cells examined for dead brood. When dead brood is found, the following important points should be determined: (1) Age of the brood when death occurred, (2) po- DIAGNOSING BEE DISEASES IN THE APIAKY 3 sition of the dead brood in the cells, (3) color of the dead brood, (4) consistency of the dead brood in different stages of decay, (5) odor coming from the combs, and (6) odor of dead larvae in different stages of decay. A chart or guide for use in diagnosing diseases of the brood of bees is given in table 1 (p. 22). It should always he kept in mind that more than one hrood dis- ease Tnay he present in a colony. Of first importance at all times is Figure 1. — Inspecting combs for brood diseases. A convenient way to hold the comb while looking for dead brood. The arrow indicates the direction of the sun's rays, which should fall on the lower side and bottom of the cells. the early discovery of American foulhrood. When a less serious hrood disease is found, it should he determined whether or not Amer- ican foulhrood also is present. AMERICAN FOULBROOD CAUSE American foulhrood is an infectious disease of the brood of bees caused by a bacterium known as Bacillus larvae. It is the most destructive of the brood diseases, is very infectious, and diseased col- onies practically always die. This bacterium causes the death of larvae and pupae by its growth and multiplication within the stomach. It also causes a typical decay of the dead brood. B. lar- vae resists drying, the action of chemicals, both high and low tem- peratures, and the dehydrating action of honey ; consequently Amer- ican foulhrood cannot be treated successfully except by burning the infected combs and bees. 4 CIRCULAR 3 9 2, U. S. DEPARTME17T OF AGRICULTURE Figure 2. — American foulbroofl : A, Healthy brood In new brood comb ; B, old brood comb with an advanced case of American foulbrood ; 0, scales of larvae dead of American foulbrood on a cross section of a new comb ; D, scales of larvae dead of American foul- brood in an old brood comb. DIAGNOSING BEE DISEASES IN THE APIARY 5 EFFECT UPON THE COLONY The strength of a recently infected colony will not be noticeably affected, and there will be only one or a few dead larvae or pupae in sealed cells with slightly (iiscolored or sunken cappings. The FiGUEB 3. — Symptoms of American foulbrood : A, Normal capping oyer healthy larva ; B—F, stages in the discoloration and removal of cappings ; O, capping removed to show healthy larva ; H-L, stages in the decay and drying of larvae killed by American foul- brood ; oral views. disease may not develop to a critical stage and seriously weaken the colony until the following year. At other times, however, the disease may advance more rapidly and seriously weaken or kill the colony the first season. If the disease has been present and active 6 CIRCULAR 39 2, U. S. DEPARTMENT OF AGRICULTURE for a considerable period, the colony will be noticeably weakened, and a large proportion of the cells (75 percent or more) will contain dead brood. All weah, infected colonies found during any time of FiGTJKB 4. — Symptoms of American foulbrood in larvae : A, Healtby larva, lateral view ; B, colled larva recently dead ; C, healthy larva, ventral view ; D—H, stages in decay and drying of larvae, ventral views. year should he hurned at once to prevent spread of the disease through rohhing. Under no circumstances should colonies he per- mitted to re^nain in the apiary until they have hecome seriously weakened hy or die of American foulhrood. DIAGNOSING BEE DISEASES IN THE APIARY APPEARANCE OF THE COMBS AND CAPPINGS In healthy brood combs, where a normal queen has been laying, there is a certain regularity in the arrangement of areas containing eggs, larvae, pupae, and emerging bees (ng. 2, A), and the cappings are convex and uniform in appearance (fig. 3, A). In a colony in- FiGOED 5. — Symptoma of American foulbrood in larva and pupae : A, healtliy pupa ; B-F, stages in the decay and drying of pupae ; O, scale of dead larva, lateral view ; H, scale of dead pupa, lateral view. fected with American foulbrood the brood is more or less irregularly arranged, depending on the degree of infection. Great irregularity, due to the intermingling of cells of healthy brood with uncapped and capped cells of dead brood and cells with punctured and sunken cappings, is sometimes spoken of as the "pepperbox" appearance (fig. 8 CIRCTJLAE 3 9 2, U. S. DEPARTMENT OF AGRICULTURE 2, 5). Dead brood in cells with discolored, sunken, or punctured cappings (fig. 3, B, C, D) should always be studied carefully to determined whether death was caused by American f oulbrood. In advanced stages of the disease many of the cappings are punctured (fig. 2, B). Cappings may also be broken away at the edge and settled down on the dead brood, appearing dark brown and shining. Cappings over dead brood are often removed by adult bees, and in advanced cases many dried scales, .as the remains of dead larvae and pupae are then called, can be seen in uncapped cells (fig. 2,,E and F). PiGDEB 6. — Symptoms of American foulbrood in p-upae : A, healthy pupa ; B-F, stages In the decay and drying of pupae, ventral views. SYMPTOMS SHOWN BY THE DEAD BROOD KIND AND A&E OF ATFECTED BBOOD Usually only worker brood is affected, but occasionally drone and queen brood are also killed. Adult bees are never affected by this disease. DIAGNOSING BEE DISEASES IN THE APIARY 9 Death occurs quite uniformly after the larvae have been capped over, have spun, their cocoons, and are fully extended on the floor of the cells, as shown by the healthy larvae in figures 3, ) ; but in new comb they are readily distinguished (fig. 2, C). During the early stages of decay the body wall is easily ruptured, and the tissues are soft and watery. Occasionally the body di- visions of the dead larva are more clearly marked than are those in healthy ones the consistency of dead brood becomes characteristically glue- like about 3 weeks after death. When a toothpick or match is thrust into a decayed larva and withdrawn, the decaying mass ad- heres and can be drawn out an inch or more in a glulike thread (fig. 1). Decayed larvae finally become dry and brittle. APPEABANOB OF THE DEAD BEOOD The appearance and position in the cells of brood killed by Ameri- can foulbrood are remarkably uniform. The dead larvae lie ex- tended along the lower side wall with their posterior ends curved 51309°— 36 2 * ,1*-, -•'•■ ^ ixy^cx^^^^- ■..-*i ^^jii-^ ^"■. ■' W -^- ..*-^' ..^ '■■-*■' ^ i "^ v'^P^ "* f^r^^'ir /^ "V"^ IBsmM^m d p^-^Xf^ ."tr^'ij J I'YYft*^ Figure 7. — American foulbrood ; ropy remains of decayed larva. 10 CIECXJLAR 3 9 2, U. S. DEPARTMENT OF AGKICTJLTURE part way up onto the bottom of the cells (fig. 5. G). There may be a small raised swelling near the head end of the scale, but this rarely is prominent. In advanced cases rows of cells contam dead larvae uniformly in this position. When scales are numerous the disease can be diagnosed from their appearance alone. Scales can be seen extended along the lower side walls when the comb is held inclined so that a bright light falls on the lower side walls and bottoms of the cells (fig. 1). Occasionally cross markings which represent the segmentation of the larvae can be seen on the scales. When completely dried the scales are brittle and adhere so tightly to the cell walls that it is difficult to remove a scale without breakmg it. When death occurs after pupation has started, the form of the pupa can be recognized in the scale (figs. 5, H, and 6, F) . The mouth parts of the dead pwpa may protrvdejrom the head of the scale and appern^ as a fme thread slanting slightly hachwards into the cell and at tirnes adhering to the upper wall (fig. 5, F). The appearance of protruding or "stuck up" tongues is one of the most dependable symptoms of American f oulbrood. ODOR OF THE DEAD BROOD In the first stages of decay, while the remains are still white, practically no odor is detectable. When the remains begin to turn brown and become ropy, however, an odor develops that is different from the typical gluepot odor characteristic of the advanced stages of this disease. In later stages, when the dead brood is brown and decidedly ropy, the familiar gluepot odor is always present, but it practically disappears when the scales are completely dry. In advanced cases, when much decaying brood is present, the gluepot odor can be detected even a foot or more from the combs. Since the odor of American foulbrood is characteristic, the use of the odor test is of considerable value in the diagnosis of doubtful cases. The odor can best be judged by holding some of the decayed remains on a toothpick at the entrance to the nostril and breathing deeply. EUROPEAN FOULBROOD CAUSE European foulbrood is an infectious bacterial disease of the brood of honeybees. The bacteria grow within the stomach of infected worker, queen, and drone larvae and cause their death, but pupae are rarely attacked. Adult bees are not affected by this disease. The earliest studies on European foulbrood seemed to indicate that it was caused by a rod-shaped bacterium, Bacillv>s alvei, which is commonly found in decayed brood. Later it was observed that lancet-shaped bacteria, different in shape and size from the rods and spores of B. alvei found in decayed brood, are usually present in large numbers in sick and recently dead larvae. This lancet-shaped bacterium, which was given the name Bacillus plwton, is now com- monly considered to be the cause of European foulbrood. It has been found recently, however, that the rod-shaped B. alvei is capable of changing its form to a lancet-shaped bacterium resembling B. DIAGNOSING BEE DISEASES IN THE APIARY 11 pluton or other forms of bacteria found in larvae affected by Euro- pean foulbrood. It seems probable, therefore, that B. atvei and B. pluton. are only different forms of the same bacterium. RACES OF BEES AFFECTED AND CONDITION OF COLONIES Common black and Italian-black hybrid bees are more frequently affected by European foulbrood than are Italians, and weak colonies are usually more seriously affected than are strong ones. This dis- ease frequently appears year after year in colonies of black or hybrid bees, and heavy losses may be suffered, but among Italian bees losses are usually unimportant. At times, however, European foul- brood spreads within strong colonies as well as within weak ones, and occasionally Italian bees are seriously affected. EFFECT UPON THE COLONY European foulbrood is most common in the spring, when brood tearing is at its height. Usually the earliest reared brood is not af- fected. Sometimes this disease appears suddenly and spreads rapidly within infected colonies. At other times it spreads slowly and does little damage. As a rule it subsides by midsummer, but occasionally it continues to be active during summer and fall, or it may reappear in the fall. A good honey flow seems to hasten recovery. In severe cases colonies are seriously weakened or killed. Usually the worker bees remove dead brood promptly (fig. 8, M) ; but in some colonies, particularly weak ones, it is allowed to accumulate. SYMPTOMS APPEABANCE OF THE COMBS In mild cases and in early stages of European foulbrood the ar- rangement of the brood in the combs is not noticeably irregular. The degree of irregularity increases with severity of the disease and the length of time it has been present. In advanced cases open cells, which may be empty or contain eggs or healthy or affected brood, are irregularly scattered among cells of capped brood (fig. 9). Cells with discolored, sunken, or punctured cappings (fig. 8, N and 0) may be present, but these are less common than in American foul- brood. Irregular arrangement of the brood is not a dependable symptom of European foulbrood, however, and final diagnosis should depend upon symptoms shown by the dead individuals. APPEAKANCB OF SICK LARVAH AND TIME OF DEATH Sick larvae lose the plumpness and glistening white color of healthy larvae and become flat white. A faint yellow color, which is an im- portant symptom, may also appear before death. Sick larvae may show abnormal movements and occupy an unnatural position in the cells. The greater number of larvae die while coiled on the bottom of open cells (fig. 8, A-I). Many larvae also die at the age when they 12 CIKCULAE 3 9 2, U. S. DEPARTMENT OF AGRICULTUBE FiGDEB 8. — Symptoms of European foulbrood. A-C, Larvae at the earliest age at which they may be attacked by the disease ; A, earliest symptoms, B, more advanced symptoms, C, scale of a larva that died at this age. D, Healthy larva of slightly older age. M, Sick larva of this age. F, Scale of larva of this age. (?, Healthy larva at the oldest age that larvae normally remain coiled on the bottom of the cells. H, I, Larvae of this age dead of European foulbrood. J, Healthy larva lust before the cell is capped, if, L, Larvae of this age dead of European foulbrood. M, Dead larva that has been partly removed by the bees. N, Discolored and sunlien capping over a dead larva. 0, Punc- txired capping over a dead larva. DIAGNOSING BEE DISEASES IN THE APIAEY 13 would normally be spinning their coccoons (figs. 8, /-Z, and 10, D-F). Comparatively few larvae die while fully extended (fig. 10, A, B, C, G, 11, 1). Pupae are rarely affected by this disease. Figure 9. — European foulbrood ; heavily infected comb showing larvae in various stages of disease and decay. Larvae dead of European foulbrood, therefore, are usually coiled on the bottom of the cells but may be irregularly twisted or fully extended. COLOB OF THE DEAD BKOOD Soon after death larvae become dull and grayish or yellowish- white. During decay the color deepens and may become brown or almost black. The tracheae, or breathing tubes, in dead larvae usu- ally show more clearly than in healthy ones (fig. 8, G and F). They appear as radiating white lines in the dead coiled larvae and as narrow white lines across larvae that die while extended. A white line which crosses the radiating white lines can frequently be seen on the side of dead larvae. The prominence of the tracheae is a valuable symptom of European foulbrood but is not strictly depend- able. An elongated, dull grayish-white or yellowish-white mass can be seen through the skin along the back of sick and recently dead larvae. This mass is within the chyle stomach and consists of a turbid fluid that contains many bacteria. In healthy larvae, pollen in the stomach can often be seen through the skin along the back (fig. 8, /) , but the color is usually of a brighter and deeper shade 14 CIECULAE 3 9 2, V. S. DEPARTMENT OF AGRICULTURE FiGnsB 10. — Symptoms of European foulbrood : A, Ventral view of an extended larva re- cently dead of European foulbrood ; B, extended larva partly decayed ; C, scale of an ex- tended larva; D, recently dead larva, and E and F, scales of dead larvae irregularly twisted ; G, oral view of recently dead extended larva ; II, partially decayed larva ; I, scale of larva that died after straightening out. DIAGNOSING BEE DXSe/sES IN THE APIARY 15 of yellow than in affected larvae. Dissecting sick or recently dead larvae and examinina- the contents of the digestive tract helps in making a diagnosis after experience has been gained. CHANGES CAUSED BY DECAY AND DRYING The appearance of the dead larvae changes gradually during de- cay and drying. The gray and the yellow colors deepen during decay, but the depth of the color in scales varies considerably. Lar- vae that die before the cells are sealed dry rapidly, and decay is soon stopped; hence these scales are usually light colored. Larvae that die after the cells are sealed usually become dark brown or nearly black. Diagnosis of European foulbrood is more difficult after the dead brood is decayed and dry. For a short time after death, larvae can be removed from the cells without tearing the sldn. Within a few days the skin and other tissues become soft; and the larvae settle against the lower wall of the cells, and appear moist, melting, and flattened. At this stage in decay, larvae are somewhat translucent and watery and cannot be removed entire. Upon drying they become pasty, sometimes ropy, and finally rubbery or brittle. Scales of European foulbrood usually do not cling closely to the cell walls and are easy to remove. Larvae that die of European foulbrood in sealed cells may become quite ropy and resemble larvae dead of American foulbrood. Since the bees remove dead brood from open cells first, it sometimes hap- pens after disease ceases to be active that the brood which died in sealed cells is all that remains in the combs. When this happens it may be difficult to tell whether American foulbrood, or European foulbrood, or both of these diseases are present. ODOR OF DEAD BROOD The odors of European foulbrood cannot be accurately described but must be learned by smelling of the dead brood. When there are many decaying larvae in the combs an odor that is characteristic of this disease can sometimes be detected. Usually the odor of recently dead larvae is slight. A sour odor is sometimes present in partially decayed larvae. Some larvae, particularly those that die after they have straightened out and the cells are sealed, develop a putrid odor resembling the odor of decayed meat. This odor is nearly always present in larvae killed by European foulbrood which in other re- spects resemble larvae killed by American foulbrood. After the odors have been learned, the odor test helps considerably in dis- tinguishing between European and American foulbrood when other symptoms overlap, PARAFOULBROOD CAUSE Parafoulbrood is caused by bacteria which resemble the bacteria of European foulbrood. Worker, queen, and drone larvae and some- times pupae are killed by the bacteria, which grow within the di- gestive tract, but adult bees are not affected by this disease. The spore stage of the bacterium found in affected brood is known as Badllv^ para-alvei. 16 CIRCULAR 3 9 2, U. S. DEPARTMENT OF AGRICULTURE DISTRIBUTION AND RACES OF BEES AFFECTED This disease has been found only in limited sections of North Caro- lina, South Carolina, Georgia, and Florida. All the races of bees common in North America are susceptible, but Italians appear to be more resistant than are common blacks and hybrids. Weak colo- nies are usually more seriously affected than strong ones, but heavy losses of brood may also occur in strong colonies. EFFECT UPON COLONIES Parafoulbrood progi'esses rapidly within some colonies and se- riously weakens or kills them. In others it progresses slowly, the colonies are not noticeably weakened, and the disease disappears of its own accord. Some colonies clean out the dead brood promptly, while in others it is allowed to accumulate. In some apiaries only a few colonies will be diseased, while in others every colony will be Figure 11. — Parafoulbrood ; a heavily infected comb showing larvae in various stages of disease and decay after death. affected. Loss caused by parafoulbrood may vary from the weak- ening of a few colonies to the loss of entire' apiaries. This disease usually appears in the spring and disappears by midsummer, but oc- casionally colonies exhibit symptoms of the disease throughout the year, or there may be a slight increase of infection in the autumn. The first brood reared in the spring is not affected. SYMPTOMS APPEARANCE OP THE COMB.S Infected combs resemble combs with European foulbrood. The brood is more or less irregular, depending upon the amount of infection and the length of time the disease has been active (fig. 11). DIAGNOSING BEE DISEASES IN THE APIARY 17 Dead brood in open cells is removed by the bees sooner than that in sealed cells. Occasionally the bees increase the thickness of the cappings over dead brood in sealed cells. Such cappings appear dark, sunken, and greasy, and are sharply depressed m the center. Dead larvae may remain in these cells for months, or even over winter. APPEiABANCE (IF SICK BROOD AND TIME OF DEATH Sick larvae change from glistening white to dull or flat white, and a slight loss of plumpness may be noticed. They move uneasily in their cells and are often found in abnormal positions. A yellow discoloration occasionally appears before the larvae die. Death from parafoulbrood usually occurs when the larvae are coiled or irregularly twisted in the cells, but many extended larvae and a few pupae are killed. The average age at the time of death is usually somewhat greater than in case of European foulbrood. APPEARANCE OF DEAD BKOOD Larvae dead of parafoulbrood are coiled, irregularly twisted, or fully extended in the cells, depending largely on the age when death occurs. Usually the nmnber of larvae and pupae that die in sealed cells is somewhat greater and the number of larvae that die while coiled is less than is the case in European foulbrood. Larvae that die in open cells dry rapidly and usually form light- colored scales, although some become light brown, reddish brown, or dark brown. Larvae that die in sealed cells dry more slowly, and decay continues for a longer time. Many of these become reddish brown during decay and form dark-colored scales. In an occasional decayed larva or scale the tracheae show clearly. In sick or recently dead larvae the stomach can be seen through the skin along the back. The content of the stomach consists of a turbid grayish or yellow- gray fluid that contains many bacteria. COKSISTEWCY OF DEAD BEOOD Dead larvae soon become soft and watery. In capped cells some become decidedly ropy during decay and form dark reddish-brown or brown scales of a leathery consistency. In open cells the larvae usually become pasty and later form light-colored brittle scales. In some dead larvae ropiness develops rapidly, while in others it develops slowly or is entirely absent. Eopiness in parafoulbrood often resembles this symptom in American foulbrood. When this occurs a distinction can usually be made by noting the color and odor of the dead brood. The scales can be removed easily from the cells. ODO'K OF DEAD BROOD Only a slight odor can be detected in recently dead brood, and most larvae have but slight odor during decay. Many dead larvae in sealed cells and also some in open cells, however, develop an intense putrid odor similar to that of European foulbrood but fre- 51309° — 36 3 18 CIKCULAK 3 9 2, XJ. S. DEPARTMENT OF AGRICULTURE quently much more intense. It can sometimes be detected as soon as a decayed larva is removed from the comb, and can also be detected in the dry scales. A reliable symptom of this disease is a reddish-brown color and ropy consistency of decayed brood, particularly when accompanied by a pronounced putrid odor. SACBROOD Sacbrood is caused by a filterable virus, an organism so small that it will pass through a porcelain filter and cannot be seen under the most powerful microscope. Infection in the case of sacbrood takes place by way of the alimentary canal. Both worker and drone brood may be affected. It has not been definitely determined FiGDKB 12.— A brooa comb heavily infected with sacbrood, showing numerous dead larvae. whether or not queen larvae are killed. Pupae are killed occasionally, but adult bees are not affected. IMPORTANCE Sacbrood is a widely distributed disease, but it usually does not cause serious losses. It is important, however, for beekeepers to recognize sacbrood so that it will not be confused with the f oulbrood diseases. Sacbrood may appear at any time during the brood-rearing season, but it IS most conmion during the first half of the season, and prac- tically always subsides after the main honey flow has started. In ordinary cases the colonies are not noticeably weakened by sacbrood but m exceptional cases, when 50 percent or more of the brood is affected, they may be considerably weakened. DIAGNOSING BEE DISEASES IN THE APIABY 19 SYMPTOMS APPEARANCE OF THE COMBS In colonies with sacbrood the brood is slightly irregular. Scat- tered here and there among the healthy brood are cells containing dead brood (fig. 12). The capping-s over dead brood are first punc- tured and later removed by the bees. The holes vary in size, and occasionally there is more than one. Sometimes the size and uniform shape of the hole indicate that the cell has never been completely capped. Dead larvae usually lie fully extended on the floor of the cell (fig. 13, B-F), showing the dark-brown heads through the openings (fig. 12). When these conditions are present the dead larvae should be studied carefully. Figure 13. — Sacbrood : A, Oral view of healthy larva at the age when death usually occurs from sacbrood ; B—F, stages in decay and drying of larvae dead of sacbrood. AGE OF AFFECTED LARVAE Death from sacbrood almost always occurs after the cell is capped and the larva has spun its cocoon and is motionless. At this stage the larva is fully extended on the floor of the cell. In heavily infected colonies a few coiled larvae may be killed. COLOR AND ODOR OF THE DEAD BROOD Shortly after death caused by sacbrood the color of the larva changes from the pearly white to a slightly yellowish color. This gradually becomes darker, beginning with the head and front third of the larva, which soon changes to a brown or grayish brown and later a dark brown. Scales are almost black for the entire length, the head end usually being darkest. 20 CIECULAE 392, r. S. DEPABTMEXT OF AGKICULTUEE There is little, if any, distinctive odor associated with sacbrood, although watery, saelike larvae in the later stages may have a slightly sour odor. COXSISTEXCY OF DEAD BROOD The skins of dead larvae remain tough, and are easily removed from the cells intact. The internal tissues at the same time become watery, but rarely show any indication of ropiness. Suspended in the waterlike liquid are numerous fiiie brown granules. '\^Tien a dead larva is removed from the cell, liquid collects beneath the skin, which resembles a sac ; hence the name sacbrood. As the larva dries, the skin becomes wrinkled, usually most noticeable in the front third (fig. 13, C-F). After thorough drying it forms a scale. PONinOX OF THE DE.U) BKOOD IX THE CELLS Larvae killed by sacbrood almost invariably lie extended lengtli- wibe with their back> on the floor of the cells (fig. 14, 6^). In con- tract with American foulbro(xl (fig. 5. (r), the head and front third of a larva dead of sacbrood is elevated while the tail end, as drying yirogresses, slumps partly down off the bottom of the cell. The raised head is a distinctive symptom of sacbrood. Since adult bees often remove recently dead larvae by biting off a piece at a time, occasional celL will be found in which only part of the dead larva remains. THE SCALES Scales of larvae dead from sacbrood can be removed from the cells with ease. They are dark grayish brown, or nearly black, and are hard and brittle with the head end turned sharply upward. The outline may be ctmiewhat wavy. The back or lower surface is smooth and polished, while the upper surface is rough and somewhat con- cave. The lower surface takes the form of the cell walls and gives the entire scale a boatlike appearance often referred to as gondola- shaped or like a Chinese shoe. INFECTION WITH TWO OR MORE BROOD DISEASES In localities where two or more brood diseases are prevalent, more than one briK^id disea>e will occasionally be found in the same colony or even in the same comb. So far as is known a single larva is never affected by more than one disease. When American foulbrood is found in the same comb with European foulbrood or sacbrood. usually one of the disea>es will be more prominent, at least in the active stages, which may catise the mixed infection to be overlooked, the beekeeper seeing only the most prominent symptoms. In cases where there is doubt or a suspicion that more than one disease may iic- present in the same colon}-, a laboratory diagnosis is desirable to prevent imj^roper treatment. Since Amtr/can fmdhrood /,y the most ift/'!oii-i. a careful search for this disease should edicai/s I/e made tcoi u'liiii another disease is kii-otcn. to he present. Table 1 gives in summary form the characters differentiating the principal brood diseases. DIAGNOSING BEE DISEASES IN THK APIARY 2i FiGUEB 14. — Symptoms of sacbrood : A., Ventral view of a healthy larva at the age when death usually occurs from sacbrood ; B-F, stages In the decay and drying of larvae dead of sacbrood, ventral views ; G, lateral view of larva recently dead of sacbrood ; B, lateral view of scale. 22 CIRCULAR 3 9 2, U. S. DEPARTMENT OF AGRICULTURE S a S H 03 tn >> ,a ■3s is ■a ^ p. £ oo 3 „" I Is 0] ■03 96P a) 0) as ffl (M fc- (0 o m o bfi « 3 P^ <5 a " 3 (i' a| I XI o ii o at M <» O H fl 'C >i o ^ ca o O P ■ffJS Sis" °.2 ft r 30 3 2" ■o 2 fl ? o H E^ s C3 O fl 4} ■g!:l r^ ® O § ftS g OJ3 9 § 3 S tsss £=.as axJ m ea a 0—0 O O §3 «■« '^ o ."tn a> 3 2 ^ b" a 36-2 Eg s !: S35-S •- « S N £ fc -3 o ^ fl3™a"B3 g oS'a.Sg'3 ^n^-a iS 0,3 M u n iS ei-'^ ft c H i> 9 .fe.S.a O [ Sc8>^BtjP Si fts s te ,. a M [ a*" >»'=3 ^ >a 3 t, Sja>-S " S^§Sfc:&la t*+^i S fl tj zz gftgt.,S4§> o 3_,,g 3 ^ 2 § D S ©fta S=3 M +j,£2 O fc- o O O 0) ■o S 0-3 So = a M'ga o"g£ §s >• .29ft h o o I « « S >i ■^all fe p.,2 f* rt K I will 2 S ® ?^ ^^' » . ° . - o „ fc a t," r3 ?T3" v^ w o fe ® J D §^ i'S.S, ■9 I rsS 3 ft^^g 5.g o aos='«3iaSfe og^S'l-sfisas ■§2p3.ISiS'ao ►^ B -4 S "^ ,t»*> 3 111 "C a » rH •coftjSu .ft°§ 3"'.&^ g >■ X ft ■S ^ a> a i>>'3 P'O P w-Ssa •3 ta ^^ 03 S.3 J=« *3 i« t-'o a?.an| oij « aj " giB MM 3 £■§-■§9 -i:s o a> o c sa ft ° ft" 03-0 0) O o P'^ S H w 13 tr _- S MO o^ a.9e|s P,ftJ3 g'O M C DIAGNOSING BEE DISEASES IX THE APIARY 23 FUNGOUS DISEASES OF BROOD CAUSES In addition to the diseases previously described, diseases of the brood of bees are caused by several different fungi. The most com- mon of these are species of Aspergillu.^. In this country A. flamis attacks brood more frequently than other fungi. In Europe a fun- gus known as Pencystis apis causes a disease of brood known as "'chalk brood." This fungus does not occur in North America. IMPORTANCE Normally only slight losses of brood are caused by fungous dis- eases. The small amount of brood that is killed is removed prompt- FiGUEE 15. — Brood comb artificially Inoculated with Aspergillus flavus, a fungus that kiUs the brood of bees. Dead larvae of different ages can be seen in the cells. ly by the worker bees and is rarely noticed by the beekeeper. Brood is most likely to become infected when moisture collects in the hive late in the winter and early in the spring, permitting fungi to grow over the combs. AGE OF BROOD AND RACES OF BEES AFFECTED Brood of all ages, and also adult bees, are susceptible to fungous diseases. After the feeding period is passed, however, and the cells have been capped, brood is less likely to become infected. All the races of bees conimon in this country are susceptible. 24 CIRCULAR 3 9 2, U. S. DEPARTMENT OF AGRICULTURE APPEARANCE OF THE DEAD BROOD A larva killed by a fungus becomes noticeably harder soon after it dies, and the glistening white changes to a dull creamy white. Later the dead larva becomes shrunken and wrinkled. The head end of a larva that dies after it has straightened out in the cell dries most rapidly and often curves upward at first but later tends to straighten out again (fig. 15). The fungus soon grows through the skin in a ring just back of the head and forms a sort of white collar. Within 1 or 2 daj^s the fungus grows over the entire larva and forms a false skin which clings closely to the true skin. The color at this stage is clialky white. The fungus produces spores on the outer surface of tlie dead larva, and the white changes to a shade of green, black, or other color, corresponding to the color of the spores. Spores form earliest and most abundantly near the head end of FiGnKE 16. — Dead isrood (mummies) killed by a pathogenic fungus. dead larvae. The color of the spores deepens as they mature and fades as they become old and dry. After dead larvae and pupae have become dry they are known as mummies (fig. 16) . In Europe the disease of bees caused by Aspergillim -flavus is called "stone brood" on account of the hard texture of the dead brood. DISEASES OF ADULT BEES WHAT TO OBSERVE WHEN LOOKING FOR DISEASES OF ADULT BEES No general rules can be given for the diagnosis of diseases of adult bees. Such diagnosis is made more difficult by the fact that at any time of the year many bees may die as a result of old age or abnormal conditions. Symptoms of the different diseases over- DIAGNOSING BEE DISEASES IN THE APIARY 25 lap, and usually a diagnosis cannot be made in the apiary. There are a few dependable symptoms of diseases of adult bees, however, which can be recognized without a miscroscope, and with good samples it is sometimes possible to make a diagnosis in the apiary. NOSEMA DISEASE CAUSE Nosema disease is caused by a minute, single-celled animal parasite known as Nosema apis. Adult workers, drones, and queens are affected. Spores of N. apis enter the body of the adult bee with food or water. They germinate within the stomach and attack the tissues which line the stomach or mid-intestine, with varying harmful effects. IMPORTANCE Nosema disease is wide-spread and under conditions favorable for its spread causes extensive losses of adult bees. When accompanied by dysentery brought on by long winter confinement, the disease may spread rapidly within infected colonies and result in the death of the colonies late in the winter or in the spring; or heavy losses from Nosema disease may continue for weeks after the bees have been flying freely and dysentery has subsided. Infected bees usually perform^ their normal duties until they are too weak to continue. The shortened life of infected bees weakens or kills the colony. SYMPTOMS SHOWN BY THE COLONY The first noticeable symptoms shown by a colony heavily infected by Nosema apis are increasing restlessness of the bees and a weaken- ing of the colony. When only a small number of bees are infected, the loss may be so gradual that it is not noticed. At other times the death rate among adult bees is very high, and the colony dwindles rapidly. The queen usually is among the last handful of bees to die. Nosema disease may appear annually at about the same time. During any time of year, however, colonies with bees infected by N. apis may be foimd that show no noticeable loss. SYMPTOMS SHOWN BY INFECTED BEES In the individual bee the symptom most commonly observed is inability to fly more than a few yards without alighting. Many bees will be seen crawling on the ground, on the bottom board, at the entrance,* and on the top of frames when the cover is removed. Sometimes infected bees crawl actively long distances from the hive, or they may crawl up blades of grassi in an effort to fly. At times they collect in small groups on the ground in front of the hive. ft is mostly the older workers that are killed, although drones, queens, and young workers may be attacked. At times the disease seems to be aggravated by periods of cold, damp weather, particu- larly in the spring when the bees cannot fly freely. The legs of affected bees may be dragged along in crawling, as if paralyzed; and the rear wings may be unhooked from the front 26 CIBCULAK 3 9 2, U. S. DEPARTMENT OF AGKICULTUEE wings and held at abnormal angles. Such bees are capable of only feeble fanning with the wings. The abdomen is often distended with feces and may appear shining or greasy. APPEARANCE OF THE INTESTINAL TKACT The intestinal tract of bees infected by Nosema apis is fre- quently swollen and discolored. When favorable specimens are at hand this symptom can be used for diagnosis in the apiary. If the bees are alive, or have just died, the entire intestinal tract can be removed as follows : Remove the head and hold the thorax with the thumb and forefinger, then grasp the tip of the abdomen with a pair of forceps and pull gently. By this procedure the entire intestinal tract can frequently be withdrawn from the abdomen. In healthy bees the long, cylindrical mid-intestine is usually of a brownish-red, vellowish. or gravish-white color. Circular constric- FlGUBE 17. — Nosema disease : A, Intestines from liealthy bees ; B, Intestines from bees infected with Nosema disease. tions show for nearly^ the entire length of the intestine (fig. 17, A), and the tissues are fairly tough and of a healthy appearance. When Nosema disease is present the mid-intestine swells (fig. 17, B) but finally shrinks to about normal size. Heavily infected intestines are usually of a dull grayish white, and some or all of the circular constrictions disappear (fig. 17, B). The tissues become soft and waterjr and are more easily crushed than are the tissues of healthy intestines. The fluid that flows from heavily infected intestines when they are crushed is whiter and more turbid than is the fluid from healthy intestines. After experience has been gained, it is often possible, when favorable specimens can be obtained, to make a diagnosis of Nosema disease in the apiary. There is considerable variation in the appearance of the mid-intestine of healthy as well as infected bees, however, and in many cases, particularly after tlio bees are dead, a microscopical examination is necessary for a diagnosis of Nosema disease. DIAGNOSING BEE DISEASES IN THE APIABY 27 ACARINE DISEASE CAUSE Acarine disease of adult honej^bees is caused by a very small mite, Acarapis woodi Remiie. This mite lives as a parasite in the anterior thoracic tracheae (breathing organs), where it feeds directly upon the tissues of the bees. Bees are not noticeably injured by one or a few mites, but the mites breed and multiply within the trachea until they become very numerous. Heavily infested bees are unable to fly and soon die. DISTRIBUTION ^ This disease of adult bees is not present in North America, but serious losses occur from it in Europe. Queens imported from Europe are sent directly upon arrival in the United States to the Government Bee Culture Laboratory at Washington. The attendant bees are examined for mites and other bee diseases. The imported ?ueens are placed in new cages with young worker bees from the rovernment apiary before they are sent to the beekeeper who pur- chased them, in accordance with an act of Congress of 1922.^ The rules and regulations and special rules incident to this act can be obtained by writing to the Bee Culture Laboratory, Bureau of En- tomology and Plant Quarantine, National Agricultural Research Center, Beltsville, Md. TRANSMISSION The mites enter the tracheae at their openings (spiracles). When a few bees, or even one, of a colony become infested with fertile female mites, acarine disease may be transmitted to other bees within the colony. The mites mate within the tracheae, and later some of the females crawl out and enter the tracheae of other bees within the hive, thereby transmitting the disease. Acarine disease is thought to be transmitted from diseased to healthy colonies by the drifting of infested workers, or drones, or by robber bees. This disease may also be transmitted by requeening a colony with an infested queen. SYMPTOMS Infested bees are unable to breathe normally, and the walls of the tracheae and other tissues are injured. Bees that contain large numbers of mites are unable to fly and are known as crawlers. Crawlers usually leave the hive, when the weather is favorable, and iThe act of Aug. 31, 1922 (Public, No. 293 — 67th Cong.), entitled "An Act To regulate foreign commerce in the importation into the United States of the adult honeybee (Apis mellifica)," provides as follows : "• * • That, in order to prevent the introduction and spread of diseases dangerous to the adult honeybee, the importation into the United States of the honeybee (Apis melli- fica) in its adult stage is hereby prohibited, and all adult honeybees offered for import into the United States shall be destroyed if not immediately exported : Provided, That such adult honeybees may be imported into the United States for experimental or scientific purposes by the United States Department of Agriculture : And provided further. That such adult honeybees may be imported into the United States from countries in which the Secretary of Agriculture shall determine that no diseases dangerous to adult honeybees exist, under rules and regulations prescribed by the Secretary of the Treasury and the Sec- retary of Agriculture. "Sec. 2. That any person who shall violate any of the provisions of this Act shall be deemed guilty of a misdemeanor and shall, upon conviction thereof, be punished by a line not exceeding S500 or by imprisonment not exceeding one year, or both such fine and Im- prisonment in the discretion of the court." 28 CIRCULAR 3 92, U. S. DEPARTMENT OF AGRICULTURE die outside. When large numbers of infested bees crawl from the hive at about the same time, the condition is known as mass crawling. Bees often continue to work for weeks after they have become infested by mites, and acarine disease may be well advanced in a colony before symptoms are noticeable. The most commonly rec- ognized symptoms are crawling and the loss of ability to fly. Crawl- ing may come on gradually when the disease spreads slowly within the colony, or it may develop rapidly and result in mass crawling. After mass crawling has occurred, the colony is freed of most of the diseased bees and may appear to recover temporarily. Mass crawling often follows a period of unfavorable weather. Crawling is frequently accompanied by retention of feces, swollen abdomens, and un jointed wings. PiGUEE 18. — Acarine disease. Discolored trachea taken from the thorax of an infested bee. The mites that cause acarine disease can be seen through the tracheal waU. Mag- nified 75 times. (Photograph by J. Rennie.) DIAGNOSIS IN THE APIARY In healthy bees the tracheae are always pure white. In heavily infested bees the tracheae become bronzed or blackened in irregular spots. The presence of these spots is used as a symptom in diagnosis. With the aid of a lens that magnifies 6 or 8 times, the dark-colored spots can be distinguished. They may be few in number, or there may be so many that the trachea appears black (fig. 18). In making examinations for acarine disease in the apiary it is best to use crawlers. The tracheae of bees killed by other disorders often DIAGNOSING BEE DISEASES IX THE APIARY 29 become black after a few days, while infested bees that are able to fly may not show the discolored spots on the tracheae. The head and front part of the thorax (prothorax) with the first pair of legs should be cut away and discarded. This will bring into view the first pair of breathing tubes, which are the ones most likely to be discolored if acarine disease is present. SEPTICEMIA CAUSE Septicemia is a slightly infectious disease of adult honeybees. It is caused W growth in the blood of infected bees of a bacterium known as BaciUtis apisepticics. This bacterium may be present in polonies, in the soil near infected colonies, or in water that has been in contact with bees killed by septicemia. Bees that become wet FiuHBE la. — SeiJticemia. Bees dead ol septicemia dismembered by slight handling. with soil water from about the hives may become infected. This is probably the most common way by which the disease spreads. The bacteria seem to enter the blood of bees by way of the breathing tubes. The presence of large numbers of bacteria in the food seems not to injure bees; but if a drop of water that contains the bacteria is spread over the entrance to the breathing organs (spiracles), dis- ease and death from septicemia usually result. The disease does not spread readily from sick or dead bees to healthy ones unless plenty of moisture is present. The bacteria are soon killed by drying, and the disease rarely occurs under dry conditions. Several other species of bacteria and yeasts cause septicemia when they are placed in the blood of bees by puncturing the body covering, but they seem to be unable to gain entrance to the blood of uninjured bees. 30 CIECULAE 3 9 2, tX. S. DEPARTMENT OF AGKICULTUEE SYMPTOMS Bees die within a few hours after they have shown the first symp- toms of septicemia. Sick bees leave the hive or are carried out by healthy workers. Sick bees resemble bees that are chilled, and their movements gradually become slower. Before death, the blood loses the normal clear, pale-brown color and becomes turbid and milky, owing to the presence of many bacteria. This symptom can some- times be used in diagnosis in the apiary. By pulling oif the head and abdomen of a dying or recently dead bee and pinching the thorax between the fingers a drop of blood oozes out that can be examined. Dead bees decay rapidly, the muscles of the thorax soon becoming soft and pasty, and the bodies have a characteristic putrid odor that is of some assistance in the diagnosis of this disease. Within 1 or 2 days the body, legs, wings, and antennae usually fall apart at the joints when the bees are handled (fig. 19). EFFECT UPON THE COLONY Only rarely are colonies noticeably weakened by septicemia, but many individual bees may be killed. Septicemia is less serious than Nosema disease or acarine disease. AMOEBA DISEASE CAUSE Amoeba disease of bees, caused by a one-celled animal parasite, V ahlkamp-fia {Malpigham.oeba) mellifica^ was discovered a few years ago in Europe. This parasite grows m the excretory organs of adult bees. In 1927 it was found in two colonies of bees in the apiary of the Bee Culture Laboratory at Somerset, Md. In 1929 it was recognized in a sample of bees sent to this laboratory from California and in 1935 in a sample from Illinois. In Europe, amoeba disease was found only in colonies with Nosema disease, and it was suspected that the two diseases were in some way related. Nosema disease was not found, however, in one of the two colonies with amoeba disease in the apiary of the Bee Culture Laboratory, nor in the sample from California. IMPORTANCE Very little is known about the disease, but the infected bees are undoubtedly injured. Its economic importance is probably negligi- ble, although this point has not been definitely determined. SYMPTOMS The disease cannot be recognized in the apiary by any symptoms. Dead bees that contain the parasites do not differ in appearance from bees dead of other causes. Field bees that appear entirely normal may also be mfected. The parasites are found, often in large num- bers, m the excretory organs (malpighian tubules) of the bees DIAGNOSING BliE DISEASES IN THE APIARY 31 FUNGOUS DISEASES OF ADULT BEES CAUSES It has been Imown for many years that in Europe a disease of adult honeybees is caused by a common fungus, Asfergillus ■fl^avus. In North America it has recently been found that this same lungus and several others attack adult bees. A. flavus has already been noted as attacking brood (p. 23). When recently emerged bees are kept at a temperature about 12° or 14° below that of the brood nest they may be attacked and killed by Mucor hiemalis, a fungus closely related to the common black bread mold. Old bees are not affected by this fungus. Spores of pathogenic fungi get into the digestive tract of bees with food or with water. If a bee comes in contact with fungus spores, some of them may cling to the mouth parts and be swal- lowed later. Nonpathogenic fungi are unable to grow within the stomach of bees, and the fungi themselves may be killed. Patho- genic fungi, on the other hand, grow readily. At first the fungus grows within the stomach, but later the muscles and other soft tis- sues are penetrated by numerous fungus brancheSj and death re- sults. When dead bees are kept under moist conditions, the fungus may grow through the body wall and form spores on the outer surface (fig. 20). FiGUEE 20. — Adult workers and a drone bee killed by Aspergillus fiavus. Spores of the fungus are seen on the bodies of the bees. IMPORTANCE Losses of adult bees caused by fungi are usually of little economic importance. When pathogenic fungi grow within the hive on combs, frames, dead bees, etc., late in the winter or early in spring, fungous diseases are most likely to^ cause significant losses. This can be 32 CIRCULAR 3 9 2, U. S. DEPARTMENT OP AGRICULTURE largely prevented, however, by providing' good wintering conditions for the bees. SYMPTOMS The first noticeable symptoms are restlessness and weakness. Weakness increases until death occurs. A few sick bees may die in or near the hive, but they usually fly or crawl from the hive and seem intent upon getting as far away as possible before they die. For this reason mycosis of adult bees is likely to be overlooked, particu- larly when only a few bees at a time are affected. By pressing the abdomen between the fingers, an increased firmness can sometimes be noticed at the time of death, but it is most notice- able a few hours later. It is unsafe to depend upon this symptom longer than about 1 or 2 days after death, since nonpathogenic fungi may produce similar symptoms in bees killed by other disorders. "PARALYSIS" CAUSE The so-called paralysis of adult honeybees appears to be a slightly infectious disease that causes weakness, trembling, and death of the affected bees. The causei of paralysis has not been definitely deter- mined, although various theories regarding the cause have been advanced from time to time. Recent work at the Government Bee Culture Laboratory indicates that paralysis is infectious. IMPORTANCE Paralysis of honeybees is a widely distributed disorder, but it causes greater losses in warm than in cold climates. Affected colonies usually recover after a short time, but in some cases the disorder continues throughout the season. In the Northern States it usually disappears or remains confined to one or a few colonies within .an apiary, but in the South it sometimes spreads and causes considerable loss. The losses range from a few bees in mild cases to most of the bees of the affected colonies in malignant cases. SYMPTOMS Owing to the fact that the appearance of the sick and dead bees is not always the same, there seems to be a difference of opinion regarding the symptoms of paralysis. Other disturbances of adult bees may also have been mistaken for paralysis. During the early stages of paralysis, affected bees remain on the combs and cannot be distinguished readily, except that the healthy bees often tug and pull at them excitedly. The sick bees make but little effort to defend themselves. Sometimes they offer food or attempt to escape by crawling away. Finally, they leave the hive and die outside or crawl into a comer of the hive or onto the top bars, where they remain until death occurs or until they are carried out of the hive by the healthy bees. Some affected bees die within a day or two after the symptoms have become noticeable, others linger for more than a week, while still others recover. The abdo- DIAGNOSING BEE DISEASES IN THE APIARY 33 mens of the sick bees are usually of normal size but often appear swollen or, less frequently, shrunken. Some of the sick bees retain their hairs until they die, whereas others become partially or entirely hairless, probably because their hairs are pulled out by healthy bees. Ix>ss of hairs is accompanied by a darkening of the abdomen and thorax and a shiny or greasy appearance. The most characteristic symptom of paralysis is weakness and a trembling or shaking movement of the body and wings, frequently accompanied by hairlessness and sprawled legs and wings. Sick bees that are motionless will sometimes show the trembling move- ments when disturbed. Some of the symptoms given here for paral- ysis are also present in other disorders of adult bees and cannot be depended upon alone for diagnosis. Trembling, weakness, and hair- lessness, particularly when accompanied by dark, shining abdomens and sprawled legs and wings, seem to be the most dependable symp- toms of paralysis. Bees in this condition tend to collect on top of the frames. A diagnosis can sometimes be made by carefully open- ing the hive, disturbing the colony as little as possible, and examin- ing the bees on the top bars of the brood nest. TRANSMISSION It has been found by beekeepers that combs of brood from colonies with paralysis can be given to healthy colonies without spreading the disturbance and that the bees emerging from these combs remain healthy. In experiments at the Bee Culture Laboratory, combs of honey and pollen from affected colonies were placed in a healthy colony without paralysis being transmitted. When all the combs of an affected Italian colony were replaced with combs of brood from a healthy Caucasian colony, paralysis appeared among the young Caucasian bees within 2 weeks after the first of them emerged. Paralysis appeared to be transmitted when sick bees and young healthy ones were confined in the same cage. When young healthy bees were wet with water containing the macerated remains of affected bees, paralysis also appeared to be transmitted. The results of these experiments seem to indicate that paralysis is slightly infec- tious and spreads directly from sick or dead bees to healthy ones. SENDING SAMPLES FOR LABORATORY EXAMINATION If only a small amount of brood or a few bees are affected, or the symptoms are unusual, it is sometimes difficult to make a definite diagnosis in the apiary. Examination by laboratory methods is then necessary. It is also desirable at times to have diagnoses made in the apiary verified in the laboratory. HOW TO PREPARE SAMPLES OF BROOD In sending samples for laboratory examination, the following in- structions should be followed: (1) Cut a sample of comb at least 4 by 5 inches in size. (2) Be sure that the sample contains as much of the dead or discolored brood as possible. (3) No honey should he present, and the comb should not be crushed. (4) Mail the 34 CIECULAR 3 9 2, U. S. DEPARTMENT OF AGBICULTUEE sample in a wooden (fig. 21) or strong cardboard box. Do not me tin, glass, or waxed paper. Smears of dead brood and small crushed pieces of comb are tre- quently unsatisfactory for diagnosis but will be examined in case the foregoing instructions cannot be followed. HOW TO SEND SAMPLES OF ADULT BEES (1) Select, if possible, bees that are sick or recently dead; bees that have been dead for some time are not satisfactory for examina- tion. (2) Send at least 50 bees in a sample; if poisoning by a.r- senicals is suspected, 200 or more bees will be needed for analysis. (3) Send bees in a wooden or strong cardboard box and not in tin or glass. Figure 21. — How to send a sample of brood for laboratory examination. HOW TO SEND SAMPLES OF TREATED COMB (1) Send a sample not less than 4 by 5 inches in size if in- fection is heavy, or an entire brood comb if infection is slight. (2) Brood remains should be present in abundance. (3) Pack the comb in a clean wooden box as soon after treatment as possible. (4) Do not send sannples that contain honey. HOW TO ADDRESS SAMPLES All samples should be addressed to the Bee Cultttee Laboratokt, Bttreatj of Entomology and Plant Quarantine, National Agri- CTJLTDRAL EeSEARCH CeNTEE, BeLTSVILLE, Md. Your name and address should be plainly written on the box. If the sample is forwarded by an inspector, his name and address should also appear on the box. ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE WHEN THIS PUBLICATION WAS LAST PRINTED Secretary of Agtieulture Henky A. Waixace. Under Secretanj Rexpord G. Tugweu,. Assistant Secretary M. L. Wilson. Director of Extension ll'orfc C. W. Wakbueton. Director of Finance W. A. Jump. Director of Information M. S. Eisenhower. Director of Personnel W. W. Stockberger. Director of Research James T. Jabdine. Solicitor Mastin G. White. Agricultural Adjustment Administration H. R. Tot.t.ey, Administrator. Bureau of Agricultural Economics A. G. Biack, Chief. Bureau of Agricultural Engineering S. H. MoCboet, Chief. Bureau of Animal Industrij John R. Mohler, Chief. Bureau, of Biological Suriey Ira N. Gabrielson, Chief. Bureau of Chemistry and Soilx Hexrt G. Knight, Chief. Bureau, of Dairy Industry O. E. Reed, Chief. Bureau of Entomology and Plant Quarantine^ Leb A. Strong, Chief. Office of Experiment Stations Jambs T. Jardine. Chief. Food and Drug Administration Walteh G. CAMPBEai, Chief. Forest Service Fiedinand A. Silcox, Chief. Grain Futures Administration J. W. T. Duvel, Chief. Bureau of Borne Economics Louise Stani,et, Chief. Library Clabibel R. Barnbtt, Librarian. Bureau of Plant Industry Feederick D. Kichet, Chief. Bureau of Public Roads Thomas H. JIacDonald, Chief. Soil Conservation Service H. H. Bennett, Chief. Weather Bureau • Wiujs R. Geegg, Chief. This circular is a contribution from Bureauof Entomology and Plant Quarantine Lee A. Stkong, Chief. Division of Bee Culture J. I. Hambleton, Principal Apv- culturist, in charge. 35 . ±>. GOVERNMENT PRINTING OTFICE: I93E For sale by the Superintendent of Documents, Washington, D. C. . . . Price 5 cents UNITED STATES DEPARTMENT OF AGRICULTURE DEPARTMENT CIRCULAR 287 Washington, D. C. November, 1923 THE OCCURRENCE OF DISEASES OF ADULT BEES, II. E. F. PHILLIPS, Apioiilturist, Bureau of Entomology CONTENTS. Page. Introduction 1 Nature of the Isle of Wight disease 2 The United States 8 The Dominion of Canada 11 Great Britain 11 France 12 Switaerland 16 Germany 19 Italy 20 Page. Denmark 21 Czechoslovakia 21 South Africa 22 Other European countries 22 Embargoes to prevent introduction of bee diseases .«. 25 Literature cited 27 Appendix 31 I INTRODUCTION. N VIEW of the wide interest of American beekeepers in the Isle of Wight disease and in the effort to prevent its introduction into the United States, it seems well to summarize the reports that have come to the attention of the Bureau of Entomology regarding its distribution throughout the world. In this paper the situation in Europe is chiefly discussed, partly because the disease is found there, but especially because importations of queenbees are desired from few, if any, countries other than those of continental Europe. There seems to have been no effort elsewhere to make such a com- pilation, yet this information must be needed by beekeepers of many countries. Under the law enacted by Congress in August, 1922, prohibiting importation of adult bees into the United States, the Secretary of Agriculture is authorized to lift the prohibition from countries in which it is determined that no disease dangerous to adult bees exists, and in the formulation of the regulations under this law information on the distribution of the Isle of Wight disease throughout the world is vitally important. It is also desirable to record the data obtained from the additional search for this disease made within the United States in 1922, since the first publication on this subject. Obviously no claim of originality is made for this 53546°— 23 1 2 Department Circular 287, U. S. Dept. of Agriculture. paper, since no cases of this disease are known to occur within the United States and the writer has not had opportunity to study it abroad. All quotations from writers in foreign languages are trans- lations for which the present writer is responsible. The writer would respectfully request the cooperation of bee- keepers in the United States and in foreign countries in furnishing information regarding the Isle of Wight disease in any part of the world, based on the finding of the mite which causes it. American beekeepers are again urged to send for examination samples of all adult bees which show any abnormality. Beekeepers of other coun- tries may during the season of 192^ send such ma/terial to the Bee Culture Laboratory, Bureau of Entomology, Washington, D. C, U. S. A., from any country where provisions are not made for such investigations. For the benefit of foreign beekeepers not familiar with the work of the bureau, it may be stated that it is not the policy of the Bureau of Entomology to publish the names of those sending beekeeping materials for examination. There is no charge for these examinations. Correspondence is preferred in English, French, or German. NATURE OF THE ISLE OP WIGHT DISEASE. In a previous publication of the bureau {J/B) ,^ in which the Isle of Wight disease was discussed, the nature of the disease was not described, since it was not anticipated at that time that the interest in this subject would be so great. It seems desirable now to sum- marize the observations on this disease and to correct certain errors that have appeared in American beekeeping literature concerning it, without any attempt to add any new facts concerning the disease. No adequate discussion of this subject has been available to Ameri- can beekeepers generally, and many inquiries have come to the Bureau of Entomology concerning it. NAME OP THE DISEASE. The Isle of Wight disease, as it is commonly called, is also some- times known in Great Britain as acarine disease, from the order name (Acarina) of the mite which causes it. The latter name was given to the disease by Eennie (5^) and is preferred by him. In German these two names are translated as " Insel-Wight-Krankheit " and " Milbenkrankheit," while in French, in addition to the translations of the common names, acariose is sometimes used. The disease was first recognized in 1904 in the Isle of Wight, hence the usual common name, and in succeeding years was reported to have spread with great rapidity throughout Great Britain. It is probable, however, that it existed in England previous to its discovery on the Isle of Wight {'29), and the Isle of Wight can not be considered as its place of origin. The name of " Isle of Wight disease " is used in this paper because it is better known to American beekeepers than " acarine dis- ease." It is preferable, in the opinion of the writer, since it is not descriptive. Descriptive names for brood diseases have proved con- fusing and it was solely to avoid this confusion that the author proposed the present generally accepted names for the two serious brood diseases in 1906. 1 Reference is made by number (italic) to " Literature cited," p. 27. The Occurrence of Diseases of Adult Bees, II. 3 GENERIC NAME OF THE MITE. Dr. John Eennie, of the University of Aberdeen, Scotland, with his associates, P. Bruce White and Miss Elsie J. Harvey, found in 1920 that this scourge is caused by a microscopically small parasitic mite,^ which was named Tarsonemus woodi {60) . In this paper it will be noted that in referring to reports from various authors the generic names Acarapis and Tarsonemus are both used for this mite. The name Tarsonemus woodi was given this mite by Eennie, and Tarsonemus is still accepted by him as the correct generic name (17). Hirst {31, 32) believes that because of certain important structural modifications, probably attributable to its parasitic habit, a new genus, Acarapis, should be erected for it. It is coming to be rather generally accepted that Hirst is correct, and his standing as a spe- cialist in this field entitles his opinion to great weight. The fact that the generic name Acarapis is composed of two appropriate words, "acarus" (a mite) and "apis" (the honeybee), makes it a fitting name for this parasitic species, but in taxonomic nomenclature fitness of the name argues neither for nor against its acceptance (^5) . The name Acarapis woodi is preferred by Dr. H. E. Ewing {25) , of this bureau, specialist in mites, and by the writer (^5). An important paper on this subject by Vitzthum {70), the well- known mite specialist of Germany, has recently appeared, in which it is shown that the mite causing the Isle of Wight disease certainly does not belong to the genus Tarsonemus, as claimed by Eennie, who discovered it, but that it properly belongs to the new genus, Acarapis, erected by Hirst. Vitzthum states that while Acarapis is related to the genus Tarsonemus, in that both genera belong to the Heterostig- mata, it is more nearly related to certain other genera containing mites parasitic on other insects. He points out that, in spite of the fact that Acarapis contains the only known species of mites which are internal parasites, the body form has not been materially modified by the parasitic habit. This taxonomic analysis by Vitzthum seems to settle conclusively the disputed question as to the correct generic name for this species. OHIGIN OP THE PARASITIC HABIT. Since the recognition of the Isle of Wight disease there has been much speculation among beekeepers as to how the parasite arose and when and by what means it assumed its parasitic habit. There is, of course, not the slightest reason to believe that this mite first in- vaded the thoraxes of bees on the Isle of Wight in 1904, or that its parasitic habit is a new one. From its specialized structure, one must conclude that the parasite has existed as such for untold centuries {of. 60). It is an interesting speculation whether this mite took on 2 Mites do not belong to the class of insects (Hexapoda), but are members of ttie class of Arachmida, to which also belong spidersi and scorpions. A prominent difference be- tween the two classes is the presence of four pairs of legs in the adult arachnids, Instead of three pairs as in insects. Mites (Acarina) are small- animals some species of which are found in great abundance. They generally hare a sacliJse, unsegmented body, usually fused with the cephalothorax, and the mouth parts form a beak. The abdomen and cephalothorax are, however, distinct and the abdomen is segmented in the Tarsonemidae, to which Acwrwpia woodi belongs. The larva on hatching from the egg usually has only three pairs of legs, but after molting has four pairs as in most, adult mites. Most species are free-living, as predacious forms or scavengers, but some species are parasitic and cause certain plant diseases. Some animal diseases are properly attributed to this group. Certain species are found in human ailments, such as itch. Aoarapis woodi seems to be the only described tarsonemid mite wliich is pathogenic on animals. 4 Department Circular 287, U. S. Dept. of Agriculture. its present parasitic habit as an invader of colonial honeybees, or whether, as Bouvier (i^) suggests, it was first a parasite of solitary bees. It has not so far been found in solitary bees. The only prac- tical bearing which this question might have is that if it were actu- ally found that the mite exists in solitary bees at this time, the con- trol of the Isle of Wight disease might be much more difficult (60) . Eennie reports (50) that he and his associates have examined a con- siderable number of other insect species but have found the tracheae always clear of mites. The parasite may also have been a plant feeder, as some have suggested, but there seems to be no evidence of it (SO) , except that other tarsonemid mites have this habit. Migratory nymph stages of related species of tarsonemid mites have been found in tracheae of other insects, but it has usually been assumed that this is a mere accidental and fatal situation for these mites, and that they have entered the spiracles after attaching themselves to the insects, as migratory nymphs of many mite species do. A modification of some such behavior may account for the acquisition of a parasitic and pathogenic habit for Acarapis woodi. The course of evolution of this mite is a question which appears to have little practical sig- nificance. METHOD OF ATTACK. Acarapis woodi apparently enters the body of the honeybee only through the first thoracic spiracles on either side of the thorax, these being larger openings than any other spiracles along the side of the body. Queens, workers, and drones seem equally susceptible to attack, and the mites may enter either one or both sides of the thorax. The structure of the parts of the bee concerned in this in- vasion has been studied by Snodgrass {56) , so that it is useless to go into detail here. All developmental stages of the mite have now been found by Eennie {50)_ and Ewing (^^), indicating that it is able to pass its entire life in this situation. No migratory nymph stage has been described for this species, such as is described for some related species. This species has not so far been found except within the thoracic trachete or as migrants from them, or on the out- side of the bee, as described by Morgenthaler (^i, 4^). It appears, therefore, that it is a highly specialized animal, both in structure and in habit, adapted to a parasitic life, and that the disease is strictly a contagious one. The word " infestation " should be used for this disease, not " infection." The means by which these imprisoned mites feed is not whoUy clear, but it is assumed, without doubt correctly, that they draw their nourishment from the blood of their host. They spot the tracheal trunks with feces and thus color the normally white walls brown or black in a characteristic fashion, so that their presence is easily detected on dissection with slight magnification. Efforts to make artificially controlled inoculations of these mites have not been especially successful {30) , and there is some uncertainty as to the manner in which they pass from one bee to another and succeed m entermg the thorax, but there can be little doubt that they do this merely by crawling out of one bee and attaching themselves to another, later to enter the spiracles. The adult and nymph stages of both sexes are capable of locomotion, but, according to Eennie {S2), only the adult fertilized female mite migrates effectively. The Occurrence of Diseases of Adult Bees, II. 5 • Eennie (62) has repeatedly seen the female mites on ihe outside of infested bees, where they have apparently migrated after mating. The mites live but a short time away from their hosts or after the death of the bees, usually not more than 24 hours. CAUSE OP SYMPTOMS. The manner m which these parasites injure their hosts and cause the Isle of Wight disease is also not entirely clear {S9, SJi). In- ability to fly, so common in advanced cases of the Isle of Wight disease, may be produced by artificial stopping of the spiracles of the thorax {59), so that "crawlers," so frequently found in badly mfested colonies, may result from mere mechanical shutting off of the air which passes into the tracheal trunks. Mites are often so numerous as to make the passage of air virtually impossible. The muscles of flight are located immediately adjacent to the tracheae which are invaded, and an infinite number of small tracheal branches permeate this musculature {66) , so that it may be assumed that the stoppage of these trunks would affect the aeration of the flight muscles, and possibly also that of the head, before any other portion of the bee's body would be affected by this mechanical stop- page. This would seem adequate explanation of the lack of power to fly so frequently seen in advanced stages of the Isle of Wight dis- ease, but since the same symptom is seen in bees affected with other diseases of adult bees, this makes the mechanical explanation less certain. The so-called bee paralysis and Nosema disease, both found in the United States, are not caused by this mite and the tracheae of bees suffering from either of these diseases are clean. In addition to the puncturing of the tracheal walls to suck the blood of the host and the stopping of air circulation, there is believed to be {69) some change in adjacent tissues. It is also assumed that these parasites may produce some material which has a toxic effect on the host, but this does not appear certain. The way in which the symptoms of this disease are produced seems to be a cjuestion of theoretical interest only, since, regardless of its method, this mite ob- viously causes the Isle of Wight disease, and ' therefore the control of the disease must rest on the elimination of the mites. SYMPTOMS. The symptom most commonly observed in a colony suffering from a severe attack of the Isle of Wight disease is that many bees crawl inactively on the ground in front of the hive, unable to fly. They are frequently seen falling from the alighting board to the ground or in cool weather gathered in little clusters on the ground in front of the hive. Such bees usually fail to return to the hive and die of hunger or exposure, probably usually of hunger, since active bees in summer live without food for only a few hours. The wings are sometimes carried in a position abnormal for walking bees, with the front and hind wings neither hooked together nor lying flat on the dorsal surface, often described as " dislocated." The badly infested bees in the spring are usually heavily laden with feces, probably because of their inability to fly during winter. Doubtless because of this accumulation of feces, attention was first given the alimentary tract as the probable location of the cause of the disease {29, JiB). 6 Department Circular 287, U. S. Dept. of Agriculture. • It should be recognized, however, that if for any reason bees are un- able to fly during the period of the year when brood is being reared, feces rapidly accumulate, so that there is no necessary significance in their accumulation. The presence of " crawlers " seems to be the only fairly constant symptom observed in the severe cases; in fact, there are no positive symptoms and there may be no external sign of any sort, so that the only way to be sure of the presence of the dis- ease at present is through the finding of mites by microscopic exami- nation. The crawling bees are often found some distance from the hive. Kennie {61, 52, 53) emphasizes the fact that crawling is an advanced symptom and that the disease may exist in a colony or apiary for months before this symptom is seen. The crawling of bees in front of the hive has also been recorded in almost all cases of the so-called bee paralysis observed by Ameri- can beekeepers and for virtually all of the known affections of adult bees. Wliether this indication of abnormality is any more prevalent for the Isle of Wight disease than for most cases of paralysis can not be stated with certainty, but to one familiar with severe cases of bee paralysis but not with the Isle of Wight disease it would seem doubtful. Rennie (5^) reports the similarity of symptoms in the various diseases of adult bees in England. The shaking of the abdomen, sometimes so prominent a symptom in bee paralysis, and the hurried climbing of grass blades in an effort to take wing are not commonly given as symptoms of the Isle of Wight disease, although they have been recorded {64)- CONFUSION DUE TO CHARACTEE. OP SYMPTOMS. Since almost the same symptoms have been described for all known diseases of adult bees, as well as for cases of arsenical poisoning, it must be concluded that information on the identification or distribu- tion of any of these diseases based only on diagnosis by symptoms is utterly valueless. Some American beekeepers were formerly led from descriptions of symptoms to believe that the Isle of Wight disease is present in the United States, and more recently certain European beekeepers have arrived at the same conclusion. On this unfounded assumption they reason {19) that the methods of treat- ment for adult bee diseases used in some cases in the United States should be helpful in the treatment of the Isle of Wight disease. If then, as appears to have been the case, there is only a slight mani- festation of the disease for a day or two after the treatment, they feel justified in rushing into print with this method as a recom- mended treatment for Isle of Wight disease. Eennie has repeatedly warned beekeepers against such reasoning, but apparently without avail. The treatments that have been described for bee paralysis in the United States were used without knowledge of its cause, and such treatments have been of doubtful benefit at best, so that it can not be reconmiended that European beekeepers look to this coxmtry for methods of controlling the Isle of Wight disease. REMEDIES. It is natural that in an epidemic such as has occurred in Great Britain a great variety of remedies should be tried and recommended. Extravagant claims have been made for certain apparently worthless The Occurrence of Diseases of Adult Bees, II. 7 proprietary compounds and drugs, and unfortunately some of these nave received the indorsement of certain prominent beekeepers, al- though Eennie has given repeated warnings against reliance on such unfounded claims. None of the claims made for chemical treatments can as yet be accepted as valid, and apparently no specific treatment has yet been found. Doctor Rennie has devised a trap for the catch- ing of crawling bees as they leave the hive, but to what extent it is beneficial can not be determined from a reading of the literature. From the nature of the cause of the disease a hopeful line of attack has been suggested through some gas which will penetrate the tra- cheae and kill the mites but leave the bees unharmed. Whether a bee is worth saving which has mites, dead or alive, in its tracheal trunks is questionable. The most promising method of attack seems to be the elimination of the infested bees and the rearing of a large force of young bees to take their places, thus reducing the infestation, as has been urged by both Eennie (5^) and Morgenthaler (4-4). Ren- nie {5S) emphasizes the necessity for swarm control measures, since swarms are especially prone to show crawlers soon after issuing. He says (p. 74) : I lay special emphasis on rational and intelligent management, because I am convinced that much of the loss which has occurred in the past has given to the disease a gravity which is not inherent in it, and has been due to lack of exact knowledge on the part of competent beekeepers in the one case and to unsatis- factory beekeeping in addition in the other. Such a method of control is far more promising for the develop- ment of beekeeping in the infested regions than one based merely on the destruction of the mites. It is identical in theory with the meas- ures for the control of European f oulbrood used in the United States. Rennie (67), in his presidential address before the Apis Club, April 7, 1923, discusses the treatment of affected colonies but does not give any definite directions. He seems to entertain the hope of the eradication of the disease from Great Britain. EFFECT OF CLIMATE. The effect of climate on the propagation of the mite Acarapis woodi has not been studied, and there are no data from which one dares draw conclusions. Since both severe and chronic cases of the disease exist, it may be concluded that some environmental factor may have an influence on the severity of the disease, but it is not indi- cated that these factors are those of climate. Severe cases of the dis- ease have now been described in Great Britain, France, and Switzer- land, and it is certain that almost any climate encountered in any of these countries may be duplicated somewhere within the borders of the United States. To assume that the disease would not do damage here if it were introduced, because of climatic differences, is ex- tremely dangerous, since absolutely nothing has been learned of the influence of climate on the disease. DAMAGE. The damage resulting from the Isle of Wight disease is a subject which it is extremely difficult to discuss at this distance. Reports have varied greatly, all the way from complete destruction of single 8 Department Circular 287, U. S. Dept. of Agriculture. apiaries and of all bees over considerable areas to relatively minor losses. In certain cases (44) to be discussed later (p. 18), the results are chiefly a reduction of the honey crop through the death of an abnormal numher of field workers. It is quite natural that early reports from Great Britain should emphasize the severe cases, especially during the period when the cause of the disease was not yet known. There are evidently chronic cases of the Isle of Wight disease in England and Switzerland, as is clear from a reading of the literature. There is also reason to suspect that such cases are com- mon in France, because of several statements that the disease is less destructive there than in England, these writers evidently having in mind only the reports of severe attacks in England. There can be no question, after reading the reports from Great Britain for a number of years, that the total losses to beekeeping have been heavy and that in many cases beekeepers have lost all their bees from this disease. Some competent beekeepers claim that the damage in Eng- land is not now so great as formerly, but such claims are always to be expected as soon as a disease begins to come under control through more knowledge, and there is no definite reason to believe that the Isle of Wight disease is becoming less capable of doing damage. An interesting paper has appeared by Anderson (61), oi the North of Scotland College of Agriculture, in which he discusses especially the apparently decreased virulence of the Isle of Wight disease in Great Britain in recent years, as well as the various methods which have been employed for its control. The reason for the assumed decrease in the damage from the disease is not clear, but it is stated that on entering new districts the disease is as serious as ever. ERRORS FROM DOUBTFUL DIAGNOSIS. A curious difficulty which one encounters in attempting to esti- mate the damage done in Great Britain by the Isle of Wight disease and the efficacy of suggested remedies arises from diagnoses by incompetent persons, who, even before the determination of the cause of the disease, published diagnoses from dead bees submitted for examination. Some British beekeepers have relied on these diag- noses and have then used them as a basis for experiments on treat- ments, and it is thus impossible to determine the value of much of this work. Many American beekeepers have discounted the reports of heavy losses in Great Britain from the Isle of Wight disease chiefly because of their lack of confidence in the diagnoses and, in fact, many were for this reason led into a sense of false security regarding the disease. Even since the discovery of the cause of the disease, such ill-advised practices seem to continue, in spite of the repeated warn- ings of Eennie. THE UNITED STATES. Immediately following the publication of the first paper (46) on this subject a conference called by the chairman of a committee ap- pointed by the apiculture section of the Association of Economic Entomologists was held at the bee culture laboratory on March 9, 1922, to consider the desirability of taking steps to prevent the intro- The Occurrence of Diseases of Adult Bees, II. 9 duction of the Isle of Wight disease into the United States. At this conference it was decided unanimously that immediate action was desirable. It was recognized, of course, that the failure to find the Isle of Wight disease in the United States during the season of 1921 was not conclusive evidence that the disease is absent from this country, but the far more important fact was to be considered that never in the history of American beekeeping has there been any dam- age from a disease of adult honeybees comparable to that reported from Great Britain. It seemed justifiable to assume that the Isle of Wight disease is not found in the United States, and therefore desirable that, if possible, this scourge be kept out of the country. Immediately following the conference, and as a result of its recom- mendations, the Secretary of Agriculture recommended to the Post- master General that the postal regulations be so amended as to pro- hibit the receipt through the mails of queenbees and their accom- panying worker bees from all foreign countries except the Dominion of Canada. It was thought that with this as a temporary precau- tionary measure it would be possible to prevent the introduction of the disease until such time as more complete protective measures were available. The revised postal regulation was announced under date of March 21, 1922. The action of the Dominion of Canada, also taken at the advice of the committee of the apiculture section, will be discussed later. This action made it possible to admit queen- bees from Canada by an exception to the postal regulation with safety. At the conference on March 9 the drafting of a bill for presenta- tion to Congress was also discussed, and as a result a bill was intro- duced into both Houses of Congress early in April to regulate foreign commerce in the importation of adult honeybees into the United States. This bill was amended at the hearing before the House of Representatives Committee on Agriculture, passed both Houses as amended, and was approved by the President August 31, 1922. The history of this bill in Congress has been recorded by Fracker, Kea, and Gooderham (27). Following the passage of the law, two conferences were held re- garding the regulations provided for by the law for the exemption of certain countries which were determined by the Secretary of Agri- culture to be free of all diseases dangerous to adult honeybees. The first was called by the American Honey Producers' League at St. Louis, Mo., February 8, 1923, and the other by the Bureau of Ento- mology at Washington on March 12. The rules and regulations (see appendix) are based on the facts regarding the distribution of the Isle of Wight disease presented in this circular. In several instances, before the cause of the Isle of Wight disease was discovered, statements appeared in bee journals of the United States to the effect that the disease is present in this country, but as these records are without value they are not specifically mentioned. The symptoms described by beekeepers of Great Britain for the Isle of Wight disease were so nearly identical with some of those observed for diseases of adult bees in the United States as to make such belief plausible. As has been explained earlier (p. 6), this can not be accepted as evidence that the disease is actually present. The failure to find any cases of the Isle of Wight disease in 1921, together with the much stronger evidence based on the lack of any 53546°— 23 2 10 Department Circular 287, U. S. Dept. of Agriculture. serious disease of adult bees in the United States, justified the De- partment of Agriculture in indorsing the bill before Congress and in taking every possible means to prevent the introduction of the disease into the country. The results of the examinations of adult bees in 1921 have been published (46) . The work of the season of 1922 in the search for the mite causing the Isle of Wight disease was done chiefly by L. M. Bertholf, under the supervision of A. P. Sturtevant. Beekeepers were again invited to send to the laboratory any adult bees which showed any abnor- mality whatever, and as a result 183 specimens were received for examination. During the season the beekeeping literature of this country and the correspondence of the bureau failed to record any instances of any disease or abnormality of adult bees of any moment, and this probably is the reason for the relatively small number of bees submitted for examination. As was the case during the season of 1921, the specimens were from widely distributed locations in the United States, which adds greatly to their value as evidence of the absence of the disease. The following table, prepared by Dr. Sturte- vant, shows the results of the examinations made in 1922 : Table 1. — Results of examinations of adult bees, 1922. State or country. Coun- ties. Towns. Nega- tive. Nosema apis. Arsenic. Total. 3 2 10 4 2 1 6 5 6 2 2 1 5 4 2 2 3 1 1 8 7 1 7 1 2 11 1 1 2 2 5 2 4 7 3 3 2 12 4 2 2 1 7 5 6 2 2 1 5 4 2 2 3 1 1 10 9 1 7 . 1 2 13 1 1 3 2 2 5 2 4 8 4 2 1 13 4 2 1 1 6 5 4 1 2 1 4 4 1 3 2 1 1 6 3 California . . . . 19 Colorado 1 Florida Georgia 1 2 Idaho rUiaois 1 Indiana 5 Iowa 3 1 7 Kentucky Louisiana 2 Maryland 1 Micni^an 1 Minnesota 4 2 1 Missouri Nebraska 1 1 3 1 1 } 1^ 9 3 14 1 2 13 1 1 4 2 , 3 8 2 4 14 Nevada New Hampshire 1 3 7 3 10 1 1 1 / '8 \ 3? 1 New York North CaroUna Ohio 4 Oklahoma Oregon 2 2 Pennsylvania 10 1 21 South Carolina South Dakota 1 1 Tennessee 3 2 1 s 1 4 7 2 2 Texas "Utah 2 Virginia Washington 1? West Virginia Wisconsin 7 1 Canada i' Foreign: Italy Camiola 4 4 131 142 123 43 8 17 183 1 2 of these arsenic and Nosema. 3 Also Nosema. 8 10 arsenic; 3 arsenic and Nosema; 4 doubtful arsenic. The Occurrence of Diseases of Adult Bees, II. 11 The bees recorded as coming from Italy consisted of two lots of worker bees which had accompanied queenbees, one lot having been shipped in 1921. The specimens from Carniola were all lots ol workers from mailing cages, the shipments being made early in 1922. During 1922 specimens were received from 36 States, Canada, Italy, and Carniola, and during 1921 and 1922 specimens have been re- ceived from 44 States and from 4 foreign countries. No cases of Isle of Wight disease have been seen in this work except the cases from Scotland discussed in the earlier publication (46). THE DOMINION OP CANADA. Following the discussion of the apparent freedom of the United States and the Dominion of Canada from the Isle of Wight disease and the danger of its introduction at the meeting of the apiculture section of the Association of Economic Entomologists in Toronto in December, 1921, and the conference of March 9 called by the com- mittee of the apiculture section, the committee recommended to the Dominion Government that immediate steps be taken to prevent the introduction of the disease into Canada. It was further recom- mended by this committee that the action of the Dominion and United States Governments be such as in no way to interfere with a free interchange of bees between the two countries. The Dominion apiarist was a member of the committee of the apiculture section. The action of the Dominion Government consisted of an order of the Deputy Minister of Agriculture dated April 22, 1922, prohibiting the importation into Canada of bees, used and second- hand hives, and raw hive goods and products, except honey and wax, from the continent of Europe. A later statement from the deputy minister includes Great Britain in this prohibition. At no time has any noteworthy disease or abnormality of adult honeybees been reported at any place in Canada, and it is believed that there is no disease within the boundaries of that country which has not been found within the limits of the United States. Bees sent to the Bureau of Entomology from various points in Canada have shown Nosema apis, which is not considered a disease danger- ous to adult honeybees under the act of Congress. Bees examined by the Dominion apiarist have failed to show the presence of Acarapis woodi. The former Dominion apiarist, F. W. L. Sladen, was familiar with the Isle of Wight disease in England, and several years before his death recommended a joint action of the Dominion of Canada and the United States to prevent the introduction of this disease, which he knew from experience to be dangerous. GREAT BRITAIN. It is unnecessary to attempt to give the distribution of the Isle of Wight disease in Great Britain, as it may be assumed to be dis- tributed throughout that country. Since the announcement of the cause of the disease, a large number of writers in England have dis- cussed this disease and its treatment, but so far no definitely specific treatment for it has been announced. Doctor Rennie has continued his observations on this disease and has issued several interesting articles {51, 52, 53) on his findings. He has also published some records of the distribution of the disease. 12 Department Circular 287, U. S. Dept. of Agriculture. Further study of the disease in Great Britain is bringing to light many interesting facts. The disease varies considerably in serious- ness from season to season and in various locations, and a study of the conditions under which it does little or no damage would seem to be the most promising field for investigation in deAdsing further control measures, just as has been done in the United States for the variable brood disease, European foulbrood. The extensive shipping of " driven bees," or bees without combs, to replace colonies dead of various causes has doubtless served to spread the Isle of Wight dis- ease, as well as Nosema disease, which is also evidently prevalent there. Since the rapid breeding up of colonies in the spring seems to be very important in eliminating the disease, it is strange that more emphasis has not been placed by British beekeepers on methods of better wintering, which are vital for success at this season in the United States. The most popular means of combating the disease in Great Britain seems to be the importation of bees from Holland and queens from Italy, and the advocates of the two races of bees have claimed for them greater power to resist the Isle of Wight disease than is pos- sessed by bees native to Great Britain. On the contrary, other bee- keepers are vehement in their denunciation of these importations and of the wide distribution of foreign races of bees, so that at a distance it is impossible to judge the merits of the effort. It seems obvious from the discussions on this subject that the only superiority which can be claimed for the two introduced races lies in their ability to breed up faster than the native bees. If this is true they may have some advantages in combating the malady. No general plan for the control or eradication of the disease under Government supervision has been announced. FRANCE. Just as Department Circular 218 was going to press (March, 1922) the first record reached the Bureau of Entomology of the finding of Acarapis woodi, the mite causing the Isle of Wight disease, on the continent of Europe. A brief footnote was added to page 7 of the circular to announce this finding. This record appeared in the Janu- ary, 1922, issue of one of the French bee journals (43). The deter- mination of the mite was made by a competent observer, L. Berland, assistant in the Museum d'Histoire Naturelle, Paris, under the super- vision of Prof. E. L. Bouvier. The mites found in these diseased bees were identified as Tarsonemus woadi after comparisons with Rennie's descriptions and illustrations. The exact location of this outbreak of the disease was not recorded,^ except that it was in the 'From a recent article by Ph J. Baldensperger [" Quelle est la patrie du Tarsone- mus?" La gazette apicole, v. 24, no. 220, p. 57-o9], It would app^r that this first outbreak was in the town of Champseur, Hautes-Alpes. He states that he took a trip of investigation through this territory with an entomologist, M. Poutiers, of Mentone, and that on this trip they were not able to And any of the mites causine the disease altlioueh they had been definitely identified from the apiaries in which tSfd^lase^s first dS covered. The apparent disappearance of the mites is not explained Other beekeeners of the region ai-e reported to have lost their bees, but the cauS of 'this loL milm^ot be „ determined from lack of material. A receAt private Communication (Apr 21! l^^iK^^T +^--=?;-„^°'^w^''^' '"entone, confirms the absence of mites from the bees ^^i ?l??»^'f *"vP--„H^® reports a heavy loss of adult bees at Mentone last December, in the trachese of which he.found Acarapis wooOi, but he is not yet entirely (^rtain that the mite cauang acarme disease in Prance is identical with that described inBneland and Scotland fee states that the disease is made more ^^ere by Spnie^ b^ tteit m-«j;S.t^^ ""** *" "^ ^ ^''^'^ ^^ '"" ^""^^^ ^"*^°- Iiivestigations at MeXnf^e ^11 in The Occurrence of Diseases of Adult Bees, II. 13 French Alps. This finding was confirmed in the report of the pro- ceedings o± the December 21, 1921, meeting of the Soci6t4 Centrale d'Apiculture de France (55). Borland (li) later discussed methods for identifying the mite. In March, Professor Mamelle (^), of I'Ecole Nationals d' Agri- culture de Grignon, asked French beekeepers to send him specimens of bees for examination whenever they find any evidence of disease, and it would appear that he took up the study of the Isle of Wight disease at about that time. At the meeting of the Societe Centrale of April 18, 1922, he reported (36) that the Isle of Wight disease had made its appearance m several Departments of France, especially in Maine-et-Loire, Cote-d'Or, and Saone-et-Loire. Whether these records include the first published record was not stated, but from their location it would appear quite improbable. Two of these Departments are not many miles from the Cantons of Switzerland in which the disease was found at about the same time. It was an- nounced (S) that a committee of four men holding the rank of pro- fessor in various French institutions had undertaken to study the diseases of bees, and in the same month Professor Mamelle, a mem- ber of this committee, addressed the Societe Centrale (26) on this subject. In June Giraud and Sevalle (^8) gave a summary of the work of Doctor Rennie and recorded some recent instances of diseases of adult bees in France, without, however, stating definitely whether the mite was found in the diseased bees. Without this information the record lacks significance in the present discussion. A later record of the distribution of the Isle of Wight disease in France is to be found in a notice (37) in the advertising section of L'Apiculteur for July, 1922, which was an announcement regarding the work of Professor Mamelle, presumably written by him or at any rate published at his request. He thanks beekeepers who have sent him several hundred specimens of adult bees and brood for ex- amination and explains his delay in transmitting his diagnoses, after which the following statement appears : " Regarding the acarine disease (Isle of Wi^t disease)' it can be affirmed that the disease is found somewhat throughout France, but does not seem to present the great injuriousness as in England." In an article written in October, 1922, Bouvier (li), another member of the committee which is investigating bee disease in France, gives some additional records of the finding of the disease in France and adds that it is almost certain that there was an outbreak of the disease in Ardennes in 1919. . He states that the disease is " found nearly throughout France," and definitely records the dis- ease as present in four Departments not previously recorded — Basses- Pyrenees (adjacent to Spain), Lot, Ain, and Hautes-Alpes (adja- cent to Italy). This makes definite records for seven Departments of that country. The definite records are all from central and southern France. Nothing has come to the Bureau of Entomology from French sources to justify a belief that the cases of Isle of Wight disease in France are directly traceable to recent importations from England, nor has any announcement of any regulatory means for the control of the disease in France reached the bureau, and seem- ingly no quarantines have been established as has been claimed (5). Bouvier is inclined to believe that Tarsonemus woodi may also be a 14 Department Circular 287, U. S. Dept. of Agriculture. parasite of certain solitary insects, but does not record any cases of the finding of the mite in such insects. Other articles oh this disease have recently appeared in variovis French bee journals, but many of them have statements regarding the disease that are not based on examinations for mites and there- fore are of little if any value in the present discussion. Since several American beekeepers claim to have used flovrers of sulphur success- fully in combating the disease of adult bees commonly, known in the United States as paralysis, certain French beekeepers are advising the use of this material in combating the Isle of Wight disease, ap- parently on the mistaken idea that paralysis and the Isle of Wight disease are identical. Statements to this effect serve only to confuse the situation and are wholly valueless without experiments to sup- port them. Rennie has repeatedly warned British beekeepers against the drawing of false conclusions of this type. In a recent paper Morgenthaler {66) has specifically warned beekeepers not to place confidence in the use of flowers of sulphur in the treatment of the Isle of Wight disease and has shown wherein they may make serious mistakes in jvidging the effects of treatments tried without proper scientific checks. An effort is being made to get more detailed information regard- ing the distribution of the Isle of Wight disease in France, as this will help in determining how widespread the disease is on the Con- tinent and will permit one to form some idea of the possible dura- tion of the outbreak and the probability that the mite is also found in adjacent countries of Europe.* Several of the statements that have been made regarding the dis- ease in France assume that it is less virulent there than in England. To what extent such statements may be accepted is uncertain, and, if such a difference exists, it may be due to the condition described by Morgenthaler {U) and discussed on a later page of this circular (p. 18). Possibly some of this opinion is due to an overestimation of the losses in Great Britain which naturally arose from the reading of some of the articles from that country which appeared before the cause of the disease was known. Those reporting the supposed dif- ference between France and England do not record whether they have seen cases of the disease in Great Britain.^ THE DUCHEMIN MITE. In the article by Giraud {28) reference is made to the finding by ]6mile Duchemin in France in 1866 of a mite on the exterior of dead bees and also on the sunflower, Helianthus annuus. This mite was mentioned by Rennie {60, p. 776) in the first announcement of the discovery of Acarapis woodi. Since this finding and the references * In a recent private communication (Apr. 3, 1923), M. Luclen Berland, of the Museum Nationale d'Histoire Naturelle, Paris, who made the first determinations of the mite Jrom the French Alps (p. 12), writes that the disease exists throughout the territory of France, not continuously, but widely distributed. He states : " It appears to me probable that the disease exists throughout Europe." He calls attention to the fact that the disease is not easily spread, for all the colonies In an, apiary are not attacked. He has not found the condition which Morgenthaler (U, U) describes of the mite indis- tinguishable from Tarsmiemus woocM, on the outside of bees. He does not believe that the spread of the disease in France is of recent occurrence. 5 A recent letter (Apr. 22, 1923) from a prominent British beekeeper states that early in that month he imported some bees from near Marseilles and that the bees of one colony began to crawl in a few days after they were received. Examination of the tracheae showed them to be crowded with young parasites, but the tracheal trunks were not yet stained. " I am. of the opinion that English bees would not crawl at this stage. The iwssibility of a very severe outbreak in this region [Marseilles] is great," The Occurrence of Diseases of Adult Bees, U. 15 made to it in recent discussions have been the cause of some con- fusion in the minds of beekeepers in America and elsewhere, it is well to record exactly what Duchemin found and to show that this had nothing whatever to do with the Isle of Wight disease. No claim is made by Giraud or Sevalle {28) that Aoarapis woodi and the Duchemin mite are the same species. In 1866 M. Duchemin published a brief article {21) in which he described his finding several years before of a mite in the apiary of a poor peasant to which Duchemin attributed the rapid death of 30 colonies of bees. On near-by flowers of the sunflower, TIelianthus annuios^ to which the bees had access, he also found a mite which he considered to be identical. He concludes from observations then made and from work which he did on this subject in 1864, several years later, that the mites inhabit the sunflower and that in this way these plants are destructive to honeybees as sources of this enemy. This finding was discussed in several succeeding numbers of the same journal by the editor, Hamet, Andre (7), and again by Duchemin {22), who replies to cricitisms of Andre. The notes by Duchemin also appear in another journal {20) . From the recent discussion it appears that this purported discovery was then discussed in other periodicals {15). After the announcement of the discovery of Acarapis woodi as the cause of the Isle of Wight disease, several writers referred to this early finding by Duchemin. The well-known German investigator Von Buttel-Eeepen {15) refers to his own finding of a mite on bees of Apis indiccu and raises the question whether Tarsonemus woodi may not be the same mite which Duchemin found in France. This article was translated in part into English and reply was made to it by the writer (^7). Von Buttel-Eeepen wrote his article before the description of Acoofapis woodi had been published. In the re- ports of M. Duchemin's discovery in certain German periodicals mention had been made of an illustration which Duchemin had pre- pared, but unfortunately this illustration was not available to V on Buttel-Reepen when he prepared his paper. The illustration was not given in the account of this mite which Duchemin published in the Comptes rendus hebdomadaires des seances de I'Academie, des sciences [Paris] {20), and Kennie {50) refers to the absence of an illustration. It appeared in L'Apiculteur, a French bee journal, for February, 1866 (perhaps also elsewhere), and the illustration was copied in the American Bee Journal for May, 1922. Ewing {25) later stated that the Duchemin mite, as determined from this illus- tration, was doubtless a nymph of a species of Trichotarsus, so there is not the slightest reason for thinking that the Duchemin record has anything to do with the Isle of Wight disease. These mites may have been injurious to the bees, as Duchemin claims, but this finding has no bearing on the present outbreak of Isle of Wight disease in France or elsewhere. A similar record of the finding of a mite on bees is mentioned by Manger {39) (see also Elsaas-Lothring. Bienenziichter, 1884 (i)), and Dennler {18) , where it is recorded that Trapp, of Strassburg, found mites in considerable numbers on the head of a bee. An illus- tration of a ventral view of this mite by Schmidt j^much better than the illustration drawn by Duchemin of his mite) is copied by Man- ger, which shows that there is no reason to believe it is any way 16 Department Circular 287, U. S. Dept. of Agriculture. related to the Isle of Wight disease or identical with Acarapis woodi. This mite is identified with a considerable degree of cer- tainty by Vitzthum (S8) as a migratory nymph of Trichotarsus vsmiae. Several species of mites have been found on honeybees and in and about beehives {11, 16, 43, 50) , and it is important that such mites shall not be confused, through careless identifications, with the one species known to be pathogenic tO' honeybees. SWITZERLAND. Since the announcement of the discovery of the cause of the Isle of Wight disease in 1920, the investigators of the Schweizerische Bakteriologischen Anstalt at Liebefeld, near Bern, and the apiary inspectors have been watching for Acarapis woodi in that country. The investigation of bee diseases for Switzerland is carried on in that laboratory, under the direction of Prof. Robert Burri, whose work on the brood diseases of bees is well and favorably known. During the year 1921 examinations were made of adult bees that were suspected of disease or which showed any abnormality, but no specimens of Acarapis woodi were found {^0). It is now known, however, that the mite was present in Switzerland before that time {U)- On February 1, 1922, Dr. Otto Morgenthaler (^i) of the station found dead mites, later identified by Rennie and himself as Tarsonemus woodi, on the outside of bees that had died during the winter, no evidence of disease having been noted previously. He then found that mites of this species, or at any rate indistinguishable by microscopic examination, were present on similar dead bees ob- tained by him from five towns in Switzerland (" wherever I looked for it ") . This finding of the mite on the outside of dead bees which had not shown any definite signs of disease led to speculation as to the possibility that Acarapis woodi is in Switzerland a harmless symbiote of the honeybee ; that it lives with the bees without either species being injured by the mutual adaptations. In an English review_ {13) of the above-mentioned paper this view is suggested, following a less definite suggestion of the same nature by Morgen- thaler himself. In reply to this review Morgenthaler (^) states, however, that this does not correctly reproduce his view and that he considered the mites found on the outside of dead bees as dead animals (parasites) which had left their hosts on the death of the hosts. In the same numbers of the two Swiss bee journals in which the report of the unsuccessful search in 1921 appeared, there was pub- lished a note by Morgenthaler {Jfi) reporting that two specimens of diseased bees had been examined in cases in which definite symptoms of disease had been noted. This indicates that the Isle of Wight disease as it is found in Great Britain is actually found in Switzer- land, and precludes the belief that Acarapis woodi is in Switzerland always a harmless symbiote. The development of the work in Switzerland on this mite and on the conditions in the colonies which it produces have added materially to our knowledge of the subject, and while there is still much to learn it is becoming more and more evident that this mite is to be considered a serious pest of the apiary in Switzerland and elsewhere, wherever it is found. In a later article (^) Morgenthaler summarizes the information on Tarsonemus woodi, gives various reports of the fiinding of mites The Occurrence of Diseases of Adult Bees, II. 17 of other species in and about beehives, and repeats the records of the finding of Tarsoncmus ivoodi in Switzerland ]iist mentioned. At the time of the writing of this article he luid been unable to find living mites in the thoracic tracheas of bees from those colonies in which they were found dead on bees which had died during the previous winter, but he specifically states that he does not question the. state- ment that it is the cause of the Isle of Wight disease. The most recent discussion of the situation, which covers the ground as it is so far understood in Switzerland, is contained in an interesting lecture {^) which Morgenthaler delivered before the Wandervei-sammlung des Vereins dcutschschweizerischer Bienen- freunde on August 20, 1922, in Brugg. This lecture deals briefly with most of the diseases of adult bees, but the portion which is of special interest is that part dealing with his results with the mite. It may be _ mentioned that he finds that the protozoan parasite Nosema apis is in Switzerland, as in America, usually a relatively harmless parasite, but that under certain circumstances it produces a serious disease. Morgenthaler is inclined to believe, so far as can be judged from his lecture, that Nosema apis and Acarapis woodi are usually about of equal destructiveness to the colonies, but it is not quite clear as yet what the circumstances are which increase their damaging characteristics. He reports that the mite has now been found throughout Switzer- land. This is a small country, enabling him in one season to make a rather complete survey, but he records finding the mite through- out the country. The virulent cases were found in the Cantons of Geneva and Vaud (adjacent to France), while the worst case was in the middle of the Canton of Valais (adjacent to Italy). He re- ports five such virulent cases, and in a private communication he reports a sixth case. Eegarding the more general distribution of the mite in the milder or chronic cases, he states (44) : And indeed these mite-infested apiaries were found scattered througli the whole country, among others also on our northern, southern, eastern, and western boundaries, so that it may be surmised that the mite is also scattered in neighboring lands and will be found as soon as it is sought. Switzerland may therefore not be considered as an especially dangerous center of infec- tion of the acarine disease. Eegarding the worst case of infection, in middle Valais, he states : The acarine disease in its virulent form has been faund in Switzerland in five apiaries =■ * * but worst in a little village in middle Valais, where all three apiaries of the place were attacked. Here the occurrence of the disease may be traced back with certainty to the year 1915, and the chief sufferer — incidentally it may be noted, an excellent beekeeper, bee inspector, and teacher of beekeeping in an agricultural school — has in this time lost 26 of his 35 colonies. Importation of bees from England has nowhere occurred. The record for Switzerland can not be interpreted as consisting of cases directly attributable to importations," and one must con- ^ Id. a recent article Fr. Leuenberger ( Jahresberlcht uber die Faulbrutversichei-ung des VEereins] D[£utsch] Slchwedzerischer] Biienenfreunde] pro 1922, in Schwelzerische Bienen-Zeitung, n. f., v. 46 (1923), no. 3, p. 115-120), who has charge of the bee disease control work in Switzerland, states the Isle of Wight disease occurs in increasing amounts in French Switzerland and that apparently it has been brought in through importation of bees from western France. No case of the disease is reported by him from German Switzei-land. He expresses the hope that the disease may be localized by energetic measures 'before it causes greater damage, but does not report the nature of the measures adopted. Morgemthalei' m) reports finding the mite on the northern boundary of Switzerland (German Switzerland). 53546°— 23 3 18 Department Circular 287, U. S. Dept. of Agriculture. elude that the infestation is at least of several years' duration. The same thing is, of course, true of the records from France, so that we dare not assume (5) that the disease in continental Europe is traceable to recent introduction from Great Britain. Since Morgenthaler has found mites on bees which were not recog- nized by their owners or by inspectors as diseased, this has given rise to the idea that the mite is often not a serious pest or that it is quite harmless in most cases. For some reason this interpretation has been put on his work, but not on that of Rennie, who has de- scribed the same things. This does not seem to agree with the present ideas of Morgenthaler in the slightest degree. It is quite true that he has found mites where he himself has not been able to detect the ordinary symptoms of the Isle of Wight disease, but so has Rennie. In a private communication (January 15, 1923) he states that he sent dead bees from the Liebefeld Station apiary on which he found mites to Rennie and that Rennie found that from 3 to 5 per cent of the bees showed mites in various developmental stages in their tracheae. I have been unable to confirm this finding through my own investigation, although I have dissected many such bees. I find the tracheae always clean. I am, however, now about to test this question with new and better material. Regarding the assumed harmlessness of these mites, Morgen- thaler has the following to say {4-4-) '■ The harmlessness of the parasite has been erroneously asserted from these findings. I may here allude to only two points which must be regarded in the examination of this phenomenon. First, the bee colony possesses a series of contrivances for defense against the parasite, so that it does not succumb without a struggle to the fljst attack. The most powerful of these means of defense is found in the constant renewing of the inmates of the colony through the death of the old infested bees and the emergence of young healthy ones. It is therefore made very difficult for the parasite to obtain a foothold, notwith- standing that it can probably be found for a long time in the colony. Only when it invades the colony in overwhelming numbers because of special cir- cumstances does it get the upper hand. Secondly, however, it is very likely that the apparently healthy colonies containing parasites after all are not entirely normal. By rigorous investi- gations it would indeed be shown that many remain in colony strength below what one would expect of them. Exact weighings and measurements * * * would show clearly that for many apiaries which outwardly do not give an impression of disease, the loss of flight bees is too great. The question would here also have to be examined whether the lack of swarming is not also due to infection, concerning wjiich many beekeepers have complained these last years. Throughout this lecture, Morgenthaler deals both with Nosema apis and Acaravis woodi as parasites which under certain circum- stances, not understood, become harmful, so that these remarks are not to be interpreted as applying solely to the mite. The experience with Nosema ajns in the United States would appear to support his contentions for that parasite. It would therefore appear from the findings in Switzerland that whereas Acarapis woodi may produce a chronic and damaging dis- ease, it can also at times become dangerous to as great a degree as has been described for England. It is also clear that the outbreaks of the virulent form of the disease can not be attributed, as some (6) have attempted to claim, to recent importations from England, for Morgenthaler specifically states regarding these severe outbreaks that " importation of bees from England has nowhere occurred." The Occurrence of Diseases of Adult Bees, II. 19 Because of the thoroughness of the search in Switzerland and the wide distribution of the mite, the supposition of Morgenthaler that the mite will doubtless be found in adjacent countries when it is sought must have greater weight than the statements which have appeared from these adjacent countries that the mite is absent, since in all the adjacent countries except France no effort has been re- ported of surveys of the kind demanded for definite statements on this point. The Government apiary inspection service of Switzerland has taken steps to prevent the spread of the Isle of Wight disease (36), and the methods employed are discussed later (p. 26). GERMANY. The situation regarding the Isle of Wight disease in Germany is yet very indefinitely known. In a publication issued in 1922, Hirst (32) states that apparently Acarapis woodi has been found only in English bees, but he adds a footnote in which he states (p. 97) : "According to Vitzthum, Doctor EUinger, of Weimar, has reported that the disease has made its appearance in Germany also (Bayer- ische Bienenzeitung, April 1922)." Count Vitzthum is a well-known specialist in mites and Doctor EUinger is the author of several papers on bee diseases. There also appeared a statement from Alfonsus, of Vienna {6), in which he states (p. 2) : "The occurrence of Tar- sonemus woodi has now also been established in Germany * * * (Archiv fur Bienenkunde, vol. Ill, 6, 1921)." The number of the Archiv fiir Bienenkunde to which Alfonsus refers contains two articles (J5, 39) on the Isle of Wight disease, but in neither case is there any statement which can be interpreted as supporting the state- ment quoted. The Bayerische Bienenzeitung to which Hirst refers is not regu- larly received in the Bureau of Entomology, but the editor, Hof mann, of Munich, kindly sent the copies of his journal which contained articles on this subject. In the article by Vitzthum (57) to which Hirst refers there is only the following statement on this subject: " Doctor EUinger of Weimar communicates that the disease has also appeared in Germany." No additional information is given and it is not stated whether this statement is based on an examination of dis- eased bees for the mites or merely on the general symptoms observed in diseased adult bees, which are known to be quite unreliable. Since this statement appeared only a few months (November to April) after Kennie's announcement of the discovery of the cause of the disease, it appears somewhat doubtful whether any search for the mite had been made, especially since these intervening months were during the winter. In order to clear up this point, a letter was written to Doctor EUinger, who replied under date of January 10, 1923 : " Your question on the occurrence of the Tarsoneonus woodi in Germany I answer as follows: It is not yet certainly found in Germany." Doctor EUinger also kindly sent some advance proof sheets of an article of his {£3) on the diseases of bees which is soon to appear in a new edition of Unsere Bienen (Ludwig, editor), in which he makes no reference to the finding of the Isle of Wight dis- ease in Germany. It would, therefore, appear that his statement to Count Vitzthum was based solely on external symptoms, although this is not stated. 20 Department Circular 287, U. S. Dept. of Agriculture. In the meantime a number of other articles {15, S3, 38, 39) have appeared in German journals regarding the Isle of Wight disease and its newly discovered cause, and in none of those which have come to the attention of the writer is there any statement regarding the occurrence of the disease in Germany. Dr. Bartholomaus Manger of Ingolstadt in a private communication writes (December 30, 1922) that he reads all the German bee journals and has seen no such state- ment. Because of the geographical position of Germany in the center of various important beekeeping regions of Europe, it seemed quite desirable to determine whether the disease is actually present in that country. Probably no American beekeeper would wish to import queenbees from Germany, almost certainly not of the race of bees native to that country. Germany has long been the home of scientific research in beekeeping and there are a large number of competent investigators in beekeeping in that countrj'. It was felt that if the disease has actually been found in Germany this fact would indicate a wider distribution than has been assumed for the mite in most of the publications on the subject. With this thought in mind, the writer addressed a considerable number of the prominent beekeeping investigators of Germany, asking whether they had any knowledge of the actual finding of the mite within the boundaries of Germany. The replies to these inquiries were uniformly to the effect that the mite has so far not been recorded in any German beekeeping periodi- cal and none of the men who replied had any knowledge of such findings. It appears from these letters that no search has been made specifically for this mite, but one of the men who wrote, a prominent investigator, states that he expects to undertake such a search during the summer of 1923. Except for the surmise of Morgenthaler, based on his finding of the mite on the northern boundary of Switzerland, adjacent to south- ern Germany, it may therefore be stated that there is so far no evi- dence of the occurrence of the Isle of Wight disease in Germany. This negative statement will be of little value in comparison with such scientific investigations as will doubtless be made in the near future. ITALY. American beekeepers are perhaps more concerned with the situa- tion as to diseases of adult bees in Italy than in any other country of continental Europe, since more importations of queenbees have been made and will be desired from that country than from any other. Several articles {8, 9, 10, ^5) have appeared in the Italian bee journal on the Isle of Wight disease and on its newly discovered cause, but they are usually a summary of the work of Eennie or an- nouncements of the finding of the disease elsewhere in England. Tlie effort of the United States Government to prevent the introduc- tion of the disease into this country has also been mentioned on sev- eral occasions, not always favorably (4). The only defiinite statement which has come to the attention of the writer is a brief one by Prof. Vincenzo Asprea, of Calabria. In discussing the United States postal regulation approved March 21, 1922, prohibiting the mailing of queenbees into the United States, he says {8) : The Occurrence of Diseases of Adult Bees, II. 21 It is strange that while tlie English Government imports a large quantity of Italian queens to combat the disease, und with good success, the Americans wish to keep It out by not importing. To tell the truth, the fear is justified by the fact that the disease has been observed In the French Alps and in near-by Italian apiaries, but since then it has not been seen further. The reference to the French Alps doubtless refers to the actual finding of the mite in diseased bees from the French Alps, previ- ously mentioned (49). It will be recalled that this first report from France did not give the exact' location of the apiary from which the diseased bees were received, and in that notice no mention was made of bees in neighboring apiaries, either in France or Italy, being diseased. The disease is recorded by Bouvier (i^) as occurring in Hautes-Alpes, France. Since the publication of this brief mention of the disease in Italy, several other articles {9, 10, 45) and notices have appeared, some of them by Asprea, which would lead one to surmise that the authors believe that the Isle of Wight disease does not occur in Italy, since they usually urge that the Italian Government shall take steps to prevent the introduction of the disease into that country. The Fe- derazione Apistica Italiana has stated in a private communication (Dec. 20, 1922) that they believe that it will be possible to furnish full assurance of the absence of the Isle of Wight disease from Italy. It may therefore be concluded that there is little reliable evidence of the presence of the Isle of Wight disease in Italy, based on ex- aminations of diseased bees. The chief reason for suspecting that the disease may be present is the fact that it has definitely been found in neighboring apiaries in France and Switzerland. From private sources the author has learned that the Italian Ministry of Agriculture has been requested by the Federazione Apistica Italiana to take steps to prohibit all importation of foreign bees into Italy, in order to prevent the introduction of the Isle of Wight disease into that country, and that in all probability a search will be made in the near future to determine whether the mite is ac- tually found in that country. The Italian beekeepers are evidently and properly anxious to prevent the introduction of the disease and ■ to keep up the standing of their stock throughout the world. The results of their efforts will be watched with keen interest in the United States. Prof. Amyandro Ghigi of the University of Bo- logna, reports in a' private communication (April 18, 1923) that he has examined bees from apiaries in that Provmce and has failed to find any Tarsonemus woodi. DENMARK. In a private communication (April 30, 1923), Dr. L. Bahr reports that Tarsonemus woodi has not been found in Denmark through the occurrence of the disease which it causes. No extensive inquiry has been made on this subject in that country, but further investigations will be made in the near future. Doctor Bahr further reports that no record of the mite in Sweden or Norway has come to his attention. CZECHOSLOVAKIA. In a letter dated May 23, 1923, from Eev. Ivan F. Kitzberger, Veleslavin, Czechoslovakia, editor of one of the leading bee journals of that country, it is stated positively that the mite causing the 22 Department Circular 287, U. S. Dept. of Agriculture. Isle of Wight disease has been found in that country. Identifica- tions of the mite were made by Blattny, an assistant in the Zoo' logical Institute at Prague, who is a specialist on mites. _ He has also found several other species of mites in and about the hives, but these are not associated with any disease and some of them have previously been found in hives. No statement was made regarding the occurrence of the Isle of Wight disease or whether the mite is found without manifestation of the disease, as has occurred in parts of Switzerland. That the Isle of Wight disease may be present to an alarming degree in Czechoslovakia is indicated by an article by Altmann (60), in which it is stated that during the spring of 1923 reports have been received by him of the heavy death of adult bees and of entire colonies, of such a character that the loss can not be attributed to poor wintering. Altmann is in charge of the bee investigations for the Deutsch. bienenwirtschaft. Landes-Zentral Verein fiir Boh- men, and asks that diseased bees be sent in for examination to. deter- mine whether Nosema apis or Acarapis woodi is the cause of the heavy losses. An even more severe loss of adult bees and of colonies has been reported from the territory of Teschen (Bohemia) byKessler (5^) , but no indication of the cause of this loss is given. SOUTH AFRICA. In a letter dated June 18, 192.3, from Dr. Otto Morgenthaler, Liebefeld bei Bern, Switzerland, whose investigations of the Isle of Wight disease have added so materially to our knowledge of this subject, it is stated that the Isle of Wight disease has become estab- lished in South Africa and that attacked colonies have been destroyed by officials in Natal. Details of this outbreak are lacking. OTHER EUROPEAN COUNTRIES. So far as has come to the attention of the Bureau of Entomology, no search has been made for the Isle of Wight disease or for the. mite which causes it in any country of Europe other than those already mentioned, nor has any definite statement been seen regard- ing its presence, based on investigations. It h?is been reported to the bureau by certain American beekeepers that they have assurance that the disease does not exist in certain countries, but so far it has been impossible to get any accurate first-hand information on these subjects, and in some cases the reported evidence has been found to consist merely of letters from some queen breeder who is eager to sell his queens in this country, but who has no means of examining bees for the presence of mites or any knowledge of its presence in his country. The finding of mites so generally in France and Switzerland, as soon as steps were taken to search for them, shows conclusively that the mite is not one which has until recently been confined to Great Britain. There is, therefore, a high degree of probability, amount- mg almost to a certainty, that the mite is present in countries other than those m -which it has been sought. The facts which Morgen- thaler {U) brings out regarding the wide prevalence of weak colonies, lack of swarming, and other evidence of abnormality in The Occurrence of Diseases of Adult Bees, II. 23 the bees of Switzerland due to the infestation indicates that the mite may be present for a considerable time without the beekeeper being aware of the fact, and, even from an experienced beekeeper, one may not accept the statement that there are no cases of the disease in his country, merely on the basis of lack of visible symptoms among his own bees. Various diseases or abnormalities of adult bees, known variously as paralysis, May disease, and under other names, have been recorded repeatedly from the various countries of Europe, and since these names mean little or nothing, the causes of these conditions being purely a matter of speculation, one is entirely unable to esti- mate the probability that some of these conditions are actually the Isle of Wight disease. It certainly can not be considered safe to accept statements of interested persons who desire to make sales of queenbees in the United States when there is no way of checking their statements. CARNIOLA. The Province of Carniola, in the Kingdom of the Serbs, Croats, and Slovenes, is the home of the Carniolan bee, which has for a num- ber of years had some ardent advocates in the United States. They are excellent bees, but have not gained the popularity in this country which Italian bees enjoy. There are only a few queen breeders in the United States who have found demand enough for these bees to make their propagation profitable, and as a result many beekeepers who prefer bees of this race have been in the habit of obtaining their queens directly from Carniola. Perhaps because of this fact, there has been more demand that this Province be excluded from the opera- tion of the law against importations and that queens from Carniola be admitted freely than has come from those who desire queens from Italy. The statement has been made by those interested in these importations that there is no Isle of Wight disease in that Province. An efPort has been made by the writer through correspondence to learn if there has been any investigation to determine whether any Isle of Wight disease or any disease of adult bees exists in Carniola, but so far without success. The president of the provincial bee- keepers' association, M. Humek, wrote under date of August 18, 1922, that he is not aware of any kind of infectious bee disease in his country. He states in his letter that certain bees had been sent away for examination and that one of the best-known queen breeders of that country would send to this Government a report which would show that the Isle of Wight disease is unknown in Carniola. So far the report has not been received, and from other correspondence it would appear that there has been some unavoidable delay in the ex- aminations, the nature of which is not clear. The Royal Depart- ment of Commerce and Industry of the Kingdom has also stated that there is no case of any disease of bees in the Kingdom. An effort is being made to obtain the data on which this statement is based. When the revised postal regulations were adopted prohibiting the mailing of queenbees through foreign mails in March, 1922, four persons who had imported queenbees from Carniola sent to the Bureau of Entomology the original cages in which imported Carnio- lan queenbees had been received, including the accompanying worker bees. These worker bees were not found to contain Aearapis woodi, but they, were heavily infected with Nosema apis. This 24 Department Circular 287, U. S. Dept. of Agriculture. parasite is found widely distributed in the United States, and under the regulations provided for the enforcement of the importation law the disease which it causes is not considered as one dangerous to adult honeybees, but the fact remains that probably never in the work of the Bureau of Entomology on this disease have bees been ex- amined which contained a larger number of these intestinal para- sites. If such a heavy infection were found in bees in this country, one would ex]Dect there would be marked symptoms of abnormality. It therefore appears strange that the beekeepers of Carniola have never noted any abnormality of adult bees. If Carniolan bees have the ability to harbor this parasite without showing any symptoms or are better able to resist it than are other bees, this has not pre- viously been proved. The presence of Nosema apis in Carniola will not serve to confuse the situation with regard to importations from that Province if the much-needed examinations of adult bees are made by competent investigators. AUSTRIA. No information has been received by the Bureau of Entomology concerning the details of any search which may have been made in Austria to determine the presence of the Isle of Wight disease. A letter has been received by the author from Alois Alfonsus, of the Austrian Department of Agriculture, Vienna, written from within the United States, in which he states that the scientific institute of that department has so far been unable to find Tarsonemus woodi in that country. He does not state how extensive a search has been made or from what parts of the country bees have been examined. No published report of this work has been reported to the bureau, Austria is immediately adjacent to Switzerland, and Morgenthaler [Ji-Ji) reports finding Tarsonemtis woodi on the contiguous border. While it is most essential that a serious effort be made to determine especially whether Italy and Carniola are free from the dangerous diseases of adult bees, it is also of the highest importance to Amer- ican beekeepers that efforts be made to determine these facts for all of Europe, just as soon as conditions are favorable for the prosecu- tion of such investigations. The shortness of the distances between important beekeeping regions in Europe and the considerable traffic in bees which has long been customary throughout Europe make it necessary that detailed studies be made on this point for each coun- try, and it will doubtless be several years before the facts are ade- quately known. Although beekeepers of all countries are at present interested in the Isle of Wight disease, the investigation of its presence in many countries is difficult or impossible because of disturbed economic conditions. Investigations on beekeeping subjects were not systematically con- ducted in most European countries before the war, and since then some of the work which had been organized has been discontinued. Several countries have so far made no provision for such work. Under these circumstances it will probably be several years before the distribution of the Isle of Wight disease on the continent of Europe is fully known. It will be recalled that the discovery of the cause of this disease was the result of work by members of a university staff, The Occurrence of Diseases of Adult Bees, II. 25 aided by private funds, and not a Government project. The interest in this subject promises to be the incentive for support for much- needed investigations, either from public or from private sources. EMBARGOES TO PREVENT INTRODUCTION OF BEE DISEASES. Several countries other than the United States have taken steps to prevent the introduction of the Isle of Wight disease and other dis- eases of bees. The embargo placed by the Dominion of Canada is well known to American beekeepers. Other British Dominions have taken similar precautions, notably the Union of South Africa, Jamaica, and Australia. There seems now to be an insistent demand from beekeepers of Italy for a strict prohibition of importations of bees into Italy. Because of the vast interest created by the work of Doctor Eennie and his associates, it is to be expected that other countries will follow the same course, and in all probability the free international shipment of bees will soon be a thing of the past. Under regulation 6 (5) of the regulations formulated for the enforcement of the act of Congress of August 31, 1922 (see appen- dix), it is provided that importations of adult bees from any coun- try other than the Dominion of Canada shall be conditioned on the determination of the Secretary of Agriculture, as a result of ade- quate scientific investigation, that no diseases dangerous to adult honeybees exist in the country in question, and that adequate pre- cautions have been taken by such country to prevent the importation of adult honeybees from countries where such dangerous diseases exist. The purpose of this regulation is solely to safeguard Ameri- can beekeeping interests, but it indicates clearly what steps are neces- sary for the protection of the beekeeping interest of any country against these dangerous diseases. This policy on the part of the United States Government will make it less probable that beekeepers of other countries wishing to ship queenbees into the United States will attempt to conceal the true situation ; rather, they wiU urge that provisions for thorough investigations be made, if their trade with this country justifies the expense and labor of such investigations. As a result of the freedom which Amercian beekeepers formerly enjoyed regarding the importation of bees, they now have three brood diseases, Nosema disease, the so-called paralysis, and perhaps other diseases of adult bees, all of which, so far as they are con- tagious or infectious, were assuredly brought to this country from abroad, since the honeybee is not native to America. The price which beekeepers of the United States are now paying for these accidental importations of diseases amounts to at least a million dollars annu- ally, a high price to pay for the privilege of buying bees wherever one wishes, without knowledge as to the safety of the transaction. Under the law now in force, such importations may be safeguarded, so far as investigation has discovered the causes of diseases which are not yet present in this country. That there is still at least one disease of adult bees which is not present in the United States seems probable, because of the inability to find it during the past two sea- sons. Why the Isle of Wight disease is not widespread in the United States is a matter of mystery, but one of purely theoretical interest. If, as is believed and hoped, the United States is free from this disease, then the door has been closed in time, and it should be 26 Department Circular 287, U. S. Dept. of Agriculture. the privilege of every American beekeeper warmly to support the enforcement of this law, and this is in fact the attitude of the vast majority at this time. In the correspondence with the Bureau of Entomology regarding the regulations for importations, the necessity for further importa- tions of queenbees is emphasized by those who wish to make sucK importations, and is minimized or denied by those who do not wish to make importations. This is a natural condition and occasions no surprise. It seems evident that there is no popular -demand or great necessity for large importations at present, and, in fact, it is not proved that any actual damage would result to American beekeep- ing from a total prohibition of importations for a time. With the safeguards which have been provided under the law, it will be pos- sible to make such importations as may be urgently needed for ex- perimental and scientific purposes. As further investigations are made in the various foreign countries, it may be found safe to allow bees to enter from them without restrictions and for any purpose. Until accurate information is available from the various countries, it is the part of wisdom to limit the importations to those for experi- mental and scientific purposes and to keep a careful watch for infor- mation from all the countries from which any importations may be desired. In addition to the countries named above as having prohibited or restricted the importation of adult bees, special attention should be drawn to the recent action of Switzerland. As has been pointed out, the Isle of Wight disease occurs in that country and in all Mor- genthaler (66) now reports 19 cases of the disease in French Switzer- land. Under these circumstances the prohibition of importations might seem unnecessary, yet the recent action of the Swiss Govern- ment puts such a prohibition into effect and outlines steps for the eradication of the disease already present. The situation with re- gard to the disease in that country is outlined in three recent articles by Leuenberger (63, 64, 65) . On April 18, 1923, the Swiss Bundesrat issued a proclamation (68) under which the Isle of Wight disease is included under the animal diseases covered by the Federal law of June 13, 1917, concerning the combating of animal diseases, and the ordinance for its execution dated August 30, 1920. Provision is also made for partial compen- sation by the Federal Government for colonies destroyed in the en- forcement of this law and the several ordinances. Under date of April 25, 1923, the details for the control of the Isle of Wight disease are set forth by the veterinary office of the Swiss Government (69), in which the work of control is assigned to the foulbrood inspectors of the several cantons and the methods of compensation for colonies destroyed are outlined. The importation of bees and combs is for- bidden from March 15, 1923, and as no exceptions to this prohibition have appeared, the prohibition seems to be absolute. It is the evident intention of the Swiss Government to eradicate the Isle of Wight disease which has already appeared in that country and to prevent any further introduction of the disease by this rigid prohibition. The Occurrence of Diseases of Adult Bees, II. 27 LITERATURE CITED. (1) Anonymous. 1884. [Article on the mite discovered by Trapp.] In Blsass-Lothring. Bipiienzilchter. Reference from Manger (39) ; original not con- sulted. 1922. Maladies des abeilles. In L'Apiculteur, v. 66, no. 3, p. 104. 1922. Maladie de I'lle de Wight. In L'Apiculteur, v. 66, no. 5, p. 172. See also no. 6, p. 192. (2) (3) (4) 1922. 11 bando auche alle api ita,liane? In L'Apicoltura italiana, v. 18, num. 9, p. 216. (Extract from weekly bulletin of the Italian Chamber of Commerce, New York, " La Kevista commerciale," April 22, 1922.) (5) [AbushAdy, A. Z.] 1922. Wisdom or panic? Editorial in The Bee World, v. 3, no. 11, p. 261-262. (6) Alfonsus, Alois. 1922. An enemy of the mites in the bee-hive. In The Bee World, v. 4, no. 1, p. 2-3, 1 fig. (Authorized translation by Herbert Scherlng.) (7) ANDRfi. 1866. Sur I'acare de I'abeille. In L'Apiculteur, v. 10, p. 174-176. (Copied from Cosmos, with note by Hamet.) (8) ASPKEA, V[INCENZ0]. 1922. tSpigolature apistlche.] In L'Apicoltura itaUana, v. 18, num. 5, p. 117. (9) 1922. [Splgolature apistiche.] In L'Apicoltura italiana, v. 18, num. 8, p. 188. (10) 1922. [Splgolature apistiche.] In L'Apicoltura italiana-L'Apicoltore, V. 18, num. 9, p. 201. (11) Banks, Nathan. 1915. The Acarina or mites. U. S. Dept. Agric, Office of the Secretary, Report 108, 158 p., 294 figs. (12) Beeland, Lucibn. 1922. M^thode pour d^celer I'existence de I'acarien agent de la maladie de I'lle de Wight. In L'Apiculteur, v. 66, no. 5, p. 172-173. (13) B[etts], a. D. 1922. Acarine disease in Switzerland. In The Bee World, v. 3, no. 12, p. 293. Review of article by Morgenthaler (40). (14) BouvtEE, E. L. 1922. Flgaux entomologiques nouveaux pour notre pays. In Les An- nates, no. 2050, 8 Octobre, p. 387-388. (15) V. Buttel-Reepen, [H.]. 1920. Die neue (?) verheerende Milbenkrankheit der Bienen. In Archiv f. Bienenkunde, v. 2, no. 8, p. 328-332. (Partial trans- lation in American Bee Journal, v. 62, p. 58.) (16) Cook, A. J. 1883. A new bee enemy. In Amer. Apiculturist, v. 1, p. 134^-136, 1 fig. (17) Dadawt, C. p. 1922. Tarsonemus not Acarapis. Editorial in American Bee Journal, v. 62, no. 6, p. 253. (18) Dennlbb. 1885. An interesting discovery. In British Bee Journal, v. 13, p. 15. (19) Dbvatjcheele. 1922. Maladie de I'lle de Wight ou acariose des abeilles. In L'Apicul- teur, V. 66, no. 9, p. 290-295. (20) Dtjchemin, Emble. 1866 Note sur les abeilles et un de leurs parasites. In Compt. Rend. Acad. Sci. [Paris], v. 62, p. 48-49. See also p. 225 and 683. (21) 1866. Sur un parasite de I'abeille. In L'Apiculteur, v. 10, p. 144-146, 1 fig. (Note by Hamet, H., p. 146.) 28 Department Circular 287, U. S. Dept. of Agriculture. (22) 1866. Sur I'acare de I'abeille. In L'Apiculteur, v. 10, p. 240-241. (23) Eliingeb. 1923. Die Erkrankungen der Honigbiene. Aus " Unsere Blenen," vou Ludwig, 4. Aufl., p. 336-362. (In press; proof sheets furnished by author.) (24) BwiNG, H. E. 1922. Studies on the taxonomy and biology of the tarsonemid mites, to- gether with a note on the transformations of Acarapis (Tar- sonemus) tooodi Ren. (Acarina). In Can. Ent., v. 54, no. 5, p. 104-113, 3 fig. (25) 1922. Concerning mites. In American Bee Journal, y. 62, no. 7, p. 317. (26) Favieb, M/UiCEL. 1922. [Comptes rendus de la] Soci6t6 centrale d'apiculture. Stance du 19. Mai, 1922. In L'Apiculteur, v. 66, no. 7, p. 253-254. (27) Fkacker, S. B., Goodeeham, C. B., and Rba, Geobge H. 1923. Protecting American bees against the introduction of the Isle of Wight disease. In Jour. Econ. Ent., v. 16, no. 2, p. 133-136. (28) GiEATjD, E., et Sevaile, E. 1922. Maladie de I'lle de Wight. In L'Apiculteur, v. 66, no. 6, p. 185-192. (29) Geaham-Smith, G. B. : Fantham, H. B. ; Poeter, Annie; Btjllamoee, G. W. ; and Malden, W. 1912. Report on the Isle of Wight bee disease (miscrosporidiosis). Sup- plement no. 8, Joum. Board Agr. [London], v. 19, no. 2, 143 p., 5 pi. (30) Hakvet, Elsie J. 1921. Isle of Wight disease in hive bees. (3) Experiments on infection with Tarsonemus tooodi, n. sp. In Trans. Roy. Soc. Edin., v. 52, pt. 4, no. 29, p. 765-767. (31) HiEST, Stanley. 1921. On the mite (Acarapis woodi, Rennie) associated with Isle of Wight bee disease. In Annals and Mag. Nat. Hist., ser. 9, v. 7, p. 509-519, 7 fig. (32) 1922. Mites injurious to domestic animals (with an appendix on the acarine disease of hive bees). Economic series no. 13, British Museum. 107 p., illus. (Portion on Acarapis woodi, p. 94^103, flg. 78-85.) (33) Hoffmann, H. 1921. Die Insel-Wight-Krankheit der Blenen. In Archiv fur Bienen- kunde, v. 3, no. 6, p. 193-196, 2 fig. (34) KiLLicK, C. R. 1923. Some aspects of the pathology of acarine disease. In The Bee World, V. 4, no. 8, p. 169-171, 7 fig. (35) Leuenbbbgee, Fe. 1922. Die Milbenkrankhelt. In Schweizerische Bienen-Zeitung, n. f., V. 45, no. 4, p. 15&-160. (86) [Mammblle.] 1922. [Maladie de I'lle de Wight en France.] In L'Apiculteur, v. 66, no. 6, p. 217. (Reported by Favier.) (37) 1922. A propos des maladies des abeilles. In L'Apiculteur, v. 66, no. 7 ; advertising section, following p. 256. (38) Mangee, [Baetholomaus]. 1921. Zur Entdeckung des Erregers der Insel-Wight-Krankheit der Honigbiene. In Bayerische Bienen-Zeitung, v. 43, no. 1, p. 11 and 13. (39) .. 1921. Tiber die Milbenkrankhelt der Bienen (Insel-Wight-Krankheit). In Archiv fiir Bienenkunde, v. 3, no. 6, p. 187-192, 2 fig. (40) Moegenthalbe, Otto. 1922. Blenenkrankheiten im Jahre 1921. In Schweizerische Bienen- Zeitung, n. f., V. 45, no. 4, p. 149-159. 1922. Maladies des abeilles en 1921. In Bulletin de la socI6t6 romande d'apiculture, v. 19, no. 3, p. 63-68; no. 4, p. 86-87; no. 5, p. llS-121. The Occurrence of Diseases of Adult Bees, II. 29 (41) (42) (43) (44) 1922. Die Milbe Tarsonemus ivoodi auch in der Sciiweiz? In Schweizer- isclie Bieneu-Zeitung, n. f., v. 45, no. 3, p. 105-106. 1922. Apparition de I'acare Tarsonemus woodi 6galement en Suisse? In Bulletin de la soci6t6 romande d'apiculture, v. 19, no. 4, p. 88-89. (Traducteur, Dr. E. Rotscliy.) 1922. Tarsonemus In Switzerland. In American Bee Journal, v. 62, no. 5, p. 193-194. (Translated from Frencli translation by C. P. Dadant.) 1922. The mite Tarsonemus tcoodi in Switzerland also? In The Bee World, V. 3, no. 10, p. 258. (Translated from German by Annie D. Betts.) 1922. Acarine disease in Switzerland. In The Bee World, v. 4, no. 1, p. 22. 1922. Zum Kapitel " Bienen und Milben." In Archiv fttr Bienenkunde, V. 4, no. 2, 1 tafel, p. 45-52. 1923. Einiges iiber die Krankheiten der erwachsenen Bienen. In Schweizerische Bienen-Zeitung, n. f., v. 46, no. 1, p. 26-28, 2 fig. ; no. 2, p. 81-85. 1922-23. Quelques observations sur les maladies des abeilles adultes. In Bulletin de la soci6t6 romande d'apiculture, v. 19, no. 12, p. 287-290, V. 20, no. 1, p. 4-10. (45) Penna, E.- 1922. Sulle malattie delle api in rapporto all'applicazione dell'attesa legge suir apicoltura. In L'Apicoltura italiana, v. 18, num. 4, p. 74-81; num. 5, p. 118-123; num. 6-7, p. 147-154; num. 8, p. 177-181, illus. (46) Phzllips, E. F. 1922. The occurrence of diseases of adult bees. TJ. S. Dept. Agr., Circ. 218, 16 p., 2 figs. (Literature, p. 15-16.) (47) 1922. The mite and the Isle of Wight disease. In American Bee Jour- nal, V. 62, no. 5, p. 211-212. (48) 1922. The Isle of Wight disease. In Gleanings in Bee Culture, v. 50, no. 4, p. 234. (49) Ranguis, AbbS. 1922. Maladie des abeilles : un nouveau et grave danger. In L'Apicul- teur, V. 66, no. 1, p. 20-23. (50) Rennie, John. 1921. Isle of Wight disease in hive bees — Acarine disease. (4). The organism associated with the disease — Tarsonemus woodi, n. sp. In Trans. Roy. Soc. Edln., v. 52, pt. 4, no. 29, p. 768-779, 1 pi., 2 figs. - (51) 1921. Tarsonemus woodi and Isle of Wight disease. In Report of Aber- deenshire and Kincardineshire Bee-Keepers' Association for 1920, p. 19-21. (52) 1921-22. Notes on acarine disease. Parts 1-13. In The Bee World, V. 2, no. 12, p. 144r-145; v. 3, no. 1-12, p. 5-7, 35-36, 66-67, 95-96, 115-117, 145-146, 180-182, 204-206, 219-221, 237-239, 262-263, 285-287 ; fig. 3-5, 68-74, 106, 113-114, 126-127, 167-168. Also reprinted as North of Scotland College of Agriculture Bee Disease Investigation Memoir 6, with title, Acarine disease ex- plained, 50 p. (no fig.), Aug., 1923 (revised and enlarged). (53) 1922. The presidential address [delivered at the third annual con- ference of the Apis Club, Reading, England, April 8]. In The Bee World, v. 4, no. 3, p. 72-74. (54) Rennie, John; White, PHn.ip Bktjce; and Habvet, Elsie J. 1921. Isle of Wight disease in hive bees. (1) The etiology of the disease. In Trans. Roy. Soc. Edin., v. 52, pt. 4, no. 29, p. 737-754, 1 pi. 30 Department Circular 287, U. S. Dept. of Agriculture. (55) [Sevalle.] 1922. [lie de Wight maladie en France.] In L'Apiculteur, v. 66, no. 2, p. 59. (Reported by Leclerc.) (56) Snodgkass, R. E. 1923. The breathing organs and the muscles of the honeybee. (Unpub- lished manuscript, with figures.) (57) ViTZTHUM, Hermann. 1921. Ein Wort iiber die Insel-Wight-Krankheit. In Bayerische Bienen- Zeitung, v. 43, no. 4, p. 82-83. (58) 1922. Die Trapp'sche Bienenmilbe. In Archly fur Bienenkunde, v. 4, no. 2, p. 61^4. (59) White, P. Betjce. 1921. Isle of Wight disease in hive bees. (2) The pathology of Isle of Wight disease in hive bees. In Trans. Roy. Soc. Bdln., v. , 52, pt. 4, no. 29, p. 755-764, 1 pi. ADDITIONAL BEFEEENCES. Since the preparation of the manuscript of this circular in the spring of 1923, several important papers on the Isle of Wi^ht disease -have appeared and some new records of the occurrence of the disease have been, published, so that it seems desirable to make additional record of these finding's as this circular goes to press. The additional literature cited is appended. The same arrangement of material is followed here as in the main list. (60) AlTMANN, RiCHAKD. 1923. Massenhaftes Bienensterben. In Der Deutsche Imker, v. 36, no. 6, p. 161. (61) Andebson, John. 1923. Isle of Wight disease in hive bees. In Scot. Jour. Agr., v. 6, no. 2, p. 183-191. (62) Kessleb, Viktok. 1923. Achtet auf die Bienenkrankheiten. In Der Deutsche Imker, v. 36, no. 6, p. 167-168. (63) Leuenbebgeb, Fb. 1923. Die schweizerische Bienenzucht in Gefahr. In Schweizerische Bienenzeitung, n. f., v. 46, no. 4, p. 162-163. (64) 1923. Zur Bekampfung der Milbenkrankheit der Bienen. In Schweizer- ische Bienenzeitung, n. f., v. 46, no. 5, p. 213-214. (65) 1923. Bericht der Konferenz der kantonalen Bieneninspektoren, den 27. und 28. April, auf dem Rosenberg, Zug. In Schweizerische Bie- nenzeitung, n. f., V. 46, no. 6, p. 275-281, no. 7, p. 326-328. (66) Mobgbnthalek, O. 1923. A propos du traltement de I'acariose par le soufre. In Bulletin de la socigtg romande d'apiculture, v. 50, no. 5, p. 115-117. (67) Rennie, .Iohn. 1923. [Presidential address before the Apis Club Conference, Bristol, England, April 7, 1923.] In The Bee World, v. 5, no. 1, p. 11-12. (68) ScHWEiz. Bundeseat. 1923. Bundesratsbeschluss betreffend Aufnahme der Milbenkrankheit der Bienen in das Tierseuchengesetz vom 13. Junl 1917 (vom 18. April 1923). In Schweizerische Bienenzeitung, n. f., v. 46, no. 5, p. 214-215. Also in Bulletin de la soci6t6 romande d'apiculture, v. 20, no. 5, p. 128-129, with title, Arr6t6 du conseil federal portant admission de I'acariose des abeilles dans la loi f6d4rale du 13 juin 1917 sur les mesures k prendre pour combattre les gpizooties (du 18 avril 1923). (69) Sohweiz. Volkswiethschaftsdepaetement. Veteeinabamt. 1923. Verfiigung des Eidgen. Veterinaramtes uber die VoUziehung des Bundesratsbeschlusses betrefCend Aufnahme der Milbenkrank- heit der Bienen in das Tierseuchengesetz vom 13. Juni 1917 (vom 18. April 1923). In Schweizerische Bienenzeitung, n. f., V. 46, no. 5, p. 216-218. (70) ViTZTHUM, HeBMANN. 1923. Der Erreger der " Insel Wight "-Krankheit. In Archiv fur Bie- nenkunde, V. 5, no. 1-3, p. 25-32, 6 figs. APPENDIX. UNITED STATES DEPARTMENT OP AGRICULTURE, OFFICE OF THE SECRETARY. SERVICE AND REGULATORY ANNOUNCEMENTS. REGULATIONS GOVERNING THE IMPORTATION OF ADULT HONEY- BEES INTO THE UNITED STATES. The Act of August 31, 1922 (Public No. 293— 67th Congress), entitled " An Act to regulate foreign commerce in the importation into the United States of the adult honeybee {Apis melUfica)" provides as follows: That, in order to prevent the introduction, and spread of diseases dangerous to the adult honeybee, the importation into the United States of the honeybee (Apis mellifica) in its adult stage is hereby prohibited, and all adult honeybees offered for import into the United States shall be destroyed if not immediately exported : Provided, That such adult honeybees may be Imjwrted into the United States for experimental or scientific purposes by the United States Department of Agriculture : And provided further. That such adult honeybees may be Imported into the United States from countries in which the Secretary of Agriculture shall determine that no diseases dangerous to adult honey- bees exist, under rules and regulations prescribed by the Secretary of the Treasury and the Secretary of Agriculture. Sec. 2. That any person who shall violate any of the provisions of this act shall be deemed guilty of a misdemeanor and shall, upon conviction thereof, be punislied by a fine not exceeding $500 or by imprisonment not exceeding one year, or both such fine and imprisonment, in the discretion of the court. In accordance with the foregoing Act, notice is hereby given that the fol- lowing rules and regulations have been prescribed by the Secretary of the Treasury and the Secretary of Agriculture, the same to become effective ou and after the fifteenth day of May, 1923. Hbnkt C. Wallace, Secretary of Agriculture. Mat 12, 1923. RULES AND REGULATIONS. The following rules and regulations are promulgated under the authority conferred by the Act of Congress approved August 31, 1922, providing for the regulation of foreign commerce in the importation into the United States of the adult honeybee {Apis mellifica) : Regulation 1. — Definition. — For the purpose of these regulations, it is under- stood that a disease dangerous to the adult honeybee is one which attacks adult honeybees, as distinguished from one which attacks the brood or de- velopmental stages of the honeybee. Such diseases of adult honeybees are un- derstood to include all diseases which attack adult honeybees, including queen- bees, worker bees, and drones or male bees : Provided, That the disease caused by the protozoan parasite, Nosema apis, sometimes known as Nosema-disease, now widespread in the United States, shall not be considered as. a disease dan- gerous to adult honeybees for the purposes of these regulations. Regulation 2. — Since, in the opinion of the Secretary of Agriculture, the importation of queenbees, vvith necessary accompanying worker bees, is the only kind which is necessary for the improvement of the stock of honeybees within the United States, it is understood that, for the purposes of these regulations, such expressions as the " importation of honeybees " or " importa- tion of adult honeybees " shall mean the importation of queenbees and the necessary accompanying worker bees, except as hereinafter provided. Regulation 3. — ^The importation into the United States of the honeybee (Apis mellifica) in its adult stage, except as hereinafter provided, is prohibited, and all adult honeybees offered for entry into the United States, except as hereinafter provided in these regulations, shall be destroyed if not immediately exported. 31 32 Department Circular 287, U. S. Dept. of Agriculture. Eegtxlatiox 4. — On representation by any person to the Department of Agrij culture that there is adequate necessity for the importation of adult honeybees for experimental and scientific purposes, from any country other than those determined by tlie Secretary of Agriculture to be free of all diseases dangerous to adult honeybees, the Department of Agriculture will undertake to import such adult honeybees under the first proviso of the Act for the purpose in- tended, when the Department shall determine that such importations can be made without risk to the beekeeping Industry of the country. (2) All shipments of adult honeybees made for experimental and scientific purposes shall be addressed to the United States Department of Agriculture, Washington, D. C, and shall be subject to such examinations and holding In quarantine as may be necessary to determine the freedom of the shipment I'rom diseases dangerous to adult honeybees. It is understood, as a further precautionary measure, that the Department of Agriculture will destroy all the worker bees accompanying such imported queenbees and will provide fresh worker bees and a fresh mailing cage for each such shipment. Any such Im- portation made for experimental and scientific purposes which is found to be infected with any disease dangerous to adult honeybees may be destroyed or returned to the country of origin, at the option of the Department of Agricul- ture and no shipment will be distributed until the Department of Agriculture is convinced that the adult honeybees therein contained are free from all dangerous diseases. Any persons receiving adult honeybees distributed by the Department of Agriculture shall agree to the re-examination of the shipment from time to time, at the option of the Department, and shall relinquish the shipment and any increase therein to the Department of Agriculture for de- struction or safeguarding, should any diseases dangerous to adult honeybees at any time develop In connection with it. Regulation 5. — In accordance with the second proviso of the Act, adult honeybees may be imported into the United States from countries in which the Secretary of Agriculture shall have determined that there exists no disease dangerous to adult honeybees. (a) The Secretary of Agriculture, having determined that no disease dan- gerous to adult honeyljees exists in the Dominion of Canada and being advised that, under order of the Deputy Minister of Agriculture of the Dominion of Canada, dated April 22, 1922, the importation of bees, used and second-hand hives, and raw hive goods and products, except honey and wax, from the conti- nent of Europe into the Dominion of Canada, is prohibited, does hereby author- ize that adult honeybees, unrestricted as to the definition thereof contained in Regulation 2 hereof, may be imported from the Dominion of Canada into the United States or any of its Territories or Districts free from any restriction whatsoever provided in these regulations, until otherwise ordered. (b) Importations under the second proviso of the Act, from any country other than tlie Dominion of Canada, shall be conditioned on the determina- tion by the Secretary of Agriculture that, as a result of adequate scientific investigations, no diseases dangerous to adult honeybees exist in the country in question and that adequate precautions have been taken by such country to prevent the importation of adult honeybees from countries where such dan- gerous diseases exist. Regulation 6. — Nothing in these regulations shall interfere with the regu- lations of any state pertaining to the control of the diseases of bees, either of the adult stage or of the brood, and a removal of the restrictions of this Act as applied to any country shall not be construed as granting permission for importations prohibited by the laws of the state into which shipment is contemplated. Henkt C. Wallace, Secretary of Agriculture. A. W. Mellon, Secretary of the Treasury. The Occurrence of Diseases of Adult Bees, 11. 33 UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY. SERVICE AND REGULATORY ANNOUNCEMENTS. SPECIAL RULES For the Importation of Queenbees for Experimental and Scientific Purpose* by the Depart- ment of AKriculture in Accordance Witli Regulation 4 of the Rules and ReguIationB Pre- scribed by the Secretary of the Treasury and the Secretary of Agriculture and Made Effective as of May 16, 1923. Regulation 4 of the rules and regulations prescribed by the Secretary of the Treasury and the Secretary of Agriculture in accordance with the Act of Au- gust 31, 1922 (Public No. 293— 67th Congress), is as follows: Rbgdlation 4. On representation by any person to the Department of Agriculture that there is adequate necessity for the importation of adult honeybees for experimental and scientific purposes, from any country other than those determined by the Secretary of Agriculture to be free of all diseases dangerous to adult honeybees, the Department of Agriculture will undertake to import such adult honeybees under the first proviso of the Act for the purpose intended, when the Department shall determine that such im- portations can be made without risk to the beekeeping Industry of the country. (2) All shipments of adult honeybees made for experimental and sciehtiflc purposes shall be addressed to the United States Department of Agriculture, Washington, D. C, and shall be subject to such examinations and holding in quarantine as may be necessary to determine the freedom of the shipment from diseases dangerous to adult honeybees. It is understood, as a furtlier precautionary measure, that the Department of Agricul- ture will destroy all the worker bees accompanying such, imported queenbees and will provide fresh worker bees and a fresh mailing cage for each such shipment. Any such importation made for experimental and scientlflc purposes which is found to be infected with any disease dangerous to adult honeybees may be destroyed or returned to the country of origin, at the option of the Depai'tment of Agi-iculture and no shipment will be distributed until the Department of Agriculture is convinced that the adult honeybees thei-ein contained are free from all dangerous diseases. Any persons receiving adult honeybees distributed by the Depai'tment of Agriculture shall agree to the re-examina- tion of the shipment from time to time, at tlie option of the Department, and shall relin- quish the shipment and any increase therein to the Department of Agriculture for de- struction or safeguarding, should any diseases dangerous to adult honeybees at any time develop in connection with it. Information is not at present available as to the number of queenbees which are urgently needed for experimental and scientific purposes, but the number of such importations will necessarily be limited by the Department's facilities for examining the imported material and for keeping the imported queenbees in quarantine for such time as may be deemed essential. In order, therefore, to limit the volume of such importations and to insure that the queenbees shall be imported solely for experimental and scientific purposes, as provided by the law, and in order adequately to safeguard the beekeeping Interests of the United States, the following special rules are announced to govern such importations : (1) Importations will be limited to the following classes of institutions and persons : (a) Public institutions, such as Agricultural Colleges, Agricultural Experi- ment Stations, and similar institutions, which desire to conduct investigations on the various races of honeybees, may obtain queenbees through importation by the Department of Agriculture for such experiments. (b) An Individual, who can show that he is engaged in some special field of experimental and scientific work in beekeeping or with honeybees, may, on a satisfactory showing of scientific training and experience requisite for such work, obtain queenbees through importation by the Department of Agriculture for that purpose, provided there is reason to believe that the proposed ex- perimental and scientific work will have value as a public service. (e) Commercial queen-breeders, who urgently need queenbees for breeding experiments, may apply to the Department of Agriculture to have the neces- sary importations made. Such an application shall contain, or be accompanied by, evidence that the applicant is engaged in the rearing of queenbees on a commercial scale and shall Indicate the purpose of, and the necessity for, the importations. If an applicant Is not well known to the Department, he may be required to submit a list of persons qualified to substantiate statements made regarding his ability and standing as a breeder of queenbees. (d) The Department of Agriculture does not consider that the experimental and scientific purpose for which importations may be made under said Regula- tion 4 includes the importation of queenbees for individual beekeepers merely for the purpose of requeening their own apiaries. In case, however, queens of 34 Department Circular 287, U. S. Dept. of Agriculture. certain races cannot be obtained in the United States, and tbe testing in tlie United States of such races would be of value to the beekeeping industry, the necessary importations will be made by the Department, provided that those who make request therefor, and to whom the queens are to be distributed for experimental and scientific purposes, will agree to report to the Department twice annually on the merits of such races in comparison with races already known in the United States. Applications for such importations must show that it is impossible to secure such queens from commercial queen-breeders in the United States. (2) Persons, institutions, and others, In urgent need of imported queenbees for experimental and scientific purposes, may submit a statement of their needs to the Department of Agriculture, giving the name and address of the foreign queen-breeder from whom the queenbees are desired, and, if approved, the Department will transmit an order to the breeder in the foreign country from which such queenbees are desired. No orders for imported queenbees will be placed by the Department of Agriculture with any but experienced and recog- nized breeders of queenbees in foreign countries and evidence must be pre- sented by the applicant that the foreign queen-breeder is qualified to rear good queenbees and to mail them in a satisfactory condition. (3) In the event that importations are made and the queenbees die enroute, the Department of Agriculture assumes no responsibility whatsoever, either in the shipment of the queenbees from the foreign country to the Department of Agriculture or in forwarding the queenbees to the person at whose request the importation was made. Every care will be exercised so that the queenbees may be safely mailed under the restrictions laid down in said Eegulation 4. All shipments of queenbees will be made in accordance with the regulations of the Post Office Department governing such shipments in domestic mails. (4) All persons receiving queenbees from foreign countries distributed by the Department of Agriculture shall agree further to cooperate with the De- partment in such additional examinations of the colonies containing the im- ported queenbees or their offspring, as shall be deemed necessary to protect the beekeeping interests of the United States from the introduction of diseases dangerous to adult honeybees. In the event that any later examination of the offspring of the Imported queenbees is deemed necessary by the Department, tlie person receiving the imported queenbees shall agree to furnish the bees de- sired promptly or to permit such examinations by a representative of the De- partment as may be deemed necessary. Any person receiving such imported queenbees from the Department of Agriculture shall further agree to notify the Department immediately if any abnormal conditions are seen in the adult honeybees in the colony headed by the imported queenbee, or in any other colony in the same apiary, so as to permit immediate examinations of any ap- parently abnormal adult honeybees. The foregoing special rules are hereby adopted and shall be in force until further notice. C. W. PUGSL,BY, Acting Secretary of Agriculture. June 19, 1923. ADDITIONAL COPIES OF THIS PUBLICATION MAT BE PKOCUEED FROM THE SUPERINTEKDENT OF DOCUIIENTS GOVERNMENT PRlNTINGr OFFICE "WASHINGTON, D. C. AT 5 CENTS PEE COPY PURCHASER AGREES NOT TO RESELL OR DISTRIBUTE THIS COPT FOR PROFIT.— PUB. RES. 57, APPROVED MAT U, 1922 V WASHINGTON : GOVERNMENT PRINTING OFFICE : 1923 Technical Bulletin No. 149 January, 1930 FUNGOUS DISEASES OF THE HONEYBEE BY C. E. BURNSIDE Assistant Aficidturist, Division of Bee Culture Bureau of Entomology United States Department of Agriculture, Washington, D. C. Technical Bull. No. 149 January, 1930 UNITED STATES DEPARTMENT OF AGRICULTURE WASHINGTON, D. C. FUNGOUS DISEASES OF THE HONEYBEE' By C. E. BuuNsiDE Assistant Apiculturist, Division of Bee Cidtiu-e, Bureau of Entomology CONTENTS Page Introduction 1 Historical _ 2 Investigations with pathogens __ _ 4 Descriptions and locations of apiaries used, i Races of bees _ __ 5 The fungi studied _ _._ 6 Collection of fungi from colonies and from ' individual bees 6 Examination of diseased bees and isolation of pathogenic fungi 7 Culture methods 8 Pago Experiments with bees 11 Preliminary inoculation with molded combs _ 11 Inoculation experiments with pure cultures. 1 2 Secondarily infected bees 16 Results of inoculations and symptoms cf the diseases produced 16 Discussion ___ 33 Summary 33 Literature cited 40 INTRODUCTION The recognition and control of bee diseases is of prime importance in the commercial production of honey. The serious bacterial dis- eases of the brood have been extensively studied, and the life histories of their causative organisms and the methods for control are fairly well understood. There exist, however, other diseases of the brood and of adult bees, seemingly of lesser consequence, the causes of which are not yet known and for which satisfactory methods of control have not been determined. The writer has shown in an earlier paper {5) ^ that a considerable number of species of fungi occur regularly on adult bees, and less frequently on their brood, which completely mummify all of the softer tissues. Most of these species of fungi are capable of establish- ing themselves on the brood combs under conditions that often pre- ' Thia bulletin, prepared in part fulfillment of the requirements for the degree of doctor of philosophy at the University of Michigan, is a joint contribution from the Bureau of Entomology, U. S. Department of Agriculture, and the University of Michigan. The experimental work was done in the laboratories of the department of botany of the University of Michigan and the bee culture laboratory of the Bureau of Entomology at Somerset, Md. The author acknowledges the valuable advice and assistance of C. H. KauCfman, of the University of Michigan, under whose direct supervision this work was done ; of H. H. Bartlett, of the University of Michigan ; of James I. Hambleton, senior apiculturist, in charge of division of bee jsulture, Bureau of Entomology ; and of B. F. Phillips, formerly apiculturist In charge of bee culture investigations. Bureau of Ento- mology, and now professor of apiculture in the New York State College of Agriculture. Special acknowledgments are due to Charles Thom and M. B. Church, of the Bureau of iChemistry and Soils, for assistance in the Identification of the forms used. ^Reference isi made by italic numbers in parentheses to " Literature cited," p. 40. 59850°— 30 1 1 2 TECHNICAL BULLETIN 14 9, U. S. DEFT. OF AGRICULTURE vail within the hive during late winter and spring. The ecology of these fungi on bees in all stages and their relation to bee diseases have never been fully worked out. During the past three years a mycological study has been made by the writer, in culture and on bees, of a considerable number of species of fungi that were isolated from adults, larvae, and combs. Experiments have been performed to determine whether the fungi that are commonly found on bees are purely saprophytic or whether, under conditions favorable for infection, they can attack and kill healthy bees or brood. HISTORICAL The existence of diseases of bees was first recorded by writers be- fore the beginning of the Christian era, but the descriptions are too meager to identify them. The more careful study in Europe of dis- eases of brood dates from the work of Schirach {21) in 1771, but for more than a century following it was quite generally believed that there was only one such disorder. Succeeding reports in Europe sup- ported this view. During the decade just preceding the twentieth century American beekeepers came to believe that more than one . disease of the brood existed, and that these diseases were of decidedly different characters. This has been conclusively proven in the United States, chiefly by the investigations of White, who describes and figures two bacterial brood diseases, a filterable virus . disease, and one protozoan disease of adult bees, in a series of papers dating from 1917 to 1920. The diseases described by him are sacbrood (^5), Nosema disease {29), American foulbrood {30), and Euro- pean foulbrood {31). More recent valuable additions to the knowl- edge of brood diseases and their control have appeared in the publi- cations of Sturtevant {22, 23), and Zander {32, 33). The study of diseases of bees due to fungi has been much later in its development than the study of those caused by bacteria. Mucli the greater share of the attention of investigators of bee diseases has been given to the two serious bacterial brood diseases, American foul- brood and European foulbrood, caused respectively by BaxMlws larvae and B. pluton. In America only two -reports are on record of diseases of bees and their brood caused by hyphomycetous fungi. In 1896 Howard {H), of Texas, described a new brood disease which he called " pickled brood or white fungus," caused by a species of Aspergillus to which he gave the name "Aspergillus polUni." Two years later he described {IS) the same disease as occurring in both pupae and adult bees and stated his belief that this disease had been mistakenly diagnosed by beekeepers as paralysis. In both reports Howard gives descriptions and illustrations of the disease, which he called " pickled brood or white fungus," which are more readily applicable to the disease known as sacbrood than to those caused by fungi. He ascribed this disease to the pathogenic Aspergillus, A. pollini. The dead larvae are described as at first white and watery, later becoming black and swollen, and finally dry- ing down to black scales. In no case was the fungus, which Howard assumes to be the cause of the disease, observed on the larvae, and isolations of the organism were not made. In adult bees the disease is described as causing black, shiny, apparently frozen abdomens. FUNGOUS DISEASES OP THE HONEYBEE 6 The affected bees become much weakened, are capable of only feeble, trembling movements, and finally die. As in the case of the brood killed by the " pickled brood " disease, the fungus was not observed on or within the tissues of the infected bees, but when cultures were pre- pared from the alimentary canals of these bees, A. ■pollmi developed constantly to the exclusion of all other fungi. It appears probable, therefore, that Howard may have observed one or more species of Aspergillus on brood combs and succeeded in culturing these, or other species, from the alimentary canals of adult bees. His technical descriptions of AspergUIm pollini are entirely too meager to make it possible to determine which of the numerous species of Aspergillus were observed. If they are patho- genic for bees, it is evident that he either did not observe a true mycosis of bees or confused the condition with other disturbances. Diseases of bees caused by fungi have not been reported from North America since then. This may probably be attributed to the fact that fungous diseases of bees appear less destructive than the common bacterial diseases of bees and seldom become epidemic. In Europe, on the other hand, two species of fungi, AspefrgiUuS flavies Link and Pericystis apis Maassen, are widely recognized as the causative organisms of diseases of brood and adult bees. Of these, A. ■flav%us is considered of the greater economic importance, since it attacks worker brood and adult bees, whereas P apis usually attacks only drone brood. The two brood diseases stone brood (Steinbrut), caused by A. flaims, and chalk brood (Kalkbrut), caused by P. apis, have received more attention from European beekeepers and investigators than has the disease of adult bees caused by A. flanms. This might readily be expected as a direct result of the nature of the diseases and the reaction of infected adult bees. Pericystis mycosis is mentioned by Claussen (■?), Bahr (^), and Morgenthaler, Kessling, and Hunselmann (in communications with Claussen) . Claussen describes it as benign and transient rather than malignant, affecting capped as well as uncapped drone brood and passing over to worker brood in severe or exceptional cases. He states that dead and diseased larvae may be thrown out of the hive by the bees or allowed to remain in the brood combs, where they become mummified after they are overgrown with white mycelium. The bees, however, usually allow any brood killed by A. fiavvs to remain in the combs for a considerable length of time, or at most only partially remove it, since destruction of the cell walls is often necessary for complete removal. Slight infection among the brood quickly attracts the attention of an observing beekeeper, whereas he may completely overlook a con- siderable number of adult bees dead of this disease, owing to the fact that during the active season worn-out field bees die normally in considerable numbers about the hive. The writer, therefore, believes that the importance of Aspergillus mycosis as a brood disease may have been overestimated in comparison with its importance as a disease of adult bees. Eecent research in Europe seems to indicate that other fungi than the foregoing may under favorable conditions infect and kill bees and their brood. Fielitz (10), working with three fungi, Tricho- derma lignorvmi Tode, Mwcor nmcedo Linne, and PeniHlUwin glaucuTn 4 TECHNICAL BULLETIN 149, TJ. S. DEPT. OF AGKICULTTJRE Link, found on mummified bees in Germany, was able to infect capped and uncapped brood and adult bees by artificial inoculation with the first two. T. Ugnorum was shown to be capable of becoming actively pathogenic when introduced into healthy colonies on brood combs. When similarly introduced, M. rrmoedo attacked and killed brood in capped cells and an occasional adult bee. In his experiments with P. glaucum neither the bees nor their brood were attacked, although it is one of the commonest organisms in the hive. While making anatomical studies of honeybees affected with con- stipation, Lardinois {17) claims to have observed SaccharoTnyces apiculat-ws (Reess) Hansen constantly associated with lesions in the tissues. The same organism occurred in the intestines of dead pupae that were thrown out of the hive, in the food of larvae, and in honey. Lardinois asserts his belief that S. apicwlMvjs is the sole cause of May disease and that disturbances commonly recognized as constipation, paralysis, convulsions, staggering, malformation, and death of brood are, in reality, all forms of the disease which he called saccharo- mycosis. He does not support these conclusions by inoculation ex- periments but draws them solely from the fact that this yeast occurs in " lesions " and in the intestine of bees affected with " May disease." Doctor Lardinois believes, however, that these conclusions may be easily verified. One is quickly led to believe that bee diseases caused by pathogenic fungi may be more widespread than is commonly supposed. A com- parison of conditions in Euro^De^ where a number of recognized fungi are known to cause diseases of bees, with conditions in this country would make it seem likely that the same fungi are capable of attack- ing bees here. The publications of Thom and Church {2J^; SS, p. 200) show that a great number of strains of Aspergillus f,avus are found in America. Mucor mvicedo and Triohoderma ligrvorwin also occur elsewhere than in Europe. Perioystis apis and P. cil/aei Betts, which, according to Claussen {8), differ in certain morpho- logical characters and in their ability to attack brood of bees, have never been reported from North America. Forms of Saccharomyces apiculatus, which Lardinois has stated cause disease of bees in France, are widely distributed in North America. The writer has frequently found these forms in North America within the alimentary canal of bees and in honey. INVESTIGATIONS WITH PATHOGENS DESCRIPTIONS AND LOCATIONS OF APIARIES USED Apiaries located in the vicinity of Bronson, Mich., were used in these investigations for securing specimens of fungi and for making tests for pathogenicity and control. These apiaries are situated on boulder clay and sandy drift formations in a general farming com- munity. Three honey flows occur annually in the region, providing a plentiful supply of stores at all times when weather conditions are favorable. The experimental apiary of the botanical laboratory of the Uni- versity of Michigan at Ann Arbor was also used in these investiga- tions. This apiary was located on the roof of the Natural Science Building, thus affording excellent conditions for recovering diseased FUNGOUS DISEASES OF THE HONEYBEE 5 bees. With the beginning of cold weather the colonies were placed in an attic where a temperature of about 65° F. was maintained, affording facilities for manipulating the bees during cold weather without danger of chilling them. The general conditions here with respect to honey flows and weather factors are essentially the same as at Bronson, excepting that here the early spring honey flow is somewhat heavier on account of the surrounding fruit farms. On the other hand, the fall honey flow from wild flowers is somewhat less. Two apiaries of the bee-culture laboratory of the Bureau of Ento- mology at Somerset, Md., were used during the summers of 1924, 1925, and 1926. One of these was located at the laboratory, while the other was about a quarter of a mile away. Forest trees, par- ticularly species of Acer and Salix, provide a source of nectar dur- ing early spring, but the main honey flow is from the tulip tree {Linodendron, tvlipifera). Occasionally basswood (Tilia), spruce (Picea), and locust {Rohinia fseudoaoacia) yield appreciable quan- tities of nectar. The fall flow here is light and of short duration. RACES OF BEES The bees used for the tests for susceptibility were for the most part Italians and Italian hybrids, including workers, drones, and queens. No other races were used for inoculation experiments. Pathogenic organisms were isolated, however, from Carniolan bees. The colonies used for inoculation experiments were, most of them, in 5-frame hives (nuclei) containing three or more frames of brood. Normal colonies in standard 10-frame hives were also occa- sionally used. THE FUNGI STUDIED The fungi used were isolated from bees collected from widely different sources. The greater number of forms were from bees from the experi- mental apiaries. The first isolations of fungi were made from dead bees and their brood collected at Bronson, Mich., during the early brood-rearing season of 1924, and others were made from bees col- lected at this same apiary during the spring of 1925 and that of 1926. During the summers of the three years that this work was in progress fungi were collected from the apiaries of the bee-culture laboratory of the Bureau of Entomology. Among the forms obtained here were duplicates of most of those collected at Bronson and elsewhere. Important collections were also made from the experimental a,piary at the University of Michigan during the academic years of 1924-25 and 1925-26. In addition to the collections from the experimental apiaries, fungi have been isolated from samples of bees or brood sent to the bee- culture laboratory from all of the important beekeeping regions of the United States. Although the forms isolated from such samples duplicated those obtained from the experimental apiaries, their presence gave an indication of the extent of the range of these forms. All of the Aspergilli in the following list appeared with sufficient regularity to indicate their distribution among bees in all parts of the United States, and two species, A. ftavus and A. fvmdgatus, were , isolated on several occasions from bees imported from Europe. The 6 TECHNICAL BULLETIN 14 9, U. S. DEPT. OF AGEICULTURE yeasts were also isolated from bees from numerous sources within the United States. Trichoderma koningi was isolated from adult bees in Maryland and from mummified larvae from Oregon. Fe7%- cystis apis was also isolated from mummified brood sent from Ji,ng- land and from Germany. . -, ij. i j The fungi included in the following list were found to attack and kill bees when the latter were inoculated experimentally. Among the Aspergilli, with the exception of Aspergillus oryBoe and A. para- siticus, many cultures other than those indicated were isolated from bees and tested for parasitic properties. This is true, especially of the A. -flavus-or^zae and the A. fwnigatus groups. Aspergillus flavus (Link), sensu Thorn and Churcli. Author's collection: 1, 3, 4, 5, 7, 9, 12, 28, 12340. Thorn and Church collection : Ao5c, 183, 108. Aspergillus oryzae, (Ahlburg, Cohn), sensu Thorn and Church. Thorn and Church collection : 113 L, Ao5b. Aspergillus effusus, VIII ; D. C. Aspergillus parasiticus Speare. Aspergillus flamis-oryzae, sensu Thorn and Church. Thom and Church col- lection : Aop, Apb, Aob, Ao5u. Aspergillus fumigatus (Fresenius), sensu Thom and Church. Author's col- lection: 1, 33, 12287, 12288. Thom and Church collection: Tates IV, 118, 4063-C-18. Aspergillus iiiduXans (Bidam), sensu Thom and Church. Author's collection: 1. Thom and Church collection : 110, 4010.4, 4415. Aspergillus oohraceus (Wilhelm), sensu Thom and Church. Author's col- lection: Conn.; D. C. Thom and Church collection: 112, 2399, 4020.4, 4065.1, 4640.476, 4640.483. Aspergillus glaucus (Link), sensu Thom and Church. Author's collection: 1, Ann Arbor, Mich. Thom and Church collection : 3528.7. Saccharompces ellipsoideus Hansen. Snccharomyces cerevisi^>^K ^ >,'f-"^^m'*'/.^" ^r Fio. 1. This illustration shows a normal comb of healthy brood. Note how compactly the brood is placed, with very few unsealed cells intervening. Fig. 2. This illustration shows a comb containing American foulbrood in an ad- vanced stage. Note the spotted appearance of the brood caused by scattered sealed cells. (Courtesy of the A. I. Eoot Co.) 6 multiply rapidlj', the larva soon showing signs of being aifected. This usually takes place soon after the larva is capped. The majority of the infected larvae die in the late larval stage, though some do not die until after transforming into the pupae. Usually the first indication that the larva is diseased is a sunken and slightly darkened appearance of the capping. The bees soon begin removing this capping. First a tiny pin-hole perforation is made, which is enlarged until finally the entire capping may be removed. The diseased larva first shows a slight brownish dis- coloration and begins to lose its normal shape. As the disease advances the color becomes darker and the deea,ying mass passes through all stages of brown, variously described as chocolate, mahogany and coffee color. The larva sinks down to the lower side of the cell, presenting a flattened, melted-down appearance. The back end of the dead larva is slightly elevated against the bottom of the cell, while the front end flattens down on the lower side of the cell, though in the earlier stages it may be slightly rounded at the front and somewhat elevated. The larvae lie in a fairly uniform position, stretched out on the lower sides of the cells. This characteristic aids in distinguishing American foul- brood from European foulbrood, in which latter disease the dead larvae are found in all shapes and positions. Fi'ijy!r''T' Fig. 3. This illustration 3 juice of the larvae at difi«rent stages of the disease, the bacillus may be detected ; 1)nt spor-)S do not 2orm till after death has occurred. The ropy mass contains large numbers of spores, as does also the dry scale. According to Cheshire (26), the bees themselves become diseased. In a number of cases he obtained the bacillus from the blood of bees from infecied hives. Hilbert's examinationin 1875 led him to declare that the mature bees in infected hives were liable to be affected. Some writers contradict the statement that the bees themselves are affected by the disease ; bat they lose sight of the fact that the bees do not die in the hive, but leave it sometime before death occurs. The queen may become infected. Cheshire (26) demonstrated the pres- ence of bacilli in the ovary of a queen ; but he did not make cultures there- from. W. G. Smith (27) reported that a queen sent to Cheshire and exam- ined by him contained B. alvei in both of her ovaries. McKenzie (28) examined five queens from infected hives and succeeded in obtaining bacilli from the ovaries of three. He thinks that their presence there is accidental, as in the case of a queen from a badly diseased hive he was unable to find the bacillus, whilst in a six weeks old queen from a hive in which there were only a few diseased cells, he succeeded in finding it. A queen sent by T. A. Oovan (29) to Cowan, the editor of the British Bee Journal, was examined, and B. alvei was found in the ovaries. F. P. Ward (30) removed a queen from a diseased hive and placed her in a strong, healthy stock, " which speedily became a maes of corruption." This operation was subsequently repeated with a like result. I have also myself examined seven queens from diseased hives, and in three cases have had no difficulty in finding the bacillus, and have obtained typical cultures therefrom. The method of examination employed has been the same as that used by McKenzie. The upper surface of the bee is ster- ilized and cut longtitudically, and all the internal organs except the ovaries are removed. The surface of the ovaries is then sterilized and a hot needle plunged into the centre and allowed to stay there until it is cold, when 6 it is withdrawn and nspd to inoculate agar cultures. According to Cheshire (26) the bacilli are found in the eggs. In one examination he says he counted nine bacilli from a half-developed egg taken from the ovary of a qneen. McKenzie (28) thinks that this statement requires confirmation, as he was not able to find any infected eggs. 1 have myself examined a very large number of eggs at various times. In these examinations three difl'<=rent methods were employed : 1. — The eggs were taken from the cells in which they were laid with sterilized forceps and washed in corrosive sublimate, 1 : 500 crushed and placed on agar plates. In many cases typical growths of B alvei developed from eggs thus treated ; but as it might be maintained that the eggs were laid in cells previously infected with B. alvei, and that the momentary immersion in corrosive sublimate failed to kill all the spores that were upon the exterior, the next lot of eggs 2. — Were crushed between cover-glasses, a small portion transferred into agar, and the remainder on the cover-glass stained by Gram's method. In several instances the bacillus was found in the crushed egg, and in every case the cultural test confirmed the microscopical examination. Again, as this method also might be criticized for the reasons above stated — (3) eggs were imbedded in parraffin and serial sections made and stained by Gram's method No cnltnral tests were made ; but in a few eggs of several hundreds sectioned a bacillus corr,!sponding in its morphological characteristics to B. alvei was found. All the eggs examined were taken from hives more or less aflected with the disease. In view of these fact s, I am of the opinion that the eggs of bees from diseased hives may in some instances be infected. Chilled Brood. Chilled brood is sometimes mistaken for foul brood ; but the appearance of the former is very different from that of the latter. In the case of chilled brood the larvae turn grey ; afterwards the colour darkens, and in the final stages of decomposition it becomes black. No ropiness develops. A number of writers in various bee periodicals have mistaken chilled brood for foul brood, or they have stated that chilled brood turns to foul brood ; but Schirach. ag long ago as 1769, clearly distinguished between the two, and McKenzie (28) also performed several experiments in refutation of the state- ment that if chilled brood is allowed to putrify foul brood may arise de novo. He endeavoured to isolate B. alvei from chilled brood, but without) success. Again, he killed perfectly healthy brood by chilling, and infected some of the cells from a pure culture of B. alvei. The chilled brood were allowed to putrify in a moist chamber for several months and examined with the same results, viz : that in the cells in which B. alvei was placed it was to be found, but not in any others I have also performed similar experiments and they fully confirm McKetizie's contention. So far B. alvei has not been isolated from chilled brood in any stage of decomposition. Canestrini (31) described a case which was in all probability chilled brood and not an infec- tious malady ; but his inoculation experiments failed to establish the patho- genicity of the bacillus, which morphologically resembled B. megatherium. Geographical Distbibdtion. It has been thought that the disease varies in different countries, that foul brood as it occurs in England is different from foul brood in America ; but as no bacteriological evidence has been produced in support of the con- tention, it is not neceBsary to argue the question. I have examined diseased larvae from Canada, from Earope (France, Switzerland, Austria, Germany, Italy and England), Cuba, and 13 States of the Union, ranging from New York to California and from Michigan to Florida, and have succeeded in isolating B. alvei from all of them. It is true that some of the cultures show certain differences, but they have not been sufficiently pronounced to consti- tute even a well marked variety of the species. The pathogenicity of the bacillus no doubt varies in different countries ; of that we have abundant evidence, and the possible explanation is given by Bertrand, who thinks that where bei s have been kept for many years the disease has existed for a long time and remains in an endemic state ; but there has been produced in these countries a race of bees which have acquired a relative immunity, which con- siderably diminishes the effects of the disease, and enables apicalturists to treat it more easily. In new countries into which the disease has been intro- duced it rages with great virulence, and remedies giving good results in the older countries are worthless in the new. As an example of this statement, we have the difierent methods of treatment used in Canada and in Europe. Bertrand (32) reports the disease as being present in every country in Earope. Benton (33) says that he has never met with the disease during the six years he has kept bees in the Orient. Delia Rocca (7) described a terrible epidemic in the Levant in 1780. Bovill (34) says that he has never seen it in Cyprus. In Africa, Feuillebois (35) reports it in Algeria, and Bochatey (81) in Tunis. In Australit it is present in all the colonies, and especially so in New South Wales (86) and South Australia (37). Brickwell (38) re- ports that New Zealand is full of the disease. The Organism Bacillus Alvei, Cheshire and W. Oheyne, 1885, from the larvse of bees suffering from the disease known as foul brood, la loque (Fr.) and faul brut (Ger.). Morphological Characteristics. — In form the organism is a slender bacil- lus, with ends slightly pointed and rounded. " In the larval juices it is about 7,5(10 of an inch in length and jojoo >n breadth. On agar the bacilli vary con- siderably in size, averaging ^jgo inch, some as small as lom ii^ch, and others as large as jjoc inch. When they have attained the latter size, division of the rod seems to begin. They are always somewhat pointed at their ends. Their average breadth is ^am inch, ranging from 35J00 to 25,000 inch (23). Klamann (25) states that a clear space often appears in bacilli with pointed ends. From agar cultures 24 honrs old, at 37° 0., the bacilli average 4 /^ in length and 1.0 fj. in breadth. On gelatine cultures, grown at 22° C, they are somewhat shorter. They grow singly, but occasionally form chains of various length. Stains. — With the ordinary aniline stains the bacilli colour rather badly — Eisenberg (39) and Klamann (25). The best stains are methelene blue and methyl violet. The bacilli accept Gram's stain, but the spores are not colored by it. I find the most satisfactory stain is methyl violet. Capsule. — No capsule has been demonstrated by Welch's method. Flagella — The bacilli are actively motile and possess a single flagellum at one pole. The motility of the bacillus is quite pronounced in fresh cultures obtained from bouillon, agar and gelatine. The flagella stain by Pitfield's, Loefflers's and Van Ermegen's method. Spore Formation. — Spores are formed by the bacillus, and are large oval bodies averaging ia lei gth isJ^ inch, and in breadth ^}oo of an inch. On agar the spores are arranged in long rows, side by side, and are greater m diameter than the cells from which they are derived The earliest appear- ance of spore formation takes place in 41 honrs, at 36° C. (Cheyne), but in some cases it is even sooner. The spores are formed in the centre of the rod, and the formation occurs as follows : The rod begins to swell and become spindle-shaped. Occasionally the swelling is more marked at one end than in the centre. The spindle-shape increases in size, and the centre of the swelling gradually ceases to take the stain. The capsule of the spore is ap- parently formed within the rod and is not merely the outer part of the rod. In three or four honrs the rod is seen to have almost or completely disap- peared, although parts of the faint outline of the ordinary bacillus may be noticed. Germination of Spores — Uuder favourable conditions the beginning of the germination of the spores takes place in about three hours. The spore loses its oval shape, becomes elongated, and is soon seen to burst through the spore capsule. It then presents the appearance of a short lod, with a pale envelope embracing one end. The rod gradually leaves the spore capsule, and then goes on multiplying as a full grown bacillus. According to Eisen- berg (39), the spores are decolorized by the tubercle bacilli stain, but prepa- rations may be obtained by using the Ziehl-Nfilsen stain and alcohol for decolorization. The spores also stain by the method of Neisser. Polymorphism. — Variations in size and shape may be brought about by growth in acid media, or in media containing different sugars. These varia- tions occur also in the same culture, subjected to exactly similar conditions of growth. Involution Forms. — Abnormal forms are especially abundant when the bacillus is grown on blood serum ; peculiar Y-like forms and clubbed shapes are of common occurrence, and relatively few spores are found. Biological Oharacters. Bouillon — " In meat infusion at the temperature of the body, they grow rapidly, causing muddiness and, after a few days, a slight but not ten- acious scum " (23). In bouillon, with a reaction of -i- .08 (57), at 37° C, there is a slight turbidity in 14 hours, especially noticeable when the tube is shaken. In 24 hours, the liquid is uniformly turbid, with a very fine sedi- ment. In 48 hours, the turbidity increases and a pellicle commences to form. Eeaction of the culture at this time, + .07. After 96 honrs the broth is clear, with a pellicle, white, rather massive, and somewhat tenacious. There is also much sediment. Reaction, after 10 days' growth, — neutral. Glycerine Bouillon — Media with original reaction of -1-.08. At 37° C, the bouillon becomes slightly turbid in 12 hours, and quite turbid in 24, with a fine, whitish pellicle on surface, which does not extend to the sides of the tube. If the culture is shaken, the pellicle deposits in flaky masses. The reaction is + 1.2. In 36 hours, the turbidity clears, leaving the media bright, with a smooth, thin, tenacious, and white pellicle on the surface. In many cases the pellicle becomes very wrinkled and greasy-looking. At the end of 8 days, the reaction is +2 2, and the bouillon is several shades darker in colour, but quite clear. The reaction after 14 day's growth is -1-4.2. At 22° 0. the same changes occur but growth is slower. The bacilli are relatively less numerous than in bouillon and are slightly shorter and thicker. Glucose Bouillon. — With a reaction of + 2.0, at 37° 0., the broth is more turbid than plain bouillon after 14 hours' growth ; and in 24 hours, the 9 sediment is heavy, and turbidity very marked, but no pallicle. In 48 hours, the media is opaque and cloudy, and the pellicle is beginoing to form. In 96 hours the broth ia less cloudy, but the sediment is heavier, and a white, thick pellicle is formed. It is often wrinkled, but not quite so much so as that on the glycerine broth. Reaction of broth after 10 days' growth, +4.6. The bacilli are occasionally clubbed and y-like forms may occur. They aver- age 6 // in length and may be slightly curved. Lactose Bouillon. — With a reaction of +1.06, at 37° C, the growth re- sembles that of plain bouillon for the first 24 hours ; but at the end of 48 hours, it is more turbid. In 96 hours, a tenacious pellicle forms, less massive than that on Glucose broth. Reaction after 10 days' growth, + 2.4, The bacilli average 3,5 /< in length. Saccharose Bouillon. — With a reaction of +1.0, at 37° 0., the turbidity and sediment are heavier than any of the other bouillons. In 48 hours the broth 'is quite opaque and whitish looking. A heavy sediment is then present and pellicle formation is just beginning. In 96 hours, the cloudiness is about the same, but there is an increase of sediment and the pellicle is thin and membranous. Reaction of media after 10 days' growth, + 4.04. The bacilli average 5 /< in length. Gelatine Plates. — At 22° C. in 24-86 hours, the colonies are small, round, oval, or lozenge-shaped, with peculiar projections or shoots from one end of the colony, giving it a pear-shaped, or tadpole-like appearance, accord- ing to the amount of development of the projection. In many cases, several of these outgrowths occur from different portions of the colony. By placing a cover glass on the surface of the gelatine and using objective 7, the bacilli may be seen moving around and around the colony and to and fro along the projections. At the end of 48 hours, the colonies are larger. Fine pro- cesses or projections are shooting out into the gelatine in all directions, form- ing peculiar figures in circles or club-line forms. " It is impossible," says Oheyne, " to give a proper idea of the appearance of the growth. The forms assumed are the most beautifully shaped I have ever seen ; but they are very numerous, always retaining the tendency to form curves and circUs." After a time the gelatine is liquefied and the beautiful appearance of the colony is deslroyed by the liquefaction of the gelatine. These peculiar shaped colonies are most typical when the germ is taken from the diseased larvae. After prolonged cultivation on various kinds of media, there is a tendency for the r^olonies to become round, and the peculiar branching forms are not seen in such numbers. The composition of the gela- tine also seems to make a difference in the appearance of the colonies. In gelatine containing 12 per cent, sfelatine the processes are not so long. The same effect may be brought about by using more peptone in the composition of the media. Gelatine Tubes. — In stick cultures at 20° C. growth occurs all along the line of puncture. On the surface, delicate branching or ramifying growth occurs in three days. These outgrowths soon run together and the gelatine is liquefied, first around the line of puncture, and in 5 days extends over the ■whole surface. The growth in the depth of the gelatine occurs as a whitish streak all along the needle track ; and from this, numerous shoots and growths branch out into the gelatine in all directions, giving a haziness to the appearance of the gelatine, which then begins to liquefy. If the inocula- tion is a heavy one, the f'hoots are coarse and may have club shaped extremi- ties, and from these swollen ends fresh shoots may start. Oheyne obtained the-' most characteristic growth in gelatine containing 3 per cent, of peptone, as well 10 as 10 per cent, gelatine. The whole tube is liquefied in from 2-4 weeks' growth. The liquid becomes yellowish in color and gives ofi a peculiar odor. Kla- maunn states that in gelatine acidified with lactic acid the gro wth is slow and l^ng threads are formed. Gelatine Streak Cultures. — In gelatine streak cultures the appearance is very similar to what one sees in stick cultures. The bacilli first grow along the line of inoculation ; and then throw out shoots into the surrounding gelatine, producing the appearance noted in the stick culture. The bacilli move to and fro along the channels of liquefied gelatine, Agar plates. — On agar plates at 37" 0., the colonies at the end of eight hours are small and burr-like, with spines protruding in all directions, giving the colony the appearance of a sea-urchin. In some cases the projections are from one side or end. At the end of 12 hours, the colonies have well-defined projections, visible to the naked eye. The colonies in the depths of the agar are more spiny, the processes being much shorter. On agar plates streaked with a light inoculation, most beautiful forms occur. The growth of the bacilli spreads over the surface and branches repeatedly, giving the appear- ance of s'aweed. Thi"; appearance is distinctively characteristic ; and as the growth is very rapid, this method commends itself for making a quick diag- nosis of the presence of the bacillus in larv% supposed to be diseased. Potato cultures. — On potatoes the growth diifers considerably, according to the reaction and age of the potato. Sometimes a brownish wrinkled growth forms, which gives oflF a peculiar odor j at other times a dryish yellow layer appears, " The bacilli grow very slowly indeed at 20" 0." (Oheyne 23.) Even at 37' C. they grow slowly. Milk. — In milk at 37° C, coagulation of the casein occurs in three days. The milk becomes yellowish and gives off a characteristic odor. After several week's growth, the curd is digested and a whey-like fluid remains. Blood serum. — On blood serum at 37° , the growth is rather slow and polymorphic forms are common. " Very long filaments are formed " (23). These long forms may be from 5 to 10 times as long as the average bacillus growing on gelatine, and consist of single cells. The filaments are often wavy or twisted and of unequal thickness. The extremities of the long, bgnt rods are often clubbed ; and y-forms are numerous. Spores are formed very sparingly, and the blood serum is liquefied. Synthetic media ( UschinsJcy). — In Uschinsky's medium no growth occurs ; but if the medium is neutralized, good growth ensues. The bacilli occur in threads and a pellicle is formed. Dunham's Solution. The bacilli are small when grown in this solution; No threads form ; but there is a slight indol reaction after nine days' growth. Relation to Free Oxygen. Cheyne states that the germs grow most rapidly on the surf«ce of agar and arrange themselves side by side ; and they produce spores in this position after a few days' growth. Eisenberg (39) says nothing under the head of aerobiosis. Howard (40) writes that, " It grows best under anaerobic conditions ; is a facultative aerobe ; grows under the mica plate ; and in the presence of oxygen the growth is slight and slow." Howard also states that under anaerobic conditions it emits a foul odour re- sembling that of foul brood. It will be thus seen that Cheyne and Howard do not agree on this point. The former author also says that the character- istic odour is given oS under aerobic conditions, whilst Howard states that this smell is emitted under anaerobic conditions. Further, Cheyne states that the bacilli grow with great rapidity on the surface of agar, whereas 11 Howard obtains his best growth under the mica plate, which does not give complete anaerobiosis. Howard's conclnsions are thus ac variance with Cheyne's, and my own results fully corroborate those of the latter author. Howard states that ihe vitality of the spores of B. alvei is destroyed when exposed to atmospheric air from 24 to 36 hours. In making his experi- ments he took sterilized road dust and mixed it with the dry foul brood masses from several cells which were previously dissolved is distilled water. The mixture was worked dry, and spread on sheets of paper, and trial cul- tures were made immediately and at intervals of every twelve hours for three days ; and according to his results no growth occurred after 36 hours la giving these results, Howard does not state whethtr he exposed the spores to sunlight or diffused light ; nor does he mention the age of the dry foal brood masses, which he used from several cells. These are points of considerable importance, for as everyone knows the disinfecting power of ditect sunlight ia much greater than diffused light, and the vitality of the spores from foul brood masses of different ages varies considerably. This, I may add, has been clearly shown by some of my experiments, subsequently described. In my experiments, the spores obtained from a pure culture on the surface of agar, were spread on cover glasses and placed in a glass chamber, so arranged that a current of air was constantly circulating over them. This chamber was exposed to the ordinary light of a room with six large windows, and a cover glass was taken out every 24 hours and tested, to see if the spores would grow. This experiment was continued for one month and at the end of that time the spores still germinated rapidly. In another experiment, spores spread on cover glasses were exposed to a very diffused light, simulating as far as possible the amount of light which would enter a hive. Oover glasses were taken oat from time to time and transferred to agar, in order to ascer- tain if the spores were alive or not. The experiment was begun two years and four months ago and from the last cover glass taken and placed upon the surface of an agar plate a copious and typidal growth of B. alvei was obtain- ed. Further, thin strips of filter paper, plunged into a bouillon culture and allowed to dry, were threaded on a wire suspended in a wire basket and so exposed that the air could freely circulate around them in the ordinary light of a room. Trial cultures were made at intervals, and at the expiration of 6 months the spores from the paper germinated when the strips were placed on the surface of agar. Again, a drop of bouillon containing spores was placed in a sterile tube and allowed to dry ; and at the expiration of 124 hours (36 of which were in sunlight at a temperature varying from 30° 37° 0) sterile bouillon was added. The tubes were then placed in the incubator, asd in less thaa 24 hours a good growth of the germs had taken place. From these experiments it will be seen that the results are directly at variance with Howard's statement, as thty go to show that the vitality of the spores of B. alvei is not destroyed by exposure to atmospheric air, with or without sunlight, for even a much longer time than 24-36 hours, With regard to the aerobiosis of this bacillus, good growth has been ob- tained in an atmosphere of hydrogen by Novy's method. Bnchner's method also gave good results. The growths in the various media are very similar to those produced under aerobic conditions, but with this difference, that the surface growths are, as a rule, whiter in the hydrogen atmosphere. In illuminating gas (water g+s) no growth occured ; but the spores were not destroyed by the action of ihe gas ; for when the gas was let out of the Novy jar, good growth ensued on all cultures. In acetylene gas, a restricted 12 growth occurred. In fermentation tubes, growth occurred both in the open and in the closed arm of the tubes. No gas was formed, the bouillon in the closed arm was uniformly turbid. Thus B alvei is a facultative anaerobe. Production of Alkali In ordinary bouillon a slight amount of ammonia is formed. Control bouillon did not give the Nesaler test. In glycerine and the sugar bouillons, there is no trace of ammonia. Cheyne's cultures are faintly alkaline, both before and after inoculation in meat infusion. Elamann states that ammonia is produced, Acids formed A varying amount of acid is formed. All the sugar bouillons give an acid reacton. Formation of Pigment. On potatoes, a yellowish growth is produced ; on all other media, the surface growth is white. Development of odours Cheyne states that gelatine cultures give off an odour of stale, but not ammoniacal urine, or what may be better described as a shrimpy smell ; and this peculiar odour has been formed by Cheshire to be distinctive of diseased larvae. Klamann and Howard both state that a peculiar odour resembling that of the diseased larvae may be noticed in arti- ficial cultures. The Efects of Desiccation I have already noticed, under the head of " Relation to Free Oxygen," that the spores of B. alvei have considerable vitality in withstanding desiccation. My experiments prove conclusively that the spores are extremely hard to kill by desiccation and in this respect resemble those of anthrax, which are known to resist thorough desiccation for a number of years. One experiment which shewed this characteristic was as follows : An agar plate completely covered with a typical growth of B. alvei was allowed to dry out completely, and was left exposed to the ordinary light of the room for 7 months, and at the end of that time, a portion of the film was scraped off with a knife, placed on suitable medium and incubated, with the result that a typical growth immediately ensued. Spores on cover glasses were exposed to September sunlight (Latitude 43) for varying periods of time, and growth occurred after 4, 6 and 7 hour's exposure. The age of the spores varied from 6 days to 18 months; and spores 3 months old were not killed by 7 hours' exposure. Thermal Eelations. Maximum for Growth. The maximum for growth is about 47°C. At 44°C., good growth occurs ; but at 50°C., growth ceases. Experiments on maximum for growth were performed on gsrms isolated from a number of different places, and little oe no difference was noticed in their behaviour when incubated at the temperatures mentioned. Optimum for Growth. The optimum for growth is about 37.5°C. for all media except gelatine. This has been determined by Cheyne & Eisen- berg (39). On gelatine the best results are, of course, obtained from higher tem- peratures ; but as 10% gelatine melts at about 24°C., 22°C cannot be exceeded. Minimum for Growth. Cheyne says that the bacilli do not grow below 16°0. I have, however, occasionally obtained growth at 14°C. on the surface of agar ; but it has been extremely slow. The spores will not germinate at this temperature. No difference, under this head, is apparent in germs obtained from different countrifs. Thermal Death Point. This is a very important matter, becauEe in the heating of wax and honey from colonies suffering with foul brood, it is neces- sary to know the temperature that will destroy spores and thus prevent the infection of other bees ; and unfortunately a considerable discrepancy exists i " " growth. 1 honr, growth. H hours, growth. 2^ " growth. 3J " DO growth. in the results of experiments to determine the thermal death point of the bacillus, accounted for in part by the different methods used by different investigators. McKenzie (28) found the thermal death point by suspending silk threads saturated in a beef broth culture of B. alvei containing spores. The threads were allowed to dry, and introduced into melted wax, and left therein for a definite time, at a fixed temperature. At the end of that time, the thread was introduced into melted agar and thoroughly shaken so as to separate the wax from the threads. The cultures thus made were rapidly cooled, and the tubes placed in the incubator at 37°0. The following are his results : At 100°C. for \ of an hour, growth. At 90°0. for J hour, growth, " " 1 " growth. " " 2 hours, growth, " " 3 " no growth. " " 4 '• no growth. A temperature of 50°C. did not dtstroy the spores in 24 hours. These experiments were repeated with the same results, which results were criti- cised by Oorneil (28), who claimed that the heat to which the bacteria were exposed in melted wax was not moist but dry heat, and consequently that the wax had to be heated to a high temperature and for a long time in order to destroy the spores. According to the testimony of two prominent founda- tion makers, the wax during the refining and purifying process reaches a temperature of quite or nearly 100°O. for a short time. During the sheet- ing, however, it does not reach a temperature much above the melting point, say 79°C. Two other foundation makers, Dadsnt & Hunt (41), state that, in refining, the wax is heated for some time to 100°C., and is kept liquid for 24 hoars ; eo McKenzie thinks that if these temperatures are reached in the making, there is little danger of foul brood from comb foundation, as the specific gravity of bacteria in the melted wax is so great that throughout the process of manufacture the bacteria tend to fall to the bottom. Stern- berg (42) states that the spores require for their destruction a temperature of 100°O. for four minutes (determined in 1887); but there is so statement as to the age of the spores, In Howard's experiments (40) tubes of liquid gela- tine containing spores of B. alvei were placed in an open vessel of boiling water and allowed to remain therein for a definite time — " in all probability the water did not reach boiling point " — and trial cultures were made at stated intervals, with the following results : After 15 minutes — growth. " 30 " " II ^g «i <(. "50 " DO growth. <■ 60 " " His trial cultures were on potato and gelatine ; but no statement is made regarding the age of the spores, where they were from, or the tempera- ture at which they were incubated. It is, however, evident that they were not given the most favourable conditions for growth. I have myself performed the following experiments on the thermal death point of the spores : Method. Test tubes containing bouillon were placed in boiling water. Three loopfuls of culture were introduced into each of the tubes ; and tubes, 14 ■withdrawn from the boiling water at stated intervals, were cooled and incu- bated. Results 1. Spores from a seven months old culture in bouillon were killed at a temperature of 100° in 1 hour and 20 minutes. 2. Spores from a 2J months old culture on agar were killed in two hours and a half. 3. Spores from agar nine days old, — -slight' growth after 2 hours and 45 minutes ; no growth after three hours. 4. Spores 14 days old and 21 days old, — in each case after two hours boiling, one of the duplicate tubes formed a growth ; another after 2| hours, whilst the remainder had no growth. All were killed in 3 hours. I used also fine capillary glass tubes. A suspension of the spores in water was drawn up into sterile tubes, which were then sealed at both ends, The tubes were placed in boiling water and withdrawn at stated intervals. The contents of the tubes were then introduced into agar, which was incu- bated at 37''0. ; and great care was taken to have a suspension *of the spores by fihering them through glass wool. The results were : With a temperature of 98°C. (about the boiling point in this locality), spores from a 7 days' old culture on agar were killed in 2f hours ; and spores from agar 9 days old were killed in 3 hours. £;;^7 Another experiment was made to determine the thermal death point in honey. The honey was of two kinds, clover and backwheat, The former had a specific gravity of 1.042 at 60°C. and contained 0.0S7% of formic acid, while the latter had a specific gravity of 1.040 at GCC. and contained 0.170% of for jiic acid. The spores used were from agar three weeks old, and three methods were followed : 1. Silk threads with dry spores thereon ; 2. Test tubes containing honey with a heavy inoculation of spores ; 3. Capillary tubes containing a suspen- sion of spores in distilled water. The spores used were not filtered through sterile glass wool, as it seemed desirable to imitate as far as possible the con- ditions met with in infected honey. The following are the results : 1. Silk threads with dried spores, from an agar culture two weeks old. Time. Temperature. Repult. 15 minutes 115''G growth. 30 " 113 " " 45 " 115 " 60 " 113 " " 1 hour 15 minutes . . . 114 " •' 1 " 30 " .. 115 " " 1 "45 " .. 115 " 2 hours 114 " " 2 " 15 minutes... 116" " 2 "30 " .. 115 " 2 " 46 " .. 115 " no growth. 2. Tubes containing honey and spores mixed together. 30 minutes 115°C growth. 45 " 114 " « 60 " 114 " " 1 hour 15 minutes . . . 114 " " 1 '• 30 " .. 114 " " 1 " 45 " .. 115 " 15 2 hours IIS'-O growth. 2 " 15 minutes ... 116 " " 2 " 30 " .. 115" no growth. 2 " 45 " .. 115 " " 3. Capillary tubes with spores in distilled water. 30 minnteB . IU°0 114 " growth. (1 1 hour 1 " 30 minutes . . . , 114 " a 2 hours 114 '■ (( 2 "15 minutes , .. , 115 " <( 2 " 30 " , 115 " If 2 " 45 . 115 " .... no srowtl The temperatures were taken in a large vessel containing 10 pounds of boiling honey. The experiment was repeated, using buckwheat honey instead of clover and with like results. Relation to Light. A few experiments were made to ascertain the behaviour of spores toward light. Coverglasses spread with spores and dried, were exposed to bright sunlight during the month of February. The expo- sure was in the opon air and the glasses were on black tile. The temperature varied from — 12" C. to — 22" After exposure, the glasses were placed film side downwards on agar plates, and then incubated at 37° C. Time. Result. Results — 3 hours sunlight. Abundant growth in 16 hours R t( l« fl It it tt g 11 c( (I i< X i( These experiments were repeated in September, when the outside tem- perature varied from 24" to 30° 0., with the result, that there was growth after 4, 6, and 7 hours' exposure. Agar plates exposed after inoculation showed great differences. Por instances, spores 21 days old was killed by 5 hours' exposure, whilst plates made the day after with spores .2 months and 21 days old, required 7 hours' exposure! Spores 10 days old showed no growth after 5 hours' exposure ; and spores 5 days old, no growth after 6 hours' exposure. From a large num- ber ot determinations, the average length of exposure necessary to kill spores within the above range of temperature was found to be 5 hours. Vitality on various media. The cultures seem to live longer on agar than in liquid media. The vitality of old gelatine and bouillon cultures seems to be lessened by the products of the bacilli growing in these media. The epores taken from these sources have also decreasi'd resisting power. Effect of growth on reaction of media. Ordinary bouillon becomes slightly more alkaline as growth proceeds, the presence of ammonia being detected by Nessler's reagent ; but control bouillon does not give the reaction. In bouillon, with the addition of glycerine and various sugars, the acidity of the media is increased, but more in the case of glucose broth than in any other. In these experiments accurate titration was made with phenolphtalein as indicator. Cheyne tried the reaction, " making the infusions faintly alkaline, and after the growth of this organism in it, it is faintly alkaline." Sensitiveness to Antiseptics and Germicides. This subject is taken up in connection with the chemical remedies used for the disease. Pathogenesis. Besides being pathogenic to the larvae of bees, Cheyne has inoculated two mice and one rabbit with spore-bearing cultivations with- out effect. " Half a syringeful of a spore-bearing cnkivation injected into the dorsal subcntaneons tiseoe of each of two mice resnlted in the death of one of them in 23 hours, while the other seemed unaffected. In the case of the monae which died, the seat of injection and the neighbouring cellular tissue was found to be very oedematous ; but no microscopic changes were apparent in the internal organs. Numerous bacilli were found in the the cedematous liquid, as also a number of spores which had sprouted ; and there were likewise a few bacilli in the blood taken from the heart. This was proved by cultivation as well as by microscopic examination. On examining sections of various organs no morbid changes were found, and only a few bacilli were seen in the blood vessels. A. syririgeful of the same cul- ture was injected into a guinea pig; and the animal died 6 days later, with extensive necrosis of the muscular tissue and skin ; and cheesy looking patches were distributed through it, but there was no true pus. On making sections of the necrosed tissue, numerous bacilli, apparently B. alvei, were seen ; but there were al°o other bacilli and micrococci. No micro-organisms were seen in the internal organs. It thus remains questionable whether the necrosis was due to B. alvei or not, more especially as I have since injected three guinea pigs snbcntaneously with spore bearing cultivation, but without effect. " The effect of feeding flies with material containing spores results in death of the flies, and bacilli were found in its juices as shewn by the micro- scopic examination and cultivation. Cockroaches were not killed " (28). Fly blow larvae fed for three days on spores were not killed. With regard to the prevalence of the disease amongst wild bees, very little can be found on this subject in bee literature, but a correspondent of the British Bee Journal (43) found the disease among wild bee larvae in a tree, recognis- ing it by the smell from the entrance and also from the appearance of the brood in the combs. The correspondent remarks that this tree had probably in former years been the cause of a great deal of trouble to neighbouring bee keepers. In all probability the disease is present among the various varieties of wild bees and wasps. Knight (54) mentions an epedemic among wasps in 1807 ; Kirby & Spence (55) another in 1815 ; and Bevan (13) one in 1824; but in none of these cases was any positive evidence given to show the epidemic was foul brood . Economic Aspects. Losses. Delia Rocca (loc. cit.) in 1790 stated that the whole of the bees in the Island of Syra were carried away during 1777 to 1780 by the disease. Dzierzon (46) relates his losses from the disease. In 1868 he lost his entire apairy of 500 colonies from it. In Switzerland, the disease, at times, is extremely bad. Bertrand's apiaries have suffered severely, and the German papers make constant reference to its devastation. In England, Oowan'(4) thinks that the "only visible hindrance to the rapid expansion of the bee industry is the prevalence of this pestilential disease which is so rapidly spread- ing over the country as to make bee-keeping a hazardous occupation "; and again, (47) " So rapidly has foul brood spread by contagion that in one sea- son, unless precautions are taken, a whole neighborhood may become seriously infected, and the chances of successful beekeeping seriously imperilled, if not utterly destroyed. miiq^jrr.- ..._ _^^bich.-v *-'—"'-«• The committee on the Beekeeping Industry and ' Foul" Brood in°"the United Kingdom, report that the destruction of stock by foul brood and the a * MI'S sa °-3 «■? S 9 Q r «8Q ■^o n 43 "oh 00 '5b •§2. 1^ 2.2 if M-l " OS -i5 M •vs oO boTS * 2 '^S o-'S' m a D » ^ 0000000000000000% Cultures of B. alvei (after Cheyne). A. Colonies on the surface of gela- tine {6 diameters). B. The same colonies 24 hours later. C. Culture tube ; gl. gelatine ; p. cotton wool plug. D. Spore becoming bacillus (1800 diameters.) E. Bacillus becoming a spore. F. Spores in line, taken from a gelatine culture. G. Colony developing. 17 discouragement arising therefrom is oae of the two influences that retard the development of the bee industry. In the United States, serious harm has been done, but no definite statis- tics can be cited. The disease causes great losses and several States have enacted laws for the prevention of the disease, making it a legal oflFence for a person to keep in his apiary a colony of bees aflfected with foul brood. In Canada, the Ontario Foul Brood Inspector (56) reports in the years 1890 1892 inclusive, 622 apiaries inspected and 2,395 cases; in the years 1893-1898, 527 apiaries inspected and the disease present in 212, or about 40 per cent. In New Zealand and Australia, the disease is looked upon as being very wide spread. It will thus be seen that wherever bees are kept, serious losses are caused annually by this disease. Natural method of Infection. "With regard to the natural methods of infection, a good deal depends on the natural prediepssition of the bees to disease and the state of health of the colony. Weak, sickly, or badly nour- ished bees are as a rule the most susceptible. We must also remember that germs themselves vary in their ability to produce di^easa As in diphvheria, we may get a light or severe type of the disease ; so also in foul brood, we may have a light or a severe attack ; but the facts demonstrating the variability of this capacity are not well known ; I have, however, noticed that after prolonged cultivation of B alvei in which more than 30 transfers have been made, and the bacteria with spores have been given to bees in syrup, the virulence of the germ has seemed to be considerably impaired. In one case the colony experimented with was rather weak, was confined to the hive all day, and allowed flight only in the evening, and the spores were given in large quantities in syrup every day, nevertheless it was several weeks before the disease established itself, and then only in a light form So we may have mild or severa epidemics and the liability to' take the disease may be increased by chillin» the bees or otherwise unfavourably modifying their metabolism ; and in all such cases, they succumb more easily to the dis- ease than when in a normal, healthy condition. With regard to the manner in which the disease is carried from hive to hive, Cheshire (26) thinks that the larvae are most usually affected by the antennae of the nurse bees, and also that the tramp of the bees frequently detaches numbers of spores, which fly about in the air and fettle here and there, often where they take eflTect. I think that in comparison with other diseases which are air borne there is usually not very much danger from this cause in the Joase of B. alvei. The spores are generally found in very sticky surroundings, which, even if dry, serves to fix and keep them in situ. Ohes- shire also states that he has not found the bacillus from honey or pollen in infected hives. This statement, however, is directly contradicted by the experience of practical beekeepers and others. I have myself repeatedly found B. alvei in capped honey cells, and in the pollen mai^ses found in diseased hives, the examination in the former case having been made by removing the capping with steriliz<;d forceps and plunging a heated platinum needle into it and then pnlting the needle into melted agar, from which plates were poured, cooled and incubated. Probably the chief method of c*rrying the disease from one hive to another is by the bees from healthy hives robbing colonies that have become weak and diseased. In such cases the robbers carry with them the germs of the disease. There is likely nothing to be feared from using wax foundation from the regular makers ; for, as we have already stated, the wax, in the 2—112 18 process of making, is snVjected to a temperature sufficiently high to kill any spores that may be present. I may add that I fooad spores of B. alvei in two samples of wax sent me by E. F. Holtermann of the Canadian Bee Journal, but both samples were from hives which were very badly infected with the disease. In 1897, about ten pounds of wax was infected -with large numbers of spores grown upon agar. The wax was cut up into small pieces, and heated at a low temperature, only just sufficient to melt it; and as McKenzie (28) had shown that the spores settled to the bottom, the wax was vigorously stirred from the time the spores were added until it had set again. The wax, thus infected, was sent to Holtermann for foundation-making. He manu- factured it by the usual process of melting and gave the foundation made from it to bees, and no foul briod developed in the colony supplied with it during the years 1897 and 1898. The probability is that the spores are fixed in the wax, and are thus unable to infect the bees. Healthy bees may pick up spores of B. alvei from flowers previously visited by diseased bees; wasps, which are noted robbers, may also carry the disease, and thus infect a locality. The very large traffic in bees and bee-keeping supplies where agriculture is carried on, probably favors the spread of the disease. In fact, many instances are cited in bee journals of infection carried from one locality to another by the importation of bees and bee supplies. Persors manipalating diseased hives and then examining healthy ones may be the means of spreading the disease. The practice of using a knife for cutting out diseased comb and then using the same knife for work amongst healthy comb (which I have seen done) is by no means wise, as the spores may thus be transferred from diseased to healthy hives. Cowan (4) observes that beekeepers who have not succeeded with their bees in consequence of foul brood have been known to sell by auction hives in which the bees have died. In such cases the purchasers are usually beginners who have no idea of the danger they are incurring. Conditions favoring the spread of the Disease, Besides the weak or badly nourished condition in which bees may be, and lack of other hygenic conditions which favour the spread of this disease, great humidity in winter is said to be favourable and probably great heat is also conducive. (45.) Predisposition of Varieties. No definite statements can be made as to the predi^po8ition of various races to this disease. Qainby (49) says that black bees are more subject to foul brood than Italians. Aspinall (51) also affirms that common bees are more liable to the disease than Italians, but de Layens (47) states that Italians are more easily infected than black bees. (See also page 17.) Remedies. Three remedies have been tried : 1. Stamping out. 2. Starvation. 3. Treatment by chemicals : (a) by feeding chemicals in food ; (b) by putting certain chemical substances into the hive and allowing them to evaporate at the temperature of the hive. This latter method may be regarded as rather preven- titive than curative. 19 1. Stamping out Method. By the stamping out method all affected bees, combs and frames are destroyed, and the hives thoroaghly disinfected. Oowan (4) thinks that if foal brood were nnder government inspection, and all oases promptly dealt -with by destruction, the disease could be stamped out. The British Bee-Keepers' Aasociation has asked the Board of A.gricul- tnre to secure legislation on this line, because it thinks that in this way the trouble would be removed and the industry would receive an impetus which would benefit bee-keepers, farmers and fruit growers. The earliest advocate of this system was Delia Bocca (18), who main- tained " in extreme cases that it was necessary to burn everything without pity, as there was no other resource.'' Since Delia Eocca's time, this method has been frequently resorted to in severe cases that would not yield to treat- ment either by starvation or by the use of chemicals ; but to have any lasting effect, it would have to be universally carried out, and would involve the difficult question of compensation. 2. Starvation Methods. The starvation method was first proposed by Schirach (3) who advised that the combs be removed and boes allovred to fast during two days, and then be placed upon clean new comb, and fed on a syrup prepared with a little hot water mixed with honey, nutmeg and saffron. Since Schirach's time different modifications of this method have been made, and it has been largely used in the United States and Canada, whilst in Europe treatment by medicated syrups has been more in vogue. In 1879 L. 0. Boot (58) gave his approval to this method, but he advised that the bees be confined in a cool, dark place for 24 hours, in order that all the honey which they carried with them might be consumed, and that the bees be then put into a hive filled with healthy comb or foundation and the condemned hive scalded with boiling water and thoroughly scraped. At a later date McEvoy (44), the Ont»rio Provincial foul brood inspector, introduced another modification and has himself described his method as follows: "In thi " honey season, when the bsea are gathering freely, remove *"ih? combs in the "evening and shake the bees into their o^n hives; give them frames with " comb foundation starters on and let them build comb for four days. The " bees will make the starters into comb during the four days and store the " diseased honey in them, which they took with them from the old comb. " Then in the evening of the fourth day take oat the new combs and give " them comb foundation to work out, and then the cure will be complete. " By this method of treatment all the diseased honey is removed from the " bees before the full sheets of foundation are worked out. All the old foul '■brood combs must be burned or made into wax after they are removed " from the hives, and all the new combs made out of the starters during the " four days must be burned or made into wax, on account of the diseased " honey that wonld be stored in them. " All the curing or treating of diseased colonies should be done in the " evening, so as not to have any robbing done or cause any of the bees from " the diseaeed colonies to mix and go with bees of sound colonies. By doing " all the work in the evening it j^ives the bees a chance to settle down nicely " before morning and then there is no confusion or trouble. " This same method of curing colonies of foul brood can be carried on at " any time from May to October, when the bees are not gathering any honey " by feeding plenty of maple syrup in the evenings to take the place of the " honey flow. " Jt will set the bees robbing and spread the disease to work with foul " broody colonies in warm d*ys,when bees are not gathering honey,and for that " reason all work must be done in the evenings, when no bees are flying. 20 " Where the diseased colonies are weak la bees, pat the bees ia two, " three or four together, so as to get a good sized swirm to start the care with, " as it does not pay to spend time fussing with little weak colonies. " When the bees are not gathering honey, any apiary can be cured of " foul brood by removing the diseased combs in the evening, and giving the " bees frames with comb foundation starters on. Then, also, in the evening " feed the bees plenty of sugar syrup, and they will draw out the foundation " and store the diseased honey which they took with them from the old combs ; " in the fourth evening remove the new combs made out of the starters and " give the bees full sheets of comb foundation and feed plenty of sugar syrup " each evening until every colony is in first-class order. " Make the syrup out of granulated sugar aod put one pound of water to "every two pouads of sugar, and then bring it to a boil As previously " stated, all the old combs must be burned or made into wax when removed " from the hives, and so must all the new combs made during the four days. " The empty hives that had foul brood in them do not need any disin- " fectant in any way. I have handled many hundreds of aolonies in the Pro- " vince of Ontario and cured them of foul brood without getting a single hive " scalded or disinfected in any way, and these colonies are cured right in the " same old hives." McEvoy positively states that " No colony can be cured of foul brood by " the use of any drug. All the old comba must be removed from every dis- " eased colony and the hive got away from the bees before brood rearing is " commenced in the new clean combs." Howard (40) is most emphatically opposed to the drug treatment. " I regard," says he, " the use of any and all drugs in the treatment of foul brood as a useless waste of time and material, wholly ineflEectual, inviting ruin and total loss of bees. Any method which has nob for its object the eitire re- moval of all infections material beyond the reach of both bees and brood will prove detrimental and destructive and surely encourage the recurrence of the disease." A. I. Root (45) says that " The starvation plan in connection with burn- ing the combs and frames and boiling the hives has worked best in treating foul brood. It never reappeared after sncH treatment, though it did in all cases where the hives were not boiled, thus confirming the theory or fact of spores." These two authors, therefore, go further than McEvoy in both advising the disinfection of the hives. McEvoy (56), however, admits that his method as described above cannot be used for every case. His reports frequently refer to burned colonies ; and he acknowledges that his method does not always cure. In 1890 he used the expression, " 600 cases of foul brood and over 360 cured " ; and again in a subsequent report, after mentioning the number of case^, he added the words, " mostly cured " In a personal commuQioation, M. Bertrand of Nyon, Switzerland, states that he does not believe in and will not recommend in his periodicil (Revue Internationale d' Apiculture) the starvation method as used in America. 3. Treatment hy Chemicals — In the treatment of bees by chemicals, we assume that such substances as are used are employed as antiseptics, and that their efficiency ia due to the fact that they destroy the bacillus or prevent the germination of the spores, and thus bring about an internal disinfection ; but we must remember that many of the substances used are more poisonous in their efi'dcts upon the cells of the bae than upon B. alvei. As is -cell known quinine is fn quently used as a specific for malaria; and ia such ca^e8 the 21 cure is effected by the intervention of the b)dy cell. The efli'otiveaess of the remerly is due to the fact that it acts aa a sticaulns and exalts the natural forces of the body. Whether the drugs uped in the treatment of foul brood act antiseptioally or by stimulating the crlls of the bee and making them more active to ward off the diKsase, is a matter of doubt ; but it mubt be admitted that certain drugs do stem to etIVct a cure, and home of them are regarded as specifics by practical beekeepers. In taking up the different methods of chemical treatment, I shall as far as possible describe them in the chronological order. (1) Carbolic acid. Carbolic acid was first proposed by Butlerow (52), who recommf nded one part of acid to 600 of syrup, this proportion being the limit in which one caa give the remedy to bees. Cech (53) in a work published in 1H77, alto recommend* d carbolic acid The Cheshire treatment (26) consists in using a treatment containixig half a decilitre of carbolic acid in a litre of wiiter, thoroughly shaking it up until the acid is enirely diFSolved, and using half a decilitre of this in a litre of syrup. In this treatment it is also necessary to reduce the infected stock to the numbfr of frames it can use, and if the queen is diseased to destroy her and substitute a healthy one. The sjrup is given by pouring it into the empty cells of the brood nest. This method of treatment his been frtqnently repcrted to be successful ; but there have been many failures, perhaps partly owing to the fact that it is difficult to get the bees to take the medicated syrup Experiments on the Antiseptic Value of Carbolic Acid According to McKei zie (28), two per cent, carbolic acid does not kill the eporesin six days. One per five hna|Jred of the acid prevented the germination of the spores, but when taken out of the solution and placed in ordinary beef broth it gave luxuriant growth. Hence McKeczie thinks that the explanation of the value of carbolated syrup in the treatment of foul bro^d consists in preventing the germination of the spores. The bee journals refer to numerous instances in which feeding carbolated syrup produced an improvement in diseased stock ; but as soon as the treatment stopped, the disease broke out afresh. Salycylic Acid. The salycylic acid treatment was first used by Hilbert in 1876. The following is the method of use : Solution of Hilbert No 1 — Pure salycylic acid, 12J grams ; alcohol, 100 grams. Solution of Hilbert No. 2 — 200 drops of solution No. 1 (about five grams) in 200 grams of distilled water or rain water. Fumigation — One or two grams of the pure acid for fumigation. Syrup — From 200 to 240 drops of Solution No. 1 (or about 5 to 6 grams) in a litre of syrup, well mixed before the syrup cools. As soon as the disease is noticed the hive is disinfected and the syrup fed ; and this treatment is also used for other coloniea as a preventive treat- ment. The fumigation is accomplished in a kind of tin lantern furnished with a small alcohol lamp, suspended over which is a small movable trough for placing the acid in. The flame of the lamp is regulated in such a manner that the acid is liquified and slowly evaporated without burning. Too great heat will decompose it and render it ineffective. The fumes of the acid spread through the hive in the form of a white vapour. Whilst the fumi- gation is in progress the entrance boards and all parts that can be disinfected are washed with No. 2 solution. Fumigation and washing are repeated every 4 or 5 days until a cure is effected. The diseased colonies receive, 22 every sec9iid evening, ^ of a litre of acid syrup ; and it is wise to £;ive the same treatment to the neighbouring hives. A cure is usually efiected in 3 or 4 weeks. If later, it is generally regarded as a sign that the queen is diseased, in which case it would be well to replace her. Occasionally the queens die during the treatment ; but this is not frequent. This treatment was very successful in diseased hives belonging to Bertrand (59). All the hives that were treated, were cured. Cowan (60), who has also used Hilbert's treatment with some slight modifications, has had the same success ; and such is his confidence in the treatment that he does not fear to introduce into his apiary foul brood colonies for treatment. Some have found the treatment ineffective ; but Bertrand thinks (59) that in all such cases there has been something la'^king ia the work, some precau- tions overlooked or neglected. Experiments on the antiseptic value of salycylie acid. Salycylic acid agar ■was made containing 5 grams of 12J per cent, solntion of salycylic acid ia one litre of agar. Petrie plates were made from this and streaked on the surface with B. alvei. At the same time control cultures on ordinary agar were made. The results were abundant growth on the control plates and good growth, (but somewhat lees than on the control plates) on the salycylic acid agar. Salycylic acid Vapour. One gram of the acid was evaporated in our laboratory according to the directions given by Bertrand (59), in a box about the same size as a hive. Agar plates streaked with spores of B. alvei were left in different parts of the box during the fumigation for 10 minutes. The plates were then taken out, the covers put on and the plates incubated at 37°C. for 48 hours. Results. Fumigated plates — no growth. Control plateE — abundant growth. From these experiments it will be seen that the vapour of salycylic acid acts antiseptically, and that the feeding of the acid in the syrup, in thepropor- 'tions specified, probably acts as a stimulant to the bees, enabling them to withstand or throw off the disease. (3) Camphor. Oasipow (61) was the first to use camphor as a crirative; and Bertrand (59) describes the use of it as follows : " There is," says he, "placed upon the battom board of the hive, enveloped in a piece of muslin, " a piece of camphor about the size of a walnut, which is replaced when it has " evaporated. The presence of the camphor permits the bees to clean out the " cells containing dead larvae and stop the development of the disease. So " long as a hive contains some of the substance foul brood will not develop, ' at least according to our experience and to that of several other beekeepers. " The first thing to do then, when one doubts the state of health of a colony, " is to emplov the Ossipow remedy before proceeding to more radical means. " One can administer o*mphor in food by dissolving it in its own weight of "alcohol." Experiments on the antiseptic value of Camphor. Sloped agar in tubes was inoculated with one loopfnl of spores of B. alvei, and a crystal of cam- phor about the size of a large pea was dropped in*o the tube. The tub-is were then capped with tin foil paper and kept at 22''0. and BT^O.; and control cultures were made at the same time. At 22°0., after two days, there was good growth in the camphor tube. At 37°0., after two days, com pared with the control tube, the camphor tube showed slight restriction of growth, the extra heat having evaporated the camphor more quickly. Another series was made by esing agar Petri plates streaked with 2 23 loopfnla o( apores. In each plate was placed a portion of camphor abont the size of a large pea ; and the plates were incubated at SZ^O. In 24 hours there was gocd growth ; but close to the lump of camphor, growth was slightly inhibited. Thus, camphor in the quantity in which it might be kept in a hive has no antiseptic effect, the amount used in the fxperiments being far larger than would be used in a hive. This substance, therefore, if it has the effect mentioned by those who have used it, must act as a stimulant, strengthening the bees to overcome the disease. f4) Thyme. Klempin (62) has used branches of dry thyme with success, burning them in the smoker for disinfecting his hives ; but their effect, like that camphor, is not radical, and beekeepers are •not all in accord as to their efficacy. (5) In connection with thyme thymol may be mentioned. Zehetmayr (63) has recommended the use of thymol, and has made a little machine by which he steams the bees with this substance. If a little of it is placed in a hive it ^ill prevent infection, because bees from' uninfected hives will not come near it, — they object to the smell, until they become accustomed to it. Blow (63) thinks it very valuable, and Jones (65) remarks that, even in great dilution, it prevents the growth of the germ ; but Cowan criticises its use, because it is disagreeable to bees, and if used in sufficient quantity, acts as a poison, and therefore cannot be good in food. Experiments on the antiseptic value df Thymol. Crystals of thymol were placed in test tubes of sloped agar in our laboratory and inoculated with one loopfnl of spores of B. alvei. These were capped with tin foil paper and incubated at 22" and 37°0. Result. Control tubes — abundant growth. Thymol tube at 22°C.— slight growth. " " 37°C.— very slight growth. Agar plates, poured and streaked with two loopfuls of spores of B. alvei, were used in another experiment ; and a piece of thj^mol the size of a large pea was placed in each plate. The plates wern incubated at 37°, along with control plates, with the following results : 24 hours, control platen-abundant growth. " thymol plates^ — good growth, but close to the lump, no growth. Hence we conclude that this substance has a very slight antiseptic effect. (6) Carbolic Acid and Tar. These substances were first used by Schreuter (66) and they are applied as follows : — " A piece of felt wool is placed in a small box, and soaked with a mixture of carbolic acid and ITor- wegan tar, in equal proportions The cover of the hive is slightly raised, in order to permit of the evaporation of the carbolic acid. The box is left upon the platform of the hive beneath the brood, and remains there permanently. The dose can be renewed as often as required. The addition of tar to the acid is for the purpose of making evaporation take place more slowly." This remedy has not been used to a very great extent. Borel (67) reports success with it ; but others have not had the same results, and it is probable that it should be used only as a preventive. Experiments on the antiseptic value of Carbolic Acid and Tar. ^ Four drops of the mixture placed on blotting paper and inserted in a Petri dish containing agar streaked with spores, inhibited growth, from which we see that the mixture is antiseptic. (7) Greolin or Phenyle. Creolin has been recommended by Cowan (68) and has been used with success by other apiculturists, 24 Recipes : Solution No 1 — for sprinkling, disinfecting, etc. — half a tea- spoonfnl of soluble creolin in a 'itre of wat^r. Solution No, 2 For washing hives, pla'.fnrnas, etc. — two teaspoonfuls of soluble creolin to a litre of water. Solution No. 3— for feeding — a quarter of a teaspoonful of soluble creolin in a litre of syrup. The water of the syrup ought always to be poured upon the top of the creolin and thoroughly mixed with it ; and the mixture should b'l well shaken before using. Use. Prepare a hive and a proper floor board, which has been washed with solution No. 2. Then, after having taken out the comb from th» infected hive, shake off the befs, and sprinkle the comb with solution No. 1. Take out all superfluous comb and spray it with solution No. 2, and extract the honey from it. The honey can then be boiled, and if it is used for feeding the bees, it can be diluted and phenol added in the proportion of one quarter to a teaspoonful to a litre of the diluted honey. The combs are then put back and the bees fed with medicated syrup. If the bees take the syrup, the dose can be gradually increased ; but we must be careful not to give more than one teaspoonful to a litre of syrnp. If the bees refuse to touch it, which is not at all improbable, if they have access to other food, pour the melicated syrup upon the neighboiing comba, when the bees will quickly become habituated to it, and afterwards will take it in the ordinary manner. The vapour of creolin also acts as a disinfectant. A small phial of concen- trated creolin may be placed in a comer of the hive, and lightly stopped with a cotton plug ; and the lower part of the cotton being in contact with the liquid, capillarity will take place and draw up the creolin, and the heat of the hive will produce the necessary evaporation. . A piece of blotting paper can be used by saturating it with creolin, and placing it upon the floor board or in a box covered with perforated zinc, so that the bees will not come into contact with the disinfectant. Creolin is neither poisonous nor corrosive for man ; but, in strong doses, it kills insests. Consequently it is necessary not to give greater strengths than those mentioned above. In the use of this remedy it is necessary to stimu- late the production of brood by feeding liberally with medicated syrnp ; if the disease does not yield to this trpatment, the queen should be removed. Experiments on the antiseptic value of creolin. a. Sloped agar — each tube, inoculated with one loopful of spores, was plugged with cotton wool, saturated with creolin, and then cipped with lead foil Tubes were kept at 22" C. and 37" C. Result : After four days at 22° C. — No growth, except beneath the condensa- tion water in the tubes. After four days at 37° C. — No growth. At the end of (his time new cotton plugs were inserted iiito the tubes in the place of the creolin ones, and the cultures again incubated, when good growth ensued in 24 hours, h. Agar platfs were made and streaked with two loopfuls of spores. In each plate was placed a square inch of thick blotting paper, with four drops of creolin on it. The plates were kept in the incubator at 37° C, and re- moved in 48 hours, when very slight growth was manifest. On removal of the creolin and further incubation of the plates, good growth was obtained. Control plates gave copious growth. These experiments were repeated with only one drop of creolin. Result, after 24 hours — abundant growth. With two drops of creolin, 25 the growth was reatrictel to the inoculation track after 43 hours at 37° 0. c. In addition to the above experiments, agar was m »de containing the same proportion of disinfectant as was used in feeding thp bees of diseased hives ; 15 c. c. of this agar was taken for making a plate culture, and several plates were streaked with two loopfuls of spores^ and incubated at 37° 0. Strength of agar, — 2 c. c. creolin to 1 litre of water, i.e., about half a tea- spoonful to a quart. Results — Creolin agar, four tests — no growth. Control agar, abundant growth. This antiseptic in the strength used by Cowan for feeding purposes, would prevent the germination of the spores ; and if there was a large amount evaporating in the hive, a slight antiseptic result would tike place. (8) Eucalyptus This substance was introduced by Beauverd (69), A small tin box, with a cover pierced with small holes, is placed upon the floor board of the diseased hive, and filled with essence of Eucalyptus. The colony receives every four or five days a litre of syrup containing a teaspoonful of tincture of eucalyptus (oil eucalyptus, 1 ; alcohol, 9). Then from time to time some drops of the same tincture are dropped into the hive. Auberson, who was the metayer of Bertrand's Apiary and was managing his own higher •up the mountains, cured a number of colonies by means of this method. He finds that there is a great difierence in the effect produced by the remedy. In some caEes, the effect follows the remedy quickly ; in others, the effect is slower. Sometimes more than a year passes without resulting in a complete cnie. When the disease is of long standing, the remedy must be proportion- ate to the gravity of the evil. When there are only a few diseased cells, Anberson simply pours some drops of the essence along the back wall of the hire. He renews the dose every eight days ; and in six weeks, sometimes sooner, the colony is cured. In cases where the hive is badly affected, he takes a clean hive and floor board and impregnates the interior, floor board, and division board with eucalyptus, and then transfers combs, brood, and bees to the new hive. He leaves the foul brood colonies their rotten combs, as this is the only bandy means of disinfecting them. Three weeks later, during which he has twice poured eucalyptus on the floor board, he examines the new brood. If.it exiats in healthy patches he simply pours a few drops of the essence on the floor board until the cure is complete. If, however, the fresh brood still disclose some diseased spots, the queen is killed and replaced by another, and every fifteen days the essence i& spread on the floor board until the cure is completed. If the colony is very weak, he strengthens it by the addition of bees and healthy brood. If he has to feed a diseased hive, he never fails to put the essence in the syrup. Besides these well authenticated cases of cure by the essence of euca- lyptus, there are a number of others, and the method has been extensively used in Europe. The great drawback to the use of this remedy is that it la liable to cause robbing. Experiments on the antiseptic value of eucalyptus, (a) Eucalyptus oil. The cotton plug of a spore-inccnlated sloped agar tube was saturated with the oil,: and incubated at 37° C. In eighty-four hours there was no growth, but a fresh plug being inserted good growth occurred in twenty-four hours. (6) Agar plates inoculated with spores and containing four drops of eucalyptus on a piece of blotting paper were incubated at 37° C. No growth formed, but when the eucalyptus was removed good growth immedi- ately ensued. On plates containing two drops the growth was restricted to the inoculation track, but when the oil was removed abundant growth took 26 place. On plates containing one drop on blotting paper there was abundant growth in twenty-four houis. (c) Eucalyptus agar was made by using a teaspoonful (4 c.c.) of tincture of eucalyptus to a litre of agar. Six plates were made with eucalyptus agar, each plate inoculated with spores, with the result that the growth on the medicated agar was only slightly less that that on the control agar. The medicated agar smelt slightly, but characteristically of eucalyptus oil. A Queensland (Australia) correspondent of the British Bee Journal (71) is of the opinion that no foul brood exists among bees in that country. The reason of this is that the honey that goes into the combs is. largely gathered from the eucalyptus, the medicinal qualities of which combat foulness in all forms. This statement, however, is not reliable, inasmuch as foul brood is known to be prevalent in Queensland. (9) Napthol Beta. Napthol Beta was first used as a remedy by Lortet (72). The treatment is as follows : The drug is administered in the food, in the proportion of one-third of a gram to a litre. This one third of a gram is at first dissolved in a little alcohol, as it is extremely insoluble in water. Afternraids it is mixed ia, a litre of water, and this liquid is used for making the syrup. In England the usage is to dissolve the napthol in the sugar, the proportion being about forty to fifty centigrammes to a kilo of sugar. It is, however, better to dissolve it in alcohol, Lortet thinks that external treatment by means of fumigation or spraying is helpful, as these methods contribute largely to the disinfection of hives, comb, etc ; but as he believes that it -is always the digestive canal of the nurse bee which is infected and that it is by the act of feeding that the adult bee infects the digestive canal of the larvae, therefore all efforts should be directed to the digestive canal of the worker bees, and the treatment ought to be internal ard as energetic as possible. He states that when administered in the proportion of 0.33 gram per 1,000 of liquid it prevents all fermentation and decomposition and other changes caused by microbes. He further maintains that in addition to the use of this preparation firat-rate hygienic conditions are necessary in order to give the bfes vitality and recuperative power, which play an important part in enabling living organ- isms to resist the inroads of virulent microbes. McKenzie found that (28) a beef broth containing one per thousand of B. Napthol prevented spores of B, alvei from germinating, and consequently had an equal value with one per five hundred of carbolic acid. This remedy has been widely used and with considerable success. Experiments on the antiseptic value of Napthol Beta. Napthol Beta agar was made in our laboratory the same strength as that recommended by Lortet for feeding, that is 0. 33 gram B. Napthol to one litre of agar. Eight tests were made in Petri dishes, inoculated with spores of B. alvei, and in no case did gro-wth result ; from which we learn that a dilution of one-third of the solution used by McKenzie completely inhibited growth. Napthol Beta agar containing 0.165 gram of the drug to a litre of agar was also tried, and the result of a number of tests was that some growth took place on the medicated plates and abundant growth on the control plates. From these experiments, also those of Lortet and McKenzie, it will be seen that Napthol Beta has a strong antiseptic action. (10) Naphthaline. This substance is regarded as a preventive rather than as a curative, although there are cases known in which it has effected a cure of diseased hives. A small quantity of the drug is placed on the floor board of the hive, a crystal about 2 c m. in diameter as far from the entrance 27 of the hive as possible. The evaporation is rapid and with very strong odour. Henoe, if too mach used, the brood will be deserted 'by the workers and death of the bees may take place. As soon as the dose has evaporated it is renewed. As a preventive, naphthaline has been very favourably reported npon by a number of writers ; and Gowan (73) states that he inspected very thoroagh- ly a hive belonging to Merney which had been cured by this substance. Experiments. In our laboratory, crystals of naphthaline about the size of a large pea were put into teat tuoes containing sloped agar, inoculated with one loopfnl of spores, capped with tin foil paper and kept at 22° and 37° C. Results. After 48 hours — good growth in all tubes. Inoculated agar plates containing a crystal of naphthaline likewise gave good growth in 24 hours at 37° , as did also the control tubes and plates. Hence, we con- clude that naphthaline ha^ no antiseptic power ; and we are forced to look upon its use rather doubtfully. It may, perhaps, act as a stimulant. (11) Formic acid. This substance was first suggested by Dennler in 1885 (74), but he did not ascertain the strength in which it could be used. Sproule (75) states, that since the year 1882 he had successfully treated foul brood with formic acid. He was the first apiculturist to use the remedy an renewed and continued every week until the cure is complete, whioh is often after the first treatment. In fact the disease rarely resists the second or third application. To hasten the cure, this remedy can be given in the food of the bees — a teasp^onful to a litre of syrup. Experiments Formic acid probably has an important rdle to play in the keeping properties of honey. As long ago as 1878, formic acid was found in honey ; and Muhlenhofi' (76) observed that when honey is not intended for im- mediate use, the bee deposits in each cell a drop of formic acid, secreted by the venom glands, and then seals the cell. Erlenmeyer (77) says that formic acid of the strength of 1.205 gr. to a thousand parts of water was antiseptic, Planta (78) refutes Muhlenhoff's idea that 100 grams of sealed honey con- tains ,0186 grams of 22% formic acid. " 100 grams is the capacity of 165 worker cells, but the smallest droplet of venom contains at least .0254 grams of formic acid, which would make for 165 cells, 4 1910 grama ; that is to say, 200 times more than there is in reality." This opinion is, however, contrary to one expressed before by the same wiiter, in the year 1884 (79). Formic acid seems to help bees to ward oft the disease, especially when we supply it to them ready made ; and that found in certain kinds of honey has probably an antiseptic efi'ect. Two samples of clover honey and two samples of buckwheat honey were analyzed in our chemical laboratory with the following results : — 1 Buckwheat honey 15 grains of formic acid in 100 grains of honey. 2 " C 17 " " " " 1 Clover honey 0579 2 " 0.C57 " " " " 28 Formic acid agar was then made containing the same proportion of formic acid as was found in the first sample of buckwheat honey, and weaker formic acid agar containing the same percentage of formio acid as was present in the first eample of clover honey; and tpires placed upon the stronger formic agar did not germinate, while on the weaker formic agar the germina- tion was only slightly retarded ; and after the weaker agar was two days in the incubator, there was a large growth. Spores transferred from the strong formic agar (after being in contact with it for six days in the incubator) failed to grow on the weaker formic agar within two days ; but after four days in the incubator they grew abundantly. The culture growing on the weaker formic agar was then transferred to the strong formic agar, to ascertain whether the germ could be accustooied to more unnatural food by previous cultivation on the weak formic agar. This transfer was, however, un- successful. The germs used in these tests were isolated from samples of diseased comb from Ontario, Austria and Florida, U.S.A. Formic acid bouillon was also made containing .15% of formic acid ; and spores kept in this brolh for eight months continued to germinate when transplanted to suitable matprial. Formic acid agar was likewise made in the same proportion as snggestt d by Bertrand (69); that is, formic acid 10, water 90; and a tablespoonful of this mixture to a litre of syrup ; but instead of syrup, agar was used. Fifteen c.c. of this acid agar was poured into each Petri plate, and the surface inocu- lated with spores. E( suits : On 14 plates, no growth. On 2 plates, very restricted growth, limited toons-eighth of an inch of the needle track (60 houre). On control plates, abundant growth. From these investigations, viz., the analysis of the honey, the experi- ments based thereon, and the tests with agar made in the proportion sug- gested by Bertrand, we would note three things : (1) That the amount of formic acid recommended by Bertrand for the cure of foul brood is almost identical with the amount found in buckwheat honey ; (2) that formic acid is a good antiseptic ; (3) that the formic acid in buckwheat honey may possibly tend more or less to ward oflf foul brood. We may add that our analysis, showing a larger proportion of formic acid in buckwheat honey than in clover honey, is an interesting explanation of a fact well known among practical bee-keepers, viz., that the sting of bees when working on buckwheat is much more irritant than when working on clover. In conclusion under this head, we may say that formic acid has given good results when used in the treatment of foul brood ; and it is in a S'inse a natural remedy, being manufactured to some extent by the bees themselves. (12) Other substances used for trealing this disease. Among other sub- stances that have been used for treating this disease are sulphuric acid, sul- faminol, various modifications of substances already mentioned, and some rec- ommended in the McLean method (80), the Muth method, and others ; but these have not had so wide application as those referred to in the preceding paragraphs. Experiments on the Use of Drugs for Combatting the Disease. I have already mentioned that, in one of my experiments, I endeavoured to find out if the viruleace of the germ was attenuated by prolonged culture in artificial media, with the result that considerable attenuation occurred 29 after a large number of transfers ; and in tLe following experiments I have endeavoured to meet any objflctiona that might be made aa to the virulence of my caltarps, by isolating B. alvei from a badly diseased hive and then growing at once sufficient spores for the purposes of the experiment. Thus but three transfers from a diseased larva were made ; aad all the spores used in the following experiments were obtained in this manner : Two small hives, each containing strong healthy swarma, were selected and placed side by side. Hive A was given spores of B. alvei in eyiup containing one-ihird of a gramme of napthol B, to a litre of syrup Hive B was given spores of B. alvei in syrup containing from 1.6 to 1.8 c c. formic acid to a litre of syrup. The spores given were scraped from the surface of an agar slope culture, put into 10 c c. of sterile water, and well shaken in order to obtain a good suspension of spores. The water and spores were poured into medicated syrup and the mixture thoroughly btirred. It was then given to the bees and was readily accppted. This procedure was continued four days a week for threp weeks, aod at the end of this time each hive had received the whole of the growth from twelve sloped agar tubes. During the feeding period the combs containing the brood were carefully examined, but none of the usual symptoms of the disease appeared, although cultures were obtained from dif- ferent parts of the hives and from the digestive tract of the workers. At the end of three weeks the medicated syrup was discontinued for a week. Then ordinary syrup containing spores was given, and at the end of ten days typical symptoms began to be noticed, and after sixteen days the disease was well establshed. Both hives, so far as I was able to judge, were the same — no disease to be seen in either whilst medicated syrup was fed, but infection manifest in both soon after the formic acid and napthol B. were discontinued. This experiment goes to prove the benefit of feeding with syrup a substance which is antiseptic and which hinders the germination of the spores. It also confirms Lortet'd opinion that the digestive canal of tha nurse bee is alone infected. I have never been able to obtain Cheshire's results, viz , the isola- tion of the bacillus from the blood of the worker, but I have frequently found it in the digestive canal of bees from diseased colonies. . From the results of the above experiments I conclude that in certain cases the use of chemicals is beneficial, but I would not say that other meas- ures, such as starvation and stamping out, should be abandoned as unneces- sary or useless. Some of the drugs used are of very lit le, if any, value ; but others, such as formic acid and napthol B , are undoubtedly very useful. In some cases, especially those in which the disease is very viralent, it may be advisable to resort to more drastic measures. Toxins. I endeavoured to find out whether or not the feeding of toxin (filtrate from a two weeks old culture of B. alvei in f accharose bouillon) mixed in syrup would enable healthy bees to withstand the disease. Small amounts of this filtrate were given in syruo to a healthy colony every other day for three weeks. The amount of filtrate fed was gradually increased, but as the amount got larger the bees refused to take it, so it had to be poured over the combs. At the end of three weeks spores of B. alvei, freshly isolated, were fed, and symptoms ot the disease followed about fourteen days later. So the toxin had little or no efltct, but further experiments are being made. 30 Legislation. In the TTnited States, six States liave laws for the suppression of foul brood among bees. These are 'New York, Wisconsin, Michigan, Utah, Col- orado and California. In Canadi the Province of Ontario has enacted a foul- brood law. In Europe Mecklenburg also has a law. These statutes difler a good deal from one another, and some of them are so drafted that evasion of the law is easy. The best are probably those of Wisconsin and Ontario, and the principal points in these acts are as follows : 1. The appointment of an inspector. 2. The inspection of all apiaries reported as diseased, and the duty of the iDspector, if satisfied that the disease is present, to give full in- structions as to treatment. 3. The enactment requiring the inspector, who is the sole judge, to make a second visit to all diseased apiaries, and, if need be, burn all col- onies and combs that he may find uncured. 4. Various penalties (fines, and, in default, imprisonment) for — (a) Selling or giving away diseased colonies or infected ap- pliances. (6) Selling bees after treatment, or exposing infected appliances, (c) Obstructing the inspector. 5. Persons who are aware of the disease either in their own apiary or elsewhere are to notfiy at once the proper authorities, and in de- fault of 80 doing shall, on conviction, be liable to a fine and costs. 6. The inspector of apiaries to make an annual report, which shall in- clude a statement of the number of colonies destroyed by his order, the localities where found, and the amount paid to him for his ser- vices. Beferkncbs. 1. Aristotle '. Historia Animalum, Book ix, oh. 27. 2. Columella, L J.M. : De Re Rustics, Book ix, ch. 13. 3. Pliny: Natural History, Book xi, ch. 19, A.D, 79. 4. Schirach: Histoire des Abeilles, ch. iii, p. 56, La Haye 1771. 5. Cowan : Journal of the Royal Agricultural Society, Vol. vi. Part iv, 1895. 6. Tessier: LEnoyclopfedie M^thodique, Abeille, p. 32, 1765. 7. Bucket : Culture des Abeilles, p. 315, Vevey, 1771. 8. Bella Rocca: Traits Oomplet sur les Abeilles, Vol. iii, p. 261, Paris, 1790 9. Wildman: Treatise on the Management of Bees, London, 1796. 10. Keys: Ancient Bee-masters Farewell, London, 1796. 11. Needham : Rhein: Brussels Memoirs, Vol. ii, 1780. 12. Reaumur: Memoirs pour Servir a I'Histoire Naturelle des Inseotes, T.V., p. 1734. 13. Bevan: The Honey Bee, London, 1827. 14. Leuchhart: Bienen Zeitung, Eichstadt, 1860, p. 232. 15. Molitor Muhlfeld: Bienen Zeitung, Eichstadt, 1868, p. 95. 16. Preuss: Bienen Zeitung, 1868, p. 225. 17. Vogel, Pollmann, Leuckhart, Geilen: Bienen Zeitung, Nos. 21 and 22 18. M. Muhlfeld: Bienen Zeitung, 1869, No. 3. 19. Lamhrecht : Hallier: Bienen Zeitung, 1870, No. 2. 31 20. Gornallia: Bienen Zeitung, 1870, No. 5. 21. Fischer: Bienen Zntungr, 1871, pp. 105125. 22. Schonfdd;Gohn, Eidam: Bienen Z-itung, 1874, pp. 201 and 261. 23. Cheshire and W. Cheyne ; Journal of tiie Royal Microscopical So- ciety, 1885, p, 381. 24. Dickel: Bienen Zeitung, 1888, p. 24. 25. Klamann'. Bienenwirtschaftliches Ojntralblatt, Hannover, 1888, pts. 18 and 19. 26. Cheshire: Bees and Beekeeping, Lnnd m, 1885, Vol. ii, p. 546. 27. Smith, W. G. : British Bee Journal, London, 1886, Vol xiv, p, 225, 28 McKenzie,J J.: Ontario Aa;riculturalOollegeReport, Toronto, 1893, 29. Oovan, T. A. ; British Bjo Journal, Vol. xxiii, p. 434. 30. Ward, F. F. : British Bee Journal, 1887, p. 396. 31. Canestrini: Atti della Society Veneto Trintina di Soienz* Naturali, Padua, 1891. 32. Bertrand: Bulletin d' Apiculture dp la Suisse Romande, i886, p. 128. 33. Benton ; Bulletin d' Apiculture, 1886, No. 4. 34 Bovill: Nicosia, Oyprus, Personal communication. 35. Feuillehois : Revue Internationale d' Apiculture, Vol. xv, p. 58. 36. BradL^: New South Wales Gazette, Sydney, 1894, p. 265. 37. " ApisLiyUjticus" : Journal of Industry and Agriculture of South Australia, Adelaide, 1897, p. 341. 38. Briehwell: British Bee Journal, 1890, p. 486, 39. Eisenherg: Bakteriologische Diagncstik, Hamburg, 1891, p. 298. 40. Howard, W. R. : Foul Brood : Its Natural History and Rational Treatment, Chicago, 1894. 41. Dadant and Hunt: American Bee Journal, 1891, p. 470. 42. Sternberg : Manual of Bacteriology, New York, 1893, p. 478. 43. British Bee Journal, 1891, Vol. 19, p. 470. 44 McEvoy: Foul Brood ; Its Cause and Oare, Trenton, N.J,, 1895. 45. Root, A. I. : Gleanings in Bee Culture, 1896, Vol. xxiv, p. 853. 46 Dzierzon: Bienen Zeitung, Nordlingen, 1860. 47. Cowan : British Bee Keeper's Guide Book, London, 14th Edition. 48 Bulletin D' Apiculture: Nyon, 1886, p 121. 49. Quinhy: Bee Keeping, New York, 1885, p. 217. 50. Bertrand: Bulletin d' Apiculture, 1882, p. 219 51. Aspinall: Eevue Internationale d' Apiculture, 1897, p. 9. 52. Butlerow: Bienen Zeitung, 1874, p, 176. 53. Cech : Phenol, Thymol, and Salioylsaure als Heilmittel der Brat Pest der Bienen, 1877. 54. Knighf Philosophical Transactions of the Royal Society, 1807, p. 243. 55. Kirhy and Spence: Introduction to Entomology, 1828, Vol. ,ii, p. 111. 56. Reports of the Bee Keepers' Association for the Province of Ontario, Toronto, 1890-1898. 57. Report of Convention of American Bacteriologists : Journal Ameri- can Publis Health Association, Vol. xxiii, 1898. 58. Root, L. 0. : Qainby's New Beekeeping, p. 218, New York, 1879. 59. Bertrand : Oonduite du Rucher, 8th Edition. Nyon, 1895, 60. Cowan: British Bee Journal, Vol. 12, p. 128. 61. Ossipow: Travaux de la Sooi^Die Eoonomique Imp6riale, St. Peters- burg, 1884. 32 62. Klempin: L'Apicoltore, Milano, 1885. p. 302 (Report by Prof. Grasfi). 63. Zehetmayr: British Bee Journal, Vol. 12, p. 60. 64. Cowan : Ibid, p. 1 29. 65. Jones, D. A.: Foul Brood; Its Management and Curp, Beeton, Cinada, 1886. 66. Schreuter: Bienen Zeitung, Dec. 1887. 67. Borel: Revue Inlernationale d' Apiculture, 1888, p. 156. 68. Cowan: Revue Internationale d' Apiculture, 1889, p. 139. 69. Bauverd: Revue Internationale d' Apiculture, 1883, p 247. 70. Auberson: Revue Internationale d' Apiculture, 1891, p. 240. 71. British Bee Journal, Vol. xxiii, p. 402. 72. Lortet : Eevue Internationale d' Apiculture, 1890, p. 50. 73. Cowan: Revue Internationale d' Apiculture, 1891, p. 165. 74. Dennler: Blsassieh Lotbringische Bienen Zuohter, Nov., No., 1885. 75. Sproule: Bee Keeper's Record, 1889, Jane No.; "Gleanings," 1890, p 596. 76. Afuhlenkoff: Eichstadt Bienen Zeitung, 1884, No. 6. 77. Erlennmeyer . S^.in:e de TAcad^mie des Sciences de Munich, 6tb Feb., 1875. 78. Planta: Schweizerische Bienen Zeitung, 1893, p. 186. 79. Planta: Schweizerische Bienen Zeitung, 1884, p. 89. 80. McLean : Report of the Department of Agriculture, Washington, 1886, p. 584. 81. Bochatey: Revue Internationa'e d' Apiculture, 1900. No. 9. Ontario Department of Agriculture FRUIT BRANCH, BULLETIN 190 BEE DISEASES IN ONTARIO Printed by L, K. CAMERON', Printer to the King's Most Excellent Majesty, TORONTO, ONT., May, 1911. BULLETIN 190.] [MAY, 1911 Ontario Department of Agriculture FRUIT BRANCH Bee Diseases in Ontario Much dissatisfaction with beekeeping as a business is caused by so- called "bad luck," which is really due to a definite bee disease which any bee-keeper can learn to cure. Bees are quite as liable to disease as any other live-stock, and to be able to treat such disease intelligently is quite necessary to success. Bee-moths are often blamed for the ravages due to disease ; but moths never destroy a healthy normal colony, as they only feed on the deserted combs after the bees are nearly all gone. Heavy winter losses can often be attributed to disease. In fact, whenever a colony is not doing well the exact cause of its failure should be carefully sought to make sure there is no bacterial disease. - On the other hand, disease often makes its first appearance in the best colonies in the apiary, because infection is usually carried by robbing, and that is generally done by strong colonies. If not checked on the start it soon spreads through the whole apiary, and from it to other apiaries in the neighborhood. The inspectors of apiaries can do a great deal for the health of bees in Ontario ; but to be of real value their work must be supplemented by the earnest efforts of the individual beekeepers. Everyone should be his own inspector, carefully examining every comb of every colony in the apiary at least once a year, remembering that it is far better to detect it on the start in strong colonies than to wait until they are practically ruined and the disease has spread through the whole neighborhood. Only one cell of infectious disease makes it necessary to treat even the best colony in the apiary. And because one has kept bees for a number of years without seeing a case of disease is no reason why it should not make its appearance this year. Plenty of people have died of smallpox after having escaped it for fifty years. When a case of infectious disease is suspected the beekeeper must first notify the Minister of Agricuture, Toronto, Ont., who will send word to the nearest inspector of apiaries ; but if the case cannot have immediate attention the beekeeper should go ahead and treat the disease according to directions given in this bulletin. Examining an Apiary for Disease. The diseases which cause the most damage in Ontario attack the developing brood, causing much of it to die in the comb, and so reducing it that the colony soon dwindles from lack of young bees to replace the old. When examining an apiary for disease the prime consideration is to avoid robbing. The best time is during a good honey flow as early as possible in the season. It is necessary to have a good smoker, a hive tool for taking out combs, and a supply of wooden toothpicks for testing the brood. In opening the hive just enough smoke should be used to keep the bees in subjection. Remove each comb in turn from the brood-chamber and examine the brood. It is best to sit on a box close to the hive with your back to the sun, and hold the comb so it will shine into the cells, and throw a strong light directly on the lower sides and bottoms of the cells. If there is no disease, the empty cells will be bright and clean, and the uncapped larvae will be plump in form and of a pearly white color. At first a number of cells of capped brood should be opened with the pick, until you are quite familiar with the outward appearance of healthy capped brood. Cappings which to any but the best-trained eye appear quite healthy often cover dead larvae. When diseased cells are present they are quite frequently found around the lower edge of the comb. If any of the brood cappings appear darker than the rest, or are flat, sunken, or perforated, they should be opened to see whether the brood they cover is dead. Healthy brood is sometimes found under flat, or per- forated cappings ; but there is a difference in appearance which experience soon teaches one to detect. Brood sometimes develops without ever being fully capped ; this is not known to be an indication of infectious disease. When each hive is finished the pick used there should be left in the hive, and if any honey is daubed on hands or tools they ipust be washed thoroughly before opening the next hive. [There are three brood diseases prevalent in the apiaries of Ontario ; American Foul Brood, European Foul Brood, and Starved or Pickled Brood. The first two are known to be infectious, the last is not so con- sidered, although its cause is not well understood. Distribution op Disease. American Foul Brood is pretty evenly distributed over that portion of Ontario lying south and west of the Trent Valley. It has cost the Province of Ontario hundreds of thousands of dollars, not only in loss of bees and honey, but in its disheartening effect on the men engaged in the industry. Much, however, is being done, and more can and will be done, by the Department of Agriculture towards restoring a well grounded confidence in beekeeping as a business by various methods of instruction. T-he greatest menace at present is European Foul Brood. This scourge is known to have practically wiped out the keeping of bees over a terri- tory of perhaps three hundred square miles around Ottawa, and five hundred square miles in Northumberland, Hastings and Prince Edward ; it has also gained a foothold at Fort Erie on the Niagara River. Much loss by this particular disease might have been saved if the beekeepers had kept Italian instead of common black bees, or if they had Italianized as soon as they were warned. As the situation now stands, it seems to be spreading from these districts like a blight, carrying all black bees before it. Only those who Italianize their bees can hope to save them, as no system of inspection can cure black bees of this particular disease. Men with the right strain of Italian bees are securing enormous yields of honey right in the centre of infected district. As the Irishman says, "It's an ill wind that blows nowheres," and there is not the slightest doubt that this wind of European Foul Brood, though ill enough, will blow money into the pockets of the men who will sit tight, get Italian bees, and weather the storm. AMERICAN FOUL BROOD. This disease is caused by bacteria known to scientists as Bacillus Larvae (not B. Alvei, as was formerly supposed). It reaches the healthy young larvae by means of infected food unsuspectingly fed to them by the nurse bees. In most cases the larvae dies when nearly ready to seal up, and most of the cells containing infected larvae are capped. The dead larva softens, settles to the lower side of the cell in a shapeless mass, at first white or yellow, changing to cofifee-color and brown. At this stage it becomes glutinous, so that if it is picked with a toothpick the contents will rope out half an inch or so when the pick is slowly withdrawn. It adheres to the cell so it cannot be lifted out entire. It has the odor of a poor quality of glue. When the larva dries it forms a tightly ad- hesive scale, of very dark brown color, which cannot be removed without tearing the cell wall. Where the infected larvae are capped the cappings turn a darker color and become flat or sunken, the workers perceiving that something is wrong usually start to tear off the capping, but, discovering the con- dition of the contents, they generally leave it with a small perforation in the centre until quite dry, then the capping is removed, and in time honey may be stored in the cells containing the scales of disease. The millions of disease spores then float out into the honey, which becomes a medium for carfying the disease to other healthy larvae by robbing, in the same or some other apiary. Some of the honey is also carried into the supers, to make room for alterations in the brood nest, and is marketed in the form of bottled or section honey. It goes into many homes, especially in towns and cities, ffhe wooden sides of the sections, and many of the empty bottles, or washings from them, are thrown out by housekeepers and cleaned up by bees of the neighborhood, and the disease is carried home to their healthy brood. This is why our inspectors find more disease in the apiaries around towns and cities than elsewhere. The Treatment. Now, to be cured of this disease a colony must be freed from all this infected brood, comb and honey. To do this we simply take it away. But in the operation some precautions are necessary. We must see that the colony will get healthy food as soon as the unhealthy food is taken away, and have means for building new comb at once. So the opera- tion should be performed during a honey flow, and to make it perfectly sure it is a good plan to insert a division board feeder of sugar syrup. We must take precautions against starting robbing, or causing the treated colony to scatter to other hives or swarm out, be lost, and carry infection to other places. So the operation should be performed in the evening, when the bees are settling down for the night, and the entrance should be covered with queen-excluding metal to hold the queen in case of swarming out the next morning. A regular queen-excluder laid on the bottom board under the brood chamber will answer the latter purpose. They should also be given a clustering space to occupy, as in the case of a natural swarm. Whenever bees are disturbed in their hives they will fill their honey sacs with honey from the comb. As this will happen when the hive is being treated, and some of this diseased honey may be stored in the new combs, it is thought best to remove these after three or four days and require them to make a second start. Method oe Treatment. When there is a good honey flow on, go to the colony in the evening, taking a set of frames with one-half inch starters of foundation in them. Take the combs out of the hives, shaking the bees from them, back into the hive. If any fresh nectar flies out, it will be necessary to brush the bees off instead of shaking them. Get these combs immediately under cover, and clean up very carefully any honey that may be about, so that robbers from healthy colonies cannot carry home disease. If the honey flow is at all uncertain, it is better to put in a feeder with thin sugar syrup. On the third or fourth evening after the first operation, remove the hive from its stand and set in its place a clean disinfected hive containing frames with full sheets of foundation. Now brush the bees from what combs have been built on the starters into the new hive. Even greater care must be taken than at first to avoid leaving any honey or bits of comb about. Positively no comb must be used or left in the Wve in either the first or second treatment. You have now made an artificial swarm of this colony. It must be given the conditions a new swarm likes, or it will leave and carry its disease to parts unknown, or perhaps into some healthy hive in the apairy. A new swarm likes plenty of ventilation and shade, and also room to cluster for awhile without having to crawl in between sheets of founda- tion at once. To satisfy this natural desire, it is best to place an empty hive under the one containing the frames of foundation. If for any reason this cannot be done, two or three frames can be left out of the brood chamber for a couple of days. CDhe bees will cluster in this at first, just as a swarm clusters on a tree; but they will soon go up and take possession of the foundation, then the empty can be taken away. This simple precaution will generally prevent the svrarming out w'hich so often happens in treating foul brood; but as an extra precaution it is best to use the excluder on the entrance as well. All combs from the supers as well as from the brood chamber of the diseased colony, together with the first set of starters and whatever comb is built on them, must be either burned or melted, and boiled thoroughly before the wax is fit to use again. The honey that is removed is entirely unfit for bee feed, even after it has been boiled for a full half hour it is not safe. The only safe way to dispose of it is to burn it, or else dig a hole and bury it deep enough to be out of the reach of any bees. If directions have been followed carefully and thoroughly, the treat- ment should be successful. To make sure, however, the brood must be examined again in about three weeks and again the following season. Please note in this connection Section 6 of the Act. Saving Brood. Brood from badly diseased colonies is of no value, and dangerous, and should be burned, buried or otherw'ise destroyed at once. Brood from colonies having only a few cells diseased may be placed over an average colony slightly diseased, and the queen caged. In ten days treat as given above. Saving Combs. It is never safe to use super-combs that have been on diseased colon- ies. Even though they may appear white and clean, germs of the disease are apt to lurk in them from year to year. To melt these down is no serious loss, as the wax will more than make foundation for new ones. Disinfecting. Hives which have formerly contained diseased colonies, or in which diseased combs have been stored or carried, should be burned over in- side with a gasoline or oil torch. EUROPEAN FOUL BROOD. Until 1907 the only infectious brood disease known to exist m Ontario was the one already described. But another then made its appearance. It is called European Foul Brood (sometimes black brood"). . • i t European Foul Brood has destroyed the apiaries in great areas ot different States in the Republic to the south of us. It is now known to be rampant in at least three sections of Ontario. In one way it is much more to be dreaded than American Foul Brood, because it runs its course and destroys an apiary much more rapidly, and because the adult bees will carry out the disease scales and scatter them in the yard and farther, to find their way into healthy colonies. In the part of Ontario where it was first discovered apiaries were wiped out at first something like this : 112 colonies reduced to 23 in two years. 180 reduced to 21 in one year. 60 colonies reduced to 44 in one year, and the balance all diseased the second year. The following report in reference to European Foul Brood, received in the fall of 1910 from one of our apiary inspectors, will give an idea of the danger : Inspector's Report for Peterboro, Northumberland, Hastings and Prince Edward. Inspector W. Scott, Wooler. "1 travelled over the same ground as last year, and found that all the bees had been treated, except one apiary, but very little Italianizing had been done, and consequently the disease returned in every apiary and destroyed some of them completely. I found the disease spreading very rapidly ; it has more than doubled since last year. The disease now covers fully 400 square miles ; besides two outbreaks in Prince Edward County, also two in Hastings County, one in Roden Township, and one in Huntingdon Township. I think if the Department could encourage the beekeepers to Italianize ahead of the disease, it would prevent a great deal of loss, as the disease does not affect the Italians nearly so badly as the blacks. I think the disease could be prevented some if the law would forbid the moving of bees except by permission of the inspector. I found three cases in the past season, where the disease has broken out, caused by the moving of bees from a diseased territory to an undiseased' one. Had this m'oving been prevented it would have taken it probably two years to travel of its own accord. "I may say that $5,000 is not too large an estimate for the loss sus- tained by disease in my district last year, but these figures will be greatly increased next year as European Foul Brood is spreading very rapidly. Last year (1909) it covered about 100 square miles. At the present date it covers 400 square miles, besides an outbreak in Prince Edward County, also in Hastings County near Ivanhoe." Symptoms of European Foul Brood. The symptoms are easily distinguished from those of American Foul Brood, a^ there is very little ropiness, and the odor is different. The larvae mostly die without uncoiling from their natural positions. The color in the earlier stage is lighter than in the American Foul Brood. The odor is very pronounced and offensive, like decayed fish; in fact, on a warm moist morning it is noticed on entering the apiary, and, when a diseased comb is held up for inspection, is almost sickening. Use Same Treatment and Italianize. The same treatment already described for American Foul Brood is effectual if applied to the whole apiary at once, even though only a few colonies show symptoms. Even then the cure is only permanent when pure-bred Italian queens are introduced to the affected colonies. It is quite impossible to cure an apiary of black bees Qi European Foul Brood without introducing pure Italian queens to all colonies. We know of no reason why this plague should not sweep over On- tario as it has over most of the United States. If it does, all apiaries of black bees will be practically destroyed within the next few years. Its progress in the districts mentioned above has been appalling. No Gov- ernment expenditure can touch the situation without the co-operation of the men themselves whose property is in danger. There is a remedy, however, right at hand. Pure-bred leather-colored Italian bees are al- most immune to^this disease, which works so much havoc among the common blacks. It is very important, then, that all apiaries, especially in or near in- fected neighborhoods, should be Italianised at once, without waiting for a destructive outbreak of disease. STARVED OR PICKLED BROOD. A disease slightly resembling Foul Brood is called by some "Starved Brood," and by others "Pickled Brood." The most positive difference in the diagnosis of this disease is the absence of ropiness and of the glue-pot smell, which are always found in American Foul Brood. In Pickled Brood the larva decays from the inside, leaving the skin tough and in its natural shape; in European Foul Brood or American Foul Brood, the skin of the larva softens as the contents become glutinous, and all the natural wrinkles become smooth as the mass settles to the lower . 8 side of the cell. In Pickled Brood the larva often dries up so as to be- come loose in the cell and fall out when the comb is inverted. In Ameri- can Foul Brood it always cements fast to the lower cell wall, so it cannot be removed without tearing the cell. European Foul Brood attacks the larva generally at an earlier stage in its existence than Pickled Brood. The cause of Pickled Brood is not definitely known. It is not con- sidered to be infectious. McEvoy asserts that it is caused by an insuffi- cient feeding of the larvae, due to a sudden check of the honey flow, or a constitutional weakness of the workers. The latter he charges to in^breeding of the queens. Re-queening with vigorous queens from other apiaries will often effect a cure, and it often disappears of its own accord. AMERICAN FOUL BROOD. From the reports of the inspectors of apiaries of recent years, we find that American Foul Brood is prevalent in the following counties and townships. This does not mean that townships not mentioned in this list are guaranteed to be free from this disease, because the apiaries of Ontario have not all been inspected as yet : Brant: Brantford, Dumfries South. BrucB: Arran, Brant, Bruce, Culross, Elderslie, Greenock, Kinloss, Saugeen. Carlbton: Goulbourn, Osgoode. DuPFERiN : Garafraxa East, Luther East, Mono. DuNDAS : Winchester. Durham : Darlington. Elgin : Dorchester South, Malahide, Yarmouth. Essex : Gosfield North. Maidstone, Rochester, Sandwich East, Sand- wich West. Frontenac: Kingston Township. Grey: Artemesia, Collingwood, Euphrasia, Glenelg, Keppel, Osprey, Proton, St. Vincent, Sarawak, Sydenham. Haldimand: Cayuga, Walpole. Halton: Esquesing, Nelson, Trafalgar. Huron: Grey, Morris, Turnberry, Wawanosh West. Kent: Harwich, Romney, Tilbury East. Lambton: Bosanquet, Moore, Warwick. Leeds: Bastard, Elizabethtown, Kitley, Yonge. Lincoi,n : Louth. Middlesex : Adelaide, Biddulph, Delaware, Lobo, London, McGill- vray, Metcalfe, Westminster, Williams East, Williams West. Muskoka: Draper, Macaulay, Muskoka. Norfolk : Charlotteville, Townsend, Walsingham, Wyndham, Wood- house. Ontario: Brock, Pickering, Reach, Scott, Thorah, Uxbridge, Whitby East. Oxford: Blandford, Blenheim, Dereham, Norwich North, Norwich South, Oxford East, Zorra West. Peel : Albion, Caledon, Chinguacousy, [Toronto. Perth : Blanshard, Downie, Easthope North, Easthope South, EUice, Elma, Fullarton, Hibbert, Mornington, Wallace. SiMcoE: Adjala, Essa, Gwillimbury West, Innisfil, Medonte, Notta- wasaga, Orillia, Tay, Tecumseth, Tiny, Vespra. Victoria: Bexley, Eldon, Mariposa. Waterloo: Dumfries North, Waterloo, Wellesley, Wilmot. Wellington : Garafraxa West, Guelph, Luther West, Nichol, Pus- linch. Wentworth : Ancaster, Barton, Beverly, Binbroc^, Glanford. York : Etobicoke, Gwillimbury East, King, Markham, Scarborough, Vaughan, Whitchurch, York. EUROPEAN FOUL BROOD. From the reports of the inspectors of apiaries we find that European Foul Brood is prevalent in the following counties and townships. As this disease is spreading rapidly, it is very likely to appear in the town- ships adjoining these during the season of 191 1. All beekeepers should be very much on the alert and examine their bees carefully for the symp- toms of this disease : CarlETOn: Fitzroy, Gloucester, Nepean. Hastings: Huntingdon, Rawdon, Sidney. Leeds : Bastard. Northumberland: Brighton, Cramahe, Murray, Percy, Seymour. Prince Edward: Ameliaslburg, Hillier. Renfrew : MacNab. Welland County: Bertie. The names of cities and towns located in these townships are omitted for brevity, but as a matter of fact, bees in cities and towns are more often diseased than in the country. 10 AN ACT FOR THE SUPPRESSION OF FOUL BROOD AMONG BEES. His Majesty, by and with the advice and consent of the Legislative Assembly of the Province of Ontario, enacts as follows : 1. This Act may be known as "The Foul Brood Act." 2. The Lieutenant-Governor in Council, upon the recommendation of the Minister of Agriculture, may from time to- time appoint one or more Inspectors of Apiaries to enforce this Act, and the Inspector shall, if so required, produce the certificate of his appointment on entering upon any premises in the discharge of his duties. And the Minister shall' instruct and control each Inspector in the carrying out of the provisions of this Act. The remuneration to be paid to any Inspector under this Act shall be determined by order of the Lieutenant-Governor in Council. 3. The Inspector shall, whenever so directed by the Minister of Agriculture, visit without unnecessary delay any locality in the Province of Ontario and there examine any apiary or apiaries to which the said Minister may direct him, and ascertain whether or not the disease known as "foul brood" exists in such apiary or apiaries, and wherever the said Inspector is satisfied of the existence of foul brood in its virulent or malignant type, it shall be the duty of the Inspector to order all colonies so affected, together with the hives occupied by them, and the contents of such hives, and all tainted appurtenances that cannot be disinfected, to be immediately destroyed by fire under the' personal direction and superin- tendence of the said inspector ; but where the inspector, who shall be the sole judge thereof, is satisfied that the disease exists, but only in milder types and in ' its incipient stages, and is being or may be treated successfully, and the inspector Tias reason to believe that it may be entirely cured, then the inspector' may, in his discretion, omit to destroy or order the destruction of the colonies and hives in which the disease exists. 4. The inspector shall have full power in his discretion, to order the owner or possessor of any bees dwelling in box or immovable frame hives, to transfer them to movable frame hives within a specified time, and in default the inspector ■may destroy, or order the destruction of such hives and the bees dwelling therein. 5. Any owner or possessor of diseased colonies of bees, or of any infected appliances for beekeeping, who knowingly sells or barters or gives away such diseased colonies or infected appliances, shall, on conviction thereof, before any Justice of the Peace, be liable to fine of not less than $50 or more than $100, or to imprisonment for any term not exceeding two months. 6. Any person whose bees have been destroyed or treated for foul brood, who sells or offers for sale any bees, hives or appurtenances of any kind, after such destruction or treatment, and before being authorized by the inspector so to do, or who exposes in his bee-yard, or elsewhere, any infected comb, honey, or other infected thing, or conceals the«fact that said disease exists among his bees, shall, on conviction before a Justice of the Peace, be liable to a fine of not less than $20 and not more than $50, or to imprisonment for a term not exceeding two months, and not less than one month. 7. Any owner or possessor of bees who refuses to allow the Inspector to freely examine said bees, or the premises in which they are kept, or who refuses to destroy the infected bees and appurtenances, or to permit them to be destroyed when so directed by the inspector, may, on the complaint of the inspector, be summoned before a Justice of the Peace, and, on conviction, shall be liable to a fine of not less than $25, and not more than $50 for the first offence, and not less than $50 and not more than $100 for the second and any subsequent offence, and the said Justice of the Peace shall make an order directing the said owner and possessor forthwith to carry out the directions of the inspector. II 8. Where an owner or possessor of bees disobeys the directions of the said inspector, or offers resistance to, or obstructs the said inspector, a Justice of the Peace may, upon the complaint of the said inspector, cause a sufficient number of special constables to be sworn in, and such special constables shall, under the direc- tions of the inspector, proceed to the premises of such owner or possessor and assist the inspector to seize all the diseased colonies and infected appurtenances and burn them forthwith, and if necessary the said inspector or constables may arrest the said owner or possessor and bring him before a Justice of the Peace to be dealt with according to the provisions of the preceding section of this Act. 9. Before proceeding against any person before a Justice of the Peace, the said inspector shall read over to such person the provisions of. this Act or shall cause a copy thereof to be delivered to such persons. 10. Every beekeeper or other person who is aware of the existence of foul brood, either in his own apiary or elsewhere, shall immediately notify the Minister of the existence of such disease, and in default of so doing shall, on summary conviction before a Justice of the Peace, be liable to a fine of $S and costs. 11. Each inspector shall report to the Minister as to the inspection of any apiary in such form and manner as the Minister may direct, and all reports shall be filed in the Department of Agriculture, and shall be made public as the Minis- ter may direct or upon order of the Legislative Assembly. 12. Chapter 283 of the Revised Statutes of Ontario, 1897, intituled "An Act for the Suppression of Foul Brood Among Bees," is repealed. Published Serial No. Date. 153 Feb. 1907 154 Feb. 1907 155 156 157 Feb. Mar. Mar. ■ i907, 1907 1907 158 June 1907 159 July 1907 160^ 161 162 July Oct. D^ec. 1907 1907 1907 163 Mar. 19i08 164 i^axc. 1908 165 166 Mar. June 1908 1908 167 Oct. 1908 168 Oct. 1908 169 Feb. 1909 170 Mar. 190i9 171 April 1909 172 May 1909 173 - Oct. 1909 174 Dec. 1909 175 Dec.. 1909 176 Dec. 1909 177 Dec. 1909 178 Dec. 1909 179 Feb. 1910 ISO April 1910 181 June 1910 182 July 1910 183 Aug. 1910 184 Nov. 1910 185 Nov. 19.10 186 Dec. 1910 187 Jan. 1911 188 April 1911 189 May 1911 190 May 1911 LIST OF BULLETINS BY THE Ontario Disfartment of Agbictjltueb, Toronto. ■y. \ Ititle. " Aufhor. , : Fertilizers and their Use; ; Jtt. Harcoilrt, ' ' . .^ , *, ■ .J - ■ f R. Harcourt. Insecticides and Fungicides ;. < ll- L Fulmer. ., Farm Forestry E. J. iZaVitz. Tillage and Rotation. ,....- W; HT. Day. . ; . Remedies for the San Jose- Scale- ■"■ ' . iSan Jose Scale Act , ; » Insects .and Fungus Diseases .Affecting f C. J. S. Bethune. Fruit Trees, Revised Dec, 1907 ..fr. D". Jarvis. Tv/i-n • 1.^ ,,• ., f H. H. Dean. MUlcmg Machines .; • ; \ s. F. Edwards. The Production, Care, and. tlses of .M;ilk ;........» The Sheep Industry in Ontario Breakfast Foods: -Their Chemical Com- fR. Harcourt. position. Digestibility and Cost \ H. L. Fulmer. Incubation of CJhickens O. A. C. Staff. Legume Bacteria;;;. :.. ! . { |; ^^^f ' Alfalfa or Lucerne. : C. A. Zavjtz. Bee-keeping in Ontario , Fruit 'Branch. Mitchell- Walker Moisture Test : . . . . | ^ "^- ^^f^g^"' '' The Perennial SOw Thistle and sijme other .'. , Weed'Pests ^. J. E. Ho-witt. Legume Bacteria: Further Studies of f „ _ _, , , Nitrogen Accumulation in the Legu- ^^ i; J^avaras. minosle . . ( B- Barlow. . Mitchell-Waiker Tesf Bottle.\ .^ { :^_^_ wa?ker!' . r Insects Affecting Vegetables '..... C. J. S. Bethiine;. I Fungus-Diseases Affecting Vegetable^. . . .■ ( l- ^- Eastham.. ■ >- , :IJ.;B. Howitx. Dairy School Bulletin (No 143 Revised) . .'.Dairy School. Birds of Ontario.. ._.-.. r C. W. Nash. Farm Underdraihage: Does it Pay?. . .\ . . . . W., H. Day. Farni Drainage Operations , . wl iH. Dfty.' Bacterial Blight of Apple, Pear and Quinge,' i Trees . . . ,. D. H. Jones. Lime-Sulphur Wash I ?' n' ^"'™®^- Character and Treatment of Swamp or f W, P. Gamble. Muck Soils I A. E. Slater. Fruits Recommended for Ont-ario; FruitEx, Stations. Flour and Breadmaking. l?-J harcourt. IM. 4. Purdy. The Teeth and Their Care. . . : Ont. Dental Soc'y. Bee-keeping in Ontario Fruit Branch. Notes on Cheddar Cheese-Making. . . .^. Dairy Brdnch. Uses of Vegetables, Fruits and Honey , .1. , Little Peach Disease .;..;.; L. Caesar. Children: Care and Training , j. j. Kelso. ' The Codling Moth .'. . .L. Csesar. ' Weeds of Ontario ; j. 'fe. Howitt ^ Farm Poultrj^ , w. I^. Graham. Bee Diseases in Ontario Fruit Branoh. July, 1926 Extension Service Bulletin No. 138 Control of Bee Diseases and Pests ■■■Ot?^ (Courtesy of Frank C. Pellett, Am. Bee Journal) Putting- combs in saclcs. IOWA STATE COLLEGE OP AGRICULTURE AND MECHANIC ARTS EXTENSION SERVICE R. K. Bliss, Director AMES, IOWA Issued and distributed under Acts of Congress of May 8 and June 30, 1914. Control of Bee Diseases and Pests Bee diseases constitute one of the chief causes of the present day inefficient production of honey. This indicates that beelteepers need a better Itnowledge of the diseases and the means of reducing the loss from them. There are two general classes of apiary diseases, those of the adult bees, and those of the young or brood. At present the brood diseases cause by far the greater portion of the loss in the apiary so more attention must be given to them. Their diagnosis and control have been carefully worked out and the practical application is merely a matter of knowledge. BEOOD DISEASES AMERICAN FOULBROOD Origin and History: American foulbrood as a distinctive disease of bees has been known for about 20 years. Prior to that time it was confused with what is now known as European foulbrood, both being included in the term foulbrood. It became evident that there were at least two distinctive diseases since beekeepers were getting differ- ent reactions to identical treatments under apparently the same con- ditions. The term "American" foulbrood does not mean that this disease originated in America, nor that its distribution is restricted to Amer- ica. It is merely a term to indicate a definite disease and distinguish it from the European foulbrood. Foreign records Indicate that this disease has been prevalent thruout most beekeeping sections much longer than it has in the United States. Distribution: American foulbrood is now known to occur quite generally in every state in the Union. Its spread has been restricted in some sections, but no extensive beekeeping region enjoys immun- ity to this brood disease. In Iowa, it has been felt that In certain restricted localities American foulbrood did not occur, but careful and extensive inspect'ons indicate that the areas where foulbrood does not exist are very small indeed. Some think that the disease is spread- ing and increasing its distribution, but the fact that it has not been recorded from a definite locality does not signify that it is not present. It probably has not been discovered, but may be found at any time. Common Names: While American foulbrood is, in a way, a common name, beekeepers often refer to this disease in a general way as "black brood". This name certainly indicates the condition of the dead larvae. In other areas it is referred to as "ropy brood," which is another term describing a stage of the disease. With the gradual disappearance of European foulbrood it has come to be a practice, among beekeepers, to refer to American foulbrood simply by the term "foul". Closely associated with this is the common term, "diseased brood"- Transmission: American foulbrood is a germ disease, caused by a specific organism known as Bacillus larvae. The disease is highly in- fectious, so that the germs will readily produce the disease when in- troduced into a healthy host. American foulbrood germs are exceed- ingly minute and they travel unobserved by the human eye. The bac- teria grow and develop in the bee larvae, but whenever they are placed in honey, they immediately develop into the spore or seed stage. This is important because it is considered that the sale of honey may be one means of spreading the disease. The bacteria of American foulbrood are hardy compared with the average forms of germs. If they were not so hardy they would not be able to continue the disease in spite of all adverse conditions to which they are subjected in attempted control measures. This point is im- portant because of its bearing upon the apparent disappearance of the disease in the treated colonies and apriary and the unexplained ap- pearance of the disease at a later time. The spores remain capable of producing bacteria for 30 or 40 years, so that when the material is once contaminated it is exceedingly difficult to say that all possible sources of re-contamination have been eliminated. One must know how the disease works and appreciate the dangers in handling infected material, in order to conduct a proper fight against the spread of the disease. It is also necessary to appreciate the ravages of the disease in order that one may be care- ful arid use the necessary precautions in the treatment of the disease and the disposal of infected material. Diagnostic Characters: The larvae which are infected with the bacteria causing American foulbrood usually reach maturity and the cells are capped. Death occurs, however, except in rare cases, before the larvae have an opportunity to change to the pupal stage. The bac- teria seem to require seven days to cause the death of the larvae. Apparntly the bees are inquisitive to learn why certain sealed cells are not giving forth adult bees. Consequently a tiny hole is gnawed in the capping, which is all that is needed for the bees to tell the condition inside. In the early stage of the disease the character which will attract the attention of the beekeeper in the colony is sunken, discolored and perforated caps on a few scattered cells thruout the brood area. If these cells are examined early in the development of the disease, it will be found that the larvae are discolored and appear a shade of chocolate. If the examination is made in this stage the larvae can be tested with a toothpick or pointed match and it will be found that "the contents will "string" or "rope" as the match is withdrawn from the larvae. This condition is very typical of the dis- ease. The odor which may be de- tected from the dead larvae is supposed to be characteristic and is described as a "gluepot o d o r," but for one un- familiar with the disease this char- acter cannot be used exclusively. If the disease has progressed Fig. i ■ Perforated cap of American foulbrood. Fig. 2. Dead lana melting- away system. from trachael for some t'me in the colony, or if the combs are exam- ined after the col- ony is dead, it will not be possible to get the character- istic ropiness of the larvae. If the lar- vae continue to dry, they eventual- ly form a scale along the lower side of the cell and cover most of the bottom of the cell. This scale is black in color and ad- heres tightly to the cell wall; in fact, it is necessary to tear down the cell in order to get the scale out of it. These scales can be seen readily by standing so that the light comes over the shoulder and by tipping the comb with the bottom bar away from the observer so as to throw the l.ght on the lower side of the cells. The black scales ban se seen readily if the light strikes them at the proper angle. Seasonal History: American foulbrood works slowly in causing the extinction of the colony. A colony may become infected during the early spring, but the disease may increase slowly thruout the summer and the colony in the fall may still be apparently strong enough to go thru the winter. Beekeepers often remark that such a colony ap- peared a little below average in strength, but apparently had plenty of stores so that it seemed it should survive the winter. Under these conditions the colony invariably fails to withstand the cold weather and dies during the winter. The stores in such a hive are unprotected during early spring and are subject to the ravages of robbers, which carry the honey to their hives. This robbing occurs very early in the spring and is usually unnoticed by the beekeeper. It is evident, then, that a colony which dies from disease may serve as a feeding ground for robber bees from several other colonies, which may then become infected from this contaminated honey, brought from the dead hive. These newly infected colonies may show the disease very soon or not until late in the season, or even perhaps not until the next season, or two seasons later. The appearance of the disease in a colony depends upon the time when the diseased honey is used for rearing brood. Small amounts of the diseased honey may be stored in the bottom of the cells and later covered up with nectar or sugar syrup, and the cells may not be emptied for a season or two. Usually disease shows up the first sea- son if robbing has occurred, for the contaminated honey is stored where it will soon be used in rearing brood. Since colonies are robbed cut early in the season, early examination should be made to deter- mine it a colony is alive or dead. It is not safe to start a diseased colony into the winter, for it is sure to be abnormally low on young bees, and the chances are decidedly against it surviving. Treatment: The method of treatment will depend entirely upon the proportion of infestation in the apiary. Fundamentally, the treatment of American foulbrood consists of separating the adult bees from the contaminated material. The adult bees when separated are put on a starvation basis for four days in order to rid them of all contaminated material which is carried in their honey sacs. This process is known as the "shaking treatment," or is sometimes referred to as the McAvoy treatment. If the disease is occurring for the first time in an apiary and but one colony is diseased, the safest and surest method is to destroy the entire hive and its contents. To do this, dig a deep pit and start a fire in it. The entire hive in which the bees have been killed during the previous night, is placed on top of the fire and after thj material has been burned thoroughly the remainder of the contents of the hive are covered with the dirt. The pit must be deep enough so that the remains of the hive are far below any ordinary disturb- ance of the soil such as spading, plowing and ordinary grading. This method is open to objections and the real d.fflculty lies in not suf- ficiently covering the remains of the colony. The honey is not dis- infected in the least for it runs out of the combs when they melt at about 3 35 degrees, whereas, to disenfect honey, it must be heated to 212 degrees and held there for 30 minutes. An actual experience will illustrate the difficulty. A diseased col- ony was killed, a pit was dug and the colony put on the fire. The fire did not completely burn the material, but the pit was refilled. In time an odor came thru the soil which attracted dogs which dug down and uncovered the unburned remains of the colony. After reaching the decaying mass they left in disgust. The bees in the apiary found this hole and had acsess to the buried honey. The slight heating had not destroyed the bacetria in the honey, the disease, in- stead of being eliminated from the apiary, spread to six or eight addi- tional colonies. It is of utmost importance to make a thoro disposal of the contaminated material in a colony in order to prevent the spread of American fourbrood. A diseased colony should be taken from the yard where the healthy colonies are located if treatment is to be given. It should be moved in the evening after all of the field bees are in the hive. On the new location, a confusion board should be put in front of the entrance so that the field bees will not tend to return to their old location. It is impossible to treat a diseased colony satisfactorily in the same yard with healthy colonies, tho where it is difficult to move a colony very far, where one has but a few colonies in a back yard, the diseased colonies should be separated as far as possible from the healthy ones before the shaking treatment. The shaking process should be given to the diseased colony between sunset and dark, and all preparations should be made in advance so that it can be accomplished quickly. The new equipment should be made ready, including three or four sheets of foundation, since the remainder of the frames need not be put in the hive until later. When the new or clean equipment is ready, the diseased colony is removed from its stand and placed with its entrance elevated 45 degrees and but a foot from the original entrance. The new equipment is placed on the old stand and a newspaper or old cloth is spread in front of the entrance. The bees should be smoked as little as possible in order to keep down the confusion among them. One by one the frames are lifted from the diseased colony and the bees brushed onto the paper or cloth. The bees from the first frame will be badly confused and a little attention may be needed to get them started toward the entrance to the new hive. If the entrance has been located, the bees from the other frames will start immediately to the entrance. Ttie process is facil- itated if the frame containing the queen can be among the first ones brushed. It does not pay, however, to spend any particular time looking for the queen. The entire brushing must be done with reasonable haste, but so as to cause as little confusion as possible. After a comb is freed from bees it should be placed in a sack. After all of the combs have been brushed the bees which are still in the hive body and on the bottom board, can be dumped in front of the entrance to the new hive. The combs may then be replaced in the hive body or left in the sack, depending on what disposal is made of this contaminated material. However, the old hive and its contents must be promptly removed from the yard. As soon as the bees going into the clean hive are off the paper or cloth on to which they have been brushed, this material should be picked up and burned Immediately, not in the yard, but in a place far removed from access by bees. It is not safe to burn any material in a furnace because the honey simply runs down on the ashes without any of the diseased spores which it con- tains being rendered inactive. Then when the ashes later are thrown out the bees will seek this infected honey. The proper disposal of the contaminated material demands some sacrifice on the part of the beekeeper. It is undoubtedly cheapest in the long run to burn the frames with their contents. For this pur- pose, the destruction by the pit method as described previously is probably the best. In this way the chief source of contamination is eliminated in such manner that it is not likely to reappear. The contaminated material must be disposed of immediately after the bees have been shaken. The practice of taking hive bodies and their contents to a bee-tight room has proven wholly ineffective in controlling the disease. The reason for this is that the normal bee-tight room is sooner or later made accessible for bees which hastily rob the contaminated honey and take it to their hives. If several colonies are to be treated at one time, it may be desir- able to shake a portion of the colonies and stack the brood chambers on two colonies in order to save the brood which will continue to emerge. These two hospital colon'es must be carefully attended be- cause their forces will be spread out in an effort to care for the im- mense increase in brood. Under these conditions the colony is subject to the attacks of robbers so that it is probably advisable to reduce the entrance to a space which will permit only one or two bees to enter at a time. These nursery colonies are then given the shake treatment in 21 days. The use of nursery colonies is suggested only for the bee- keeper who is thoroly familiar with disease treatment, for they re- quire very struct attention and if neglected in any manner will spread disease faster than the beekeeper can check it. When shaking the nursery colony it will be necessary to place in the clean brood cham- ber all of the frames with foundation, as the population will be so great that a few frames will not serve to accommodate all the bees. Bees shaken onto foundation must immediately build comb in which the queen can lay eggs, and in which honey can be stored. The honey which they have brought over from the old hive is soon digested and converted into comb-building material. It is this pro- cess which apparently destroys the disease germ. If the shaking (Courtesy of Frank C. Pellett, Am. Bee Journal) Fig. 3. 'Wreck of a neglected apiary. has been carefully done the chances are that the bees will use up all of their contaminated honey in making comb for the new hive or at least before any of the eggs hatch and the larvae need to be fed. Then it should not be necessary to shake again. Occasionally disease will reappear and a "double shake" will be necessary. After the colonies have recovered and have a supply of open brood in the combs, they must be provided with stores as they may not be able to get sufficient stores In the field. In case the "shaking" was made on four frames of foundation, additional frames can be added as they are needed by the colony. In a nor- mal colony two additional frames of foundation can be added each week, but the beekeeper must decide if more are needed. Careful inspection should be made before additional foundation is made to determine if the disease is re-occurring or not. It is not advisable to attempt to salvage old brood combs contain- ing honey. Some beekeepers feel that inasmuch as wax is worth considerable money, it may be best to render up such combs and sell the wax. This practice is to be discouraged because during the time when combs are held before treatment, they are apt to be robbed; during the treatment there is a tendency for the bees to get at the contaminated honey, and after the treatment there is still the problem of disposing of the contaminated honey The salvage process is expensive and the amount of wax recovered will not pay for the labor involved. It does not pay to attempt to save the honey even tho this honey is not in any way injured for human con- sumption. iSuch disease laden honey may so easily become attrac- tive to robber bees and the disease spread in this way. Recently beekeepers have been able to buy disinfecting materials which render combs fit for re-use in the bee-hive. The first question for the beekeeper to decide is whether the combs are worth treat- ing. It is a growing practice among modern beekeepers to replace their brood combs every three to five years. This eliminates the amount of drone brood and also pollen-clogged combs from the brood chamber. Therefore any brood combs which are old are apt to be so imperfect that it is not advisable to spend much time and money in fitting them for re-use in the beehive. Consequently, only combs which are really worth usins again in the brood chamber should be treated. These combs must be free from honey and all of the cells must be uncapped for perfect results. The process of treating combs consists of immersing them in a tank containing the disinfecting material for a period of 48 hours. After this time the combs are extracted in order to free them from the disinfecting material. Then these combs are aired for a short period after which they may be used again in the hives. The real service of the disinfecting materials comes in treating super or ex- tracting combs. These combs are usually very good and are well worth saving. Extracting combs may be treated and after short airing used again for the storage of surplus honey. Having disposed of the frames and their contents, attention should be given next to disinfecting the bottom board, hive body, inner cover and outer cover. A highly satisfactory method is to immerse this equipment in a tank of boiling lye water. This not only kills the disease spores but it cleans the equipment of wax and propalis. The process requires but a relatively short time. After the ma- terial is taken out of the lye solution, it should be rinsed quickly witn cold water and then stacked so as to air slowly. The lye solutio.T is made at the rate of one can of lye to ten gallons of water. The hives can be painted again and are practically as good as new equipment in the yard. Other methods of disinfecting this equip- menet are not advised. EUROPEAN FOULBROOD Origin and History: European foulbrood has been known to bee- keepers for a long time. In the early development of beekeeping there was apparently only one foulbrood d:sease and consequently when the disease was introduced in this country it was known as European foulbrood. Only in the last 20 years has European foul- brood been differentiated from American foulbrood. There ha.T been considerable confusion in regard to European foulbrood for under certain conditions it may slightly resemble American foulbrood. Distribution: European foulbrood was very serious in the east at the time American foulbrood was determined to be a distinct disease. Since that time European foulbrood has been very devastating in California apiaries. There probably is no definite territory in tho United States which is free from disease. In Iowa, it occurs In practically every section, but is decidedly more pronounced in some areas than in others. It is worst where the black bee prevails and it frequently occurs in those areas where there is usually a deficiency of spring honeyflow. There may be out-breaks in unusual territory in times of extremely adverse spring weather. Common Name: This disease is usually referred to as foulbrood and the designating term "European" is not often used. It is some- times called "melting" brood. These common names are not entirely sufficient to distinguish it from American foulbrood. Transmission: European foulbrood is an infectious disease of the bee larvae, caused by a germ called Bacillus pluton, and is vrans- rnissable from one colony to another. The chief mode of trans- mission seems to be thru robbing out a diseased colony. There is Fig Movement prior to foulbrood. death in European not so much likeli- hood that it is car- r.'ed by the bee- keeper on his hands and clothing as is the case with American foul- brood, nor does empty equipment seem to spread the disease as readily as is the case with American foul- brood. Diagnostic Char- acters: Any weak colony in the spring may be sus- pected and should be carefully exam- ined for this dis- ease. Its presence or absence cannot be determined by the appearance of the adult bees. The infection of the lar- va probably takes place during the second day of its life, when the in- fected material is given to the larva with its feed by the nurse bees. The action of the disease is very rapid and the larva dies in about two days after the germ has been introduced into its body. Thus most of the larva die before they have completed the fourth day of growth, or before they have left the bottom of the cell, which means that they are still curled. The appearance of a dead larva is very characteristic. Its general color is that of putty; the larva appears to have melted down, and it has lost Its usual shining, glistening and pearly white ap- pearance. After the tissue melts down the tracheal or breathing system of the larva seems to stand out in relief and usually is observed by the beekeepers for the first time. As the larva dries down it develops into a scale, which never has any suggestion of chocolate color as is true in American foulbrood. The scale is loose in the cell so that it can be shaken out by tipping up the comb. The odor of the larva is not particularly characteristic, but is described as "sour". The body contents of the larva are simply "watery" and not "ropy". Some toher organisms, but principally Bucillus alvel, may be found at most any time along with European foulbrbood. These are called sec- ondary invaders and are germs which live upon decaying tissue, after the larvae have died from European foulbrood. Their presence may cause an unusually dark color and there may be a tendency to "ropi- ness"- These secondary germs make a condition midway between those typical of European and of American foulbrood. In such cases it is necessary to use extreme care in making the diagnosis. Seasonal History: This disease seems to be most prevalent in the later spring, particularly when the weather is exceedingly unfavorable, with cold, damp, raw days prevailing. The disease is not commonly observed after weather conditions become settled in the late spring. Occasionally cases are evident thruout the early summer, but these 10 are exceptions to the rule. The disease works very rapidly and a colony may become extinct in ten days. Very often a colony dies be- tween the inspections made by the beekeeper in his regular apiary management. If the disease is not severe it may seriously reduce a colony and sometimes there is a tendency to diagnose the condition as physiological rather than pathological. If the disease is not serious, the colony sometimes survives and may build up slowly with the com- ing of favorable weather in the late spring. European foulbrood is especially severe among the black and hybrid bees. Formerly the Italian race of bees was found to be more resistant and certain strains were evidently more resistant than others. At the present time ap- parently all strains of Italian bees are really resistant. Weak colonies need special attention, especially the entrance should be reduced so that it will be difficult for robb:ng to get started. Treatment: Inasmuch as this disease seems to be more prevalent with the black and hybrid bees, the first step in eliminating European foulbrood is to Italianize all colonies by introducing a queen of a good strain. Wherever colonics are found to be struggling in an effort to rid themselves of the disease, it is advisable to requeen. If the requeen- ing is done in the spring it is well to give a thin feed to further stim- ulate brood rearing by the new queen. Colonies which have been re- queened need special attention and it may be advisable to add a frame of sealed brood from a healthy colony. Since there is not the likeli- hood of transmitting the disease by the interchange of equipment, it is much easier to handle this disease than American foulbrood. Fur- thermore, the bees will readily clean out the scales, and cells are ap- parently made clean enough by the bees so that succeeding larva will rot contract the disease. European foulbrood is no longer feared by beekeepers and its chief control measure is "better beekeeping". This term includes the items of introducing better stock and of giving bet- ter management in the form of protection and ample stores. Frequent examinations are also a part of bet- t e r management and enable the bee- keeper to detect the presence of dis- ease before it gets a start. SACBROOD Early History: Sacbrood has been known for a good many years, but this name has been applied to the dis- ease in only recent years. It was con- fused with a condi- tion called "pick- led" ^'•"1d and was not easily differen- tiated from what is now known as "starved" brood, and "chilled" Fig. 5. Tongue adhering- to roof of cell. brood. 11 Distribution: Sacbrood has occurred quite generally in all localities of the United States and Canada. Its effects have never been serious and consequently it has not received the attention that it really de- serves. It occurs frequently in Iowa, with no partciular regard to lo- cality. It is more commonly associated with European foulbrood than ■with American, but is not so restricted to black bees. Common Name: The name is quite distinctive of the condition In ■which the larvae are found after death. The appearance of the dead larvae has been likened to a sack of wheat tightly tied at the top end. Diagnosis: Sacbrood is infectious, altho the specific organism has not been isolated. It is transmitted by the nurse bees in feeding the young larvae. Sick larvae when used experimentally will produce the disease again in healthy larvae. The larvae die usually after the cell is sealed, but very often the bees have entirely taken away the capping, so it appears that the larvae die before they are capped. The larvae swell until there is no trace of the segments or divisions of the hody. Their color might be termed gray, altho in some instances there is a tendency to a yellowish tint, but hardly enough to be called brown. The tightly tied sack appearance is very characteristic. Another characteristic of this disease is that the skin of the larva becomes al- most parchment like in texture. Therefore, it is very easy to remove the larva from its cell without puncturing the body wall; in fact, if the comb is tipped up and shaken, the larva will drop out. The con- tents of the larva are very watery in consistency. These symptoms are very characteristic and are sufficiently different from either Amer- ican or European foulbrood so that it should be possible to make a diagnosis. Seasonal History: Sacbrood seems to be more common during the spring and especially when unfavorable weather conditions prevail, as is the case with European foulbrood. It is more common with the black and hybrid bees than with the Italian bees, altho the correlation is not as positive as is the case with European foulbrood. It works much more slowly in the colony than European foulbrood and more often a colony will overcome the disease and perhaps regain in strength by the end of the honeyflow. It is seldom found during the summer honeyflow. Treatment: Better management seems to lend itself exceedingly well to control of sacbrood. The first considdration is to re-queen the colony which is affected. This can be accompanied with a stimulative feed and it may be advisable to add a frame or two of sealed brood from some strong colony. Under these conditions a colony will re- cover very' rapidly and the disease will probably not appear again in that colony. MISCELLANEOUS DISEASES Closely associated with American and European fulbrood and Sac- brood are minor disease and physical conditions which may be con- fused in making a diagnosis for a brood disease. Before sacbrood was definitely named, beekeepers frequently referred to a so-called dis- ease, "pickled" brood. With the coming of better methods of beekeep- ing this so-called disease has practically disappeared from the apiary. Occasionally brood will decay in a manner which is not typical of any other brood diseases which have been described. This may be the result of a specific organism, but more often it is a condition rather than a disease. In the majority of cases the term "pickled" brood has been applied to sacbrood and as a specific disease is not of Importance. 12 In Iowa where spring conditions are especially erratic, the bee- keeper may find a condition in the brood nest which is a result of chilling or desertion. A colony may be ready to build up and when a cold spell occurs it is necessary to draw in the cluster so that some of the brood is left to exposure. This brood dies and the beekeeper may be led to feel that a disease is present in the hive and under those conditions the situation will appear more serious than it really is. It is necessary for the beekeeper to keep clearly in mind the typical characteristics of American, European and Sacbrood. ADULT DISEASES The diseases of the adult bee have been considered as unimportant to the beekeeper. There are times, however, when conditions develop within the colony which are contusing and the cause of some little loss. Now there is more need for the beekeeper to understand thoroly adult bee diseases than was true formerly. DYSENTERY Dysentery is not a disease, but more properly a disorder, of adult bees. It has been frequently referred to in beekeeping literature and in a good many instances it has been confused with true diseases of the adult bee. It is the result of an accumulation of undigested mat- ter which accumulates when the bee is not able to fly. Therefore, this condition is most often observed in bees which have been con- fined in the cellar for the winter, or in cellar wintered bees which have been placed out of doors before a rather cold wave in the spring. It Is evident by the so-called spotting of the hives. Colonies seldom die from dysentery, but inasmuch as the adult bees which have suf- fered from this condition soon die, the adult population becomes ma- terially reduced in a short time. Inasmuch as this is most prevalent in the spring, a reduction of the adult population is a serious handicap to the colony. The relief is a flight for the bees and in some instances it may be well to requeen in order that new vigor may be introduced into the hive. Usually it is well to give a stimulative feed in order to TABLE OF DISEASE CHARACTERISTICS American European Sac Cause Bacillus larvae Bacillus pluton Filterable virus Age After capping 3-4 days fully grown Color Brown Gray-yellow Yellow-brown Position Lengthwise Curled on bottom Lengthwise Consistency Ropy SLmy, moist Watery Odor Glue pot Sour None Trachae Not apparent Outstanding Not apparent Skin Disintegrates Tough Parchment Cappings Sunken, perforat- ed, discolored None Open Pupa Tongue adhering to roof of cell None None Scales Adheres, dark Thin gray, loose Brown-tree Casts Worker, mostly All Worker Season Summer and fall Spring Spring-summer Spread Moderate Very rapid Moderate Treatment Shaking Requeen Requeei. 13 encourage brood rearing to the fullest extent. Colonies which are ^iven this treatment recover very rapidly, and often make fine colonies for honey production. PARALYSIS Occasionally a beekeeper will observe adult bees on the alighting board that are trembling. If this condition is in an advanced stage the bees are rather shiny in appearance and the body is quite swollen. The bees seem to be making an effort to fly, but are unable to do so. Then they crawl around on the ground in front of the hive in an ef- fort to get on some high point to make another effort to take wins. Being unable to fly, these bees soon develop dysentery and then the disorder above is outstanding. It seems that paralysis is more pre- valent among black bees than among Italian. The relief measure is to requeen with a good strain of Italian bees and give a stimulative feed. If this condition has seriously depopulated the colony it is advisable to give a frame of hatching brood from a healthy colony. NOSEMA This disease has been confused with the conditions just described and in a good many instances it is quite likely that nosema was the cause of the trouble. It is caused by a specific organism, which is infectious and probably is carried by bees in collecting water for the supply inside the hive. Robbing of diseased colonies is apparently a source in the spread of nosema. It works slowly within the hive and it is quite likely that a good many colonies recover from the disease before it is observed by the beekeeper. The organism which causes nosema attacks the digestive tract of the adult bee, causing a break- ing down of tissues and resultant inability to digest food. In a colony ■which is badly attacked the most evident condition is that of dysentery. Probably the most satisfactory program of relief is to requeen the •colony and give additional brood. This will enable the colony popula- tion to develop more rapidly than the disease will attack and the chances are that a cure can be effected. ISLE-OF-WIGHT This disease does not occur in the United States, as far as the records show at present. However, it may appear and the beekeeper should be alert to detect its presence. This disease has been very severe in Europe and the British Isles. The cause of this disease is a small mite or "chigger", which gets inside of the bee at the breath- ing pores and there attaches itself and reproduces very rapidly. In time the bee is unable to supply itself with the needed oxygen and in- asmuch as it is not able to fly, the dysentery condition readily de- velops. Affected bees in the last stages are inhabited by countless numbers of the mites. Any suspected material should be examined Tery carefully. PESTS Bee Louse. The bee louse pest has recently become of economic im- portance in a few eastern sections of the United States. There are records of its occurrence in the west, but apparently it has not estab- lished itself very extensively. The beekeeper should be on the look- out for it and slaould be familiar with the precautions and treatment It has been quite a serious pest in Europe, where it is necessary for the beekeeper to constantly fight to save colonies from its ravages. 14 The lice attach themselves to the hacks of the bees, usually one louse to each bee, and takes its nourishment from the body of the bee. A large population of the worker bees of a colony may be infested with; the lice thruout the season. The louse is more apparent in weak colonies and seemingly more abundant in poor seasons. This must be considered as relative, since a strong colony will survive a more se- vere attack of the pest than a weak colony, and of course all colonies- are handicapped in gaining strength during unfavorable weather con- ditions. The lice work sometimes on the drones, but not nearly as extensively as on the workers and the queen. Frequent examination of a colony should enable the beekeeper to determine the presence of this pest. At the present time, the control measures are rather in- direct and not entirely satisfactory. When the infestation is slight,. it seems advisable to destroy the adults of the colony. The medical treatments which have been proposed do not seem to be wholly satis- factory. Bee Moths. The moths have been a pest of bees since the' earliest beekeeping times. They are found wherever bees are kept. It is in- teresting that a pest which is so old as the bee moth should persist for centuries without any apparent natural factors of control. There are two kinds of moths, but the greater bee moth is by far the more common than the lesser. Their habits vary and of course their size should enable the beekeeper to determine which species is prevalent. Greater Bee Moth: This is now considered mostly as a pest of poorly kept apiaries. The bee moth is not the cause of any condition in the apiary, for it appears after other facts or mismanagement have occurred. Weak colonies are subject to the attack of this pest and black bees seem to be more susceptible than Italian bees. The pest is considered as a secondary invader of the beehives and consequently the first factor in the control is to have all colonies with Italian bees and as strong as possible. The moth flies entirely at night, generally during the evening twi- light. At this time it is possible for the moth to gain entrance to the hive, especially in a weak colony which is poorly guarded. The eggs- are preferably laid on the comb and when the young larvae hatch they burrow thru the cell wall to the mid-rib of the comb. Here they are quite secure from the attacks of the bees and their work can progress, with little disturbance. Their feeding areas are well protected with a. very tough silken thread, so that when they are once established in a comb it is very difficult for the bees to reach them to fight and destroy them. The larvae, when their growth is completed, may leave the comb and spin their cocoons between the" end bar and the hive body,. or perhaps on the underside of the inner cover. These cocoons are parchment like and serve as a most excellent protection. When the combs become badly infested the bees apparently lose all hope of the fight and in some cases leave the combs to the ravages of the worms. In well managed apiaries the bee moth is able to maintain itself only thru its work on stored combs. It seems to preserve its numbers so that whenever a colony is weakened it is ready prey for the moth. The control measures are first to keep all colonies in strong condi- tion and in numbers and then direct the energy toward protecting the- stored combs. The combs which are not on the beehive should be examined to detect the early work of this pest. The combs can be fumigated and for this purpose carbon bisulphide has been quite satis- factory. This material is effective against all stages of the moth, hut it must be remembered that this material is inflammable and every possible precaution is necessary to keep it away from all possible sources of flre. One should be more careful even than in handling: 15 gasoline. The fumigation should not take place in any residence or outbuildings which carry insurance, for fire which may result from the use of carbon bisulphide will nullify the insurance policy. Carbon bisulphide is not effective in average doses at temperatures below 70 ■degrees, and loses most all of its effectiveness below 50 degrees. Carbon bisulphide is a liquid which evaporates very rapidly and the resulting gas being heavier than air goes to the bottom of the container. All material which is to be fumigated should be stacked in bodies or supers and the bisulphide should be poured on a towel or cloth placed on the top bars of the top body of the stack. A safe rule Is to use one-half ounce of carbon bisulphide for each 10 frame Langstroth hive body. Of course the pile should not be over eight bodies high and as many of the cracks should be eliminated as possible. The lesser bee moth is primarily a pest of stored combs and espe- cially of honey in storage. This moth works primarily on the capp'ngs. All stages of this pest can be controlled by fumigation with carbon bisulphide in the manner just discussed. Ants. There are at least three general classes of ants common to the bee yard. A large black ant is sometimes found between the inner and the outer cover of a colony. They apparently do not molest the bees in any way, but seek these locations in order to take advantage of the escaping heat from the bee hive to assist in developing their young. A small black ant and a small brown ant at times enter the hives in search of food, which is usually honey. These ravages are not severe, but it is well to destroy the ants if they are numerous or likely to prove a nuisance. The best method of destruction is to trace the ants to their nest. The entire population can be destroyed by fumigating with carbon bisulphide. Fumigation should be done late in the evening, when most of the ants are normally in the nest. A sharp stick can be thrust into the center of the nest and the carbon bisulphide poured into this hole. The amount will depend upon the size of the nest, but two or three ounces will be sufficient for the average sized nest. To make the fumigation more effective it is well to place a damp sack over the nest to further direct all of the fumes down into the nest. In handling carbon bisulphide, use the necessary precautions to prevent fire. A relief measure is to construct legs for the hive stand and place these legs in a small tin cup which contains a heavy oil. This pre- vents the ants from gaining entrance to the hive, but of course it does not reduce the supply of ants in the apiary. Mice. Many beekeepers do not seem to appreciate the loss which is caused by mice getting into the hives. The mice cause the greater portion of their damage by getting into the colonies during the fall and spending the winter in the hive for whatever protection may be afforded. When the mice get inside of the hive they make very care- ful preparations to construct an efiicient nest for winter protection, usually in one of the lower back corners of the hive. At this time of the year the bees are not very vigorous in their efforts to repel the invaders. The damage caused by the mice is in comb destruction,, probably to secure feed. If supers are stored out-of-doors the mice may establish themselves in these combs during the summer. Precaution should be taken to pre- vent the mice from getting into the hives during the early fall. For this purpose it is well to insert in the entrance of the colony a piece of hardware cloth, which can be lightly tacked in place during the early fall period. This will prevent the mice from getting into the hive, but will provide ample facilities for the bees to fly. 16 BEE-YARD SANITATION Every practice about the apiary at all times should be such as to prevent robbing. At no time should frames of honey be taken from a colony which may be diseased and given to another colony for food. If there is any possibility of robbing, one should never open a dis- eased colony. It is best to remove diseased colonies from the apiary as soon as discovered. Carelessness is the cause of most of the spread of these diseases. There are some who feel that if a frame does not contain diseased larvae it is safe for use in another colony. Never use combs which have come from a region where foulbrood is known to exist. No beekeeper can expect to eradicate American foulbrood from his apiary who is careless and neglects at any time to remove all possible sources of infected honey or combs. Failure to eradicate may be attributed to: 1. Indifferent manipulation during the treatment. 2. Improper attention to hospital colonies. 3. Failure to remove all infected honey from equipment. 4. Exposure of diseased combs of honey to robber bees. The beekeeper can save much trouble and expense by using every precaution to prevent disease from gaining a foothold in the apiary. Honey should never be fed if there is any question as to whether it came from a disease-free colony. Bees should never be purchased without an inspection certificate. A weak or dead colony should not be robbed out at any time. It may contain disease, but even if it doesn't, robbing is a serious handicap to get started in the yard. The use of old combs and second hand equipment is a dangerous practice. Second hand hives and equipment should be treated in hot lye water before they are used for disease-free colonies. FOULBROOD LAW The law which was passed by the 37th General Assembly of Iowa was amended by the 41st General Assembly. It permits the state apiarist to make inspection of bees and buildings which may harbor equipment suspected of containing disease material. The law provides that directions shall be left for the treatment or destruction of disease material. If such treatment is not completed within ten days the same can be made by the state apiarist and the cost charged and collected as taxes. The law is sufficiently clear that disease can be fought In a territory with an expectation that the disease can be eliminated. Copies of the law may be secured upon request. Those interested in Inspection should communicate with the State Apiarist, Ames, Iowa. (MARCH, 1937 Ontario Department of Agriculture ONTARIO AGRICULTURAL COLLEGE BEE DISEASES E. J. DYCE Professor of Apiculture E. C. MARTIN Demonstrator in Apiculture '~*-T 7T, X a VIEW OF APIARY ONTARIO AGRICULTURAL COLLEGE CONTENTS Page Introduction 3 Examination of Colonies 3 Normal Development of Healthy Brood — 4 American Foulbrood 5 (a) Symptoms 5 (b) Cause 7 (c) Method of Infection 7 (d) Method of Spread 7 (e) Immunity to American Foulbrood 7 (f) Eradication 7 European Foulbrood 11- (a) Symptoms 11 (b) Cause 14 (c) Methods of Spread 14 (d) Immunity to European Foulbrood 14 (e) Eradication 15 Sacbrood 15 (a) Symptoms 15 (b) Cause 18 (c) Spread of Sacbrood 18 (d) Control Measures 18 Comparison of Brood Diseases 17 Fungous Diseases 18 Diseases of Adult Bees 18 (a) Isle of Wight Disease 19 (b) Nosema Disease 19 (c) Dysentery 19 (d) Paralysis 20 (e) Spring Dwindling 20 Sending Samples for Diagnosis 20 Pests of the Apiary 20 (a) Greater Wax Moth 20 (b) Other Moths 22 (c) Skunks :__ 22 (d) Mice 23 (e) Other Pests 23 Permits to Move or Sell Bees 23 Precautions 24 The Bee Disease Act 25 BEE DISEASES INTRODUCTION Diseases of bees are divided into two main classes, namely, brood diseases and those which attack the adult bee. Although adult bee diseases have caused great damage in sections of Europe, so far they have not been serious in any parts of North America. The three important brood diseases of Ontario are American Foul- brood, European Foulrood and Sacbrood. In general these diseases have quite similar characteristics. Whereas European Foulbrood and Sacbrood are easily controlled, there is no cure for American Foulbrood. To avoid serious losses it is essential that beekeepers become sufficiently informed to correctly diagnose all three diseases. Through the destruction of infected colonies American Foulbrood causes an annual loss of about four thousand colonies in Ontario. Besides the loss in bees, honey and equipment, many beekeepers become discour- aged and sell their bees or, which is worse, neglect their colonies, leaving them as a source of infection to other people's bees. Although some parts of the Province have suffered from disease more than others, no area is outside their range. Due to the inspection system in Ontario, disease has been held in check and beekeeping on a commercial scale is made possible. Complete eradication, however, can never be attained until every beekeeper becomes his own inspector. It is the aim of this bulletin to place before the bee- keeper in a clear and simple form the essential facts regarding identi- fication and control of bee diseases» Do not let disease destroy your colonies while waiting for the inspector. Learn symptoms and keep the colonies disease-free at all times. If not sure of your diagnosis, send a smear to the Apiculture Department, as explained later. e;xamination of colonies Control of disease is best effected by giving the colonies two thorough inspections each year. The first examination should be made in the spring at the beginning of the fruit bloom and dandelion flow before the first super is added. If disease is controlled at this time the loss of one or more supers is avoided. In addition to this, the disease does not get a chance to spread to other colonies and cause trouble later on in the season when there is surplus honey in the hives. Any infection picked up by bees robbing diseased colonies during the fall and early winter is usually evident and may be brought under control before it spreads too far. A second thorough examination is advisable during the summer just prior to the removal of the light honey crop, particularly where disease is suspected in the supers. When the bee escapes are being placed on the hives, very little additional effort is required to examine the brood combs. Finding disease at this time allows the beekeeper to keep diseased supers away from clean equipment. Colonies having disease during the fall will likely die before spring and act as a potent source of infection to other colonies. Detection of disease is much easier on a bright day. When examining a frame shake most of the adhering bees into the hive. By standing in such a manner that the sun shines into the base of each cell, little difficulty will be experienced in detecting abnormalities of the brood. A thorough examination in the above manner should always be given before inter- changing frames of brood or honey with another colony. Prevention of robbing from dead and weak colonies in the early spring is one of the most important duties of a beekeeper in the control of disease. All dead colonies should be examined on the first visit to the yard, and diseased colonies destroyed. If this is not possible, dead colonies should be made absolutely bee-tight, or, more preferably, removed from the yard. It is believed that much disease is spread by robbing from dead colonies in the spring. It is impossible to control disease if colonies are kept in box hives Infection cannot be detected unless combs are removable. Box hives are therefore a menace to the industry. The Foulbrood Act demands that all bees in such hives be transferred to movable frame hives. NORMAL DEVELOPMENT OF HEALTHY BROOD There are two types of insect development. In the case of the grass- hopper for example, the egg hatches into a young insect or nymph very similar in appearance to the adult. This is called incomplete metamor- phosis. The second type of development, under which the honey-bee is classed, is called complete metamorphosis. The honey-bee egg develops into a small white grub or larva. This later spins a fine cocoon and goes into the pupal or transition stage from which it emerges a fully developed bee. In order to detect unnatural conditions of the brood the beekeeper must know the life history and the appearance of healthy brood at every stage of its development. A study of the life history chart in conjunction with Figure 1 will give the story of this development. Average Life Cycle of the Honey Bee I^^Se Queen Worker Drone ^gg 3 days 3 days 3 days ^arva 5% days 6 days 6V2 days Pupa 7y, ^ays 12 days 14% days Total 16 days 21 days 24 days The egg of the bee is a small, white cylindrical object about 1110 of an inch long, somewhat larger at one end (future head end) and slightly f."«WH 'f deposited on the base of the cell by the queen and is fastened m place by a secretion. The larva at first very small, grows rapidly, and in a few davs occupies the whole of the base of the cell. The healthy larva beforl bdng sealed lies curled up m the base of the cell and is a glistening pearfy whTt- colour with the segmentation of the body clearly shown T^e or four tL'^u^ll *^" ^^'^^ ^^*"^"y transforms to the puSrstage the cell is ^^.X^^' ""''' ^'''- ^^' ^"^^^ l^r^^ straightens out fn the cell and "a'^and "^' cocoon preparatory to transformation to a pupa Figures th^lS^al^andtu^p^^LlfsTf^eS^^^^ --- ''^" ^^^"^" Fig. I. The honey bee: a, .Egg; b, young larva; c, old larva; d, pupa. Three times natural size. (U.S. Dept. of Agr. Farm flBul- letin 447.) AMERICAN FOULBROOD The term "American" is used simply to differentiate this disease from other brood diseases. It does not imply either origin or location of the disease. (a) Symptoms Appearance of diseased comb. If disease has been present in a colony for some time the comb will assume a mottled or pepper-box appearance. This is caused by open cells containing dead larvae or scales being interspersed with normal brood. This mottled effect is also char- acteristic of other diseases. Condition of cappings. Normal healthy cappings are light brown in colour, becoming slightly darker with age. They are somewhat rounded or convex in shape. If a diseased larva or pupa is present in the cell the capping frequently becomes sunken and dark brown in colour. Many cappings are perforated with one or two small holes by the bees as though they were investigating the tardy emergence of the brood. Time of death. Death occurs from American Foulbrood almost invariably two days before or two days after the transition to the pupal stage at which time the cells are capped. Position in the cell. Larvae lie curled in the cell at the time capping takes place. After the cell is sealed the larva straightens out, lying flat along the bottom of the cell. Three to four days after capping the change to a pupa takes place. It is within two days either before or after this transformation that death occurs. Due to the uniform position in the cell at this time the decaying brood settles in a regular uniform mass, to the bottom of the cell. This characteristic is contrasted to European Foul- brood where the decaying brood is usually twisted in the cell. Colour changes. Healthy larvae are pearly white in colour. Decay- ing larvae dead of American Foulbrood are first a li&ht yellow brown As they settle down in the cell the colour changes gradually to a dark, coffee brown, which is of uniform shade over the entire body surtace. Avvearance of affected pupae. Pupae dead of the disease go through the same process of decay and settling and similar colour changes as the larvae. The tongue of the pupa is nearly always extended towards the top wall of the cell. This is a definite sign of American Foulbrood. Ropiness of American Foulbrood. American Foulbrood destroys the larval or pupal tissue. The body wall soon becomes soft and easily rup- tured. Dead brood passes through several stages of decay, each stage varying in colour, shape and consistency. After about three weeks of decay, until the scale is formed, a characteristic ropiness is exhibited. During this period a toothpick inserted into the mass will draw out a fine gluey thread of decaying matter. (Fig. 2.) The ropiness of American Foulbrood (U.S. Dept. Ag., Far. Bui. 442) Although this ropiness is a definite characteristic of American Foul- brood, it should be remembered that it only occurs at a certain stage of putrefaction. When a suspicious cell is found during inspection, all other symptoms should be carefully observed before inserting a toothpick and destroying the larva or pupa for further inspection. Too many beekeepers have the habit of inserting a toothpick immediately they see a suspicious cell. This destroys other important symptoms. American Foulbrood odour. In the early stages of American Foul- brood no odour is evident. In advanced stages, however, where much brood is dying and the disease has been present for weeks, the odour is quite distinct. The smell is characteristic, but is probably best described as resembling that of heated glue. The value of odour is overlooked by many experienced beekeepers. Even when a small amount of infection is observed a diseased larva may be removed on a match or toothpick and an effort made to detect the characteristic odour. Some people have a very highly developed sense of smell and with such this point is a great help in diagnosis. Formation of a Scale. After four to five weeks the decaying brood dries down to a hard dark scale. American Foulbrood scales are char- acteristically uniform in shape, covering the greater portion of the lower cell wall and extending part way up the back wall. The scale adheres tightly to the cell and cannot usually be removed without tearing the cell wall. 7 (b) Cause of American Foulbrood In 1902 Dr. G. F. White, United States Department of Agriculture, iirst demonstrated that the disease was caused by a bacteria which he named Bacillus larvae. Certain bacteria have the ability to form a protective covering around themselves. In this stage, called spores, they are capable of existing for long periods away from their host. Bacillus larvae is a spore forming bacteria. It will live for years in honey and will often withstand boiling in water for twenty minutes. This spore forming characteristic makes B. larvae very difficult to exterminate. (c) Method of Infection When honey is stored in cells containing scale the spores of American Foulbrood become dispersed through the honey. Larvae fed with this diseased honey become infected and death occurs a few days later. Thus the cycle is maintained. (d) Methods of Spread American Foulbrood is spread chiefly by robbing infected honey from diseased colonies, either dead or alive. Great care should be taken to see that dead or weak colonies are not robbed out in the spring. The entrances of these colonies should be closed or the hives removed to a bee- tight dwelling about the first of April. This precaution is extremely important. It is possible for one diseased colony to infect a whole apiary. Infection may also be carried from one hive to another by drifting of the nurse bees, especially during manipulation of the colony. Great care should be taken in interchanging combs from one hive to another. Stray swarms should be hived on foundation rather than on drawn comb. Then if diseased honey is present in the crops of the swarming bees it is used up in drawing out the foundation, rather than being stored and later fed to the larvae. Bee trees are often given too much credit for the spread of disease. Although colonies in trees may spread American Foulbrood, such swarms which become weakened and die from disease do not long remain a menace. They are soon cleaned up by wax moths, ants and other insects or animals. (e) Immunity to American Foulbrood Although workers are most commonly infected, drones and queens are also susceptible to the disease. From the results of experimental inoculations and beekeepers' experience it appears that no race of bees possess any marked immunity to the disease. (f ) Eradication of American Foulbrood The old shaking treatment, whereby the bees of a diseased colony are shaken on to foundation and thus saved can no longer be advocated. In fact, it has been found that the treatment in general tends to spread disease rather than eradicate it. When combs are shaken there is a tendency for young nurse bees, not having marked their location, to _ fly into nearby hives carrying diseased honey with them. Another criticism of the treating system aside from general spread of disease at the time of treatment is that diseased combs are often stored for some time before Plate 1. American Foulbrood death occurring in the larval stage: 1. Healthy capped cell; 2, 3. Capped cells containing dead larvae; 4, Healthy larva; 5, 6, 7, 8. Progressive decay; 9, 10. dry scales. — (Reproduced from U.S.D.A. Bulletin 809.) Plate 2. American Foulbrood, death occurring in the pupal stage: 1. Healthy pupa; 2, 3, 4, 5, 6, 7. Progressive decay of dead pupae; 8, 9. Dry scales. — ^Reproduced from U,S.D.A. Bul- letin 809.) 10 being rendered. This is very dangerous as there is always a grave danger of infection by bees robbing stored combs. The treating plan proved to be false economy. It is cheaper to kill diseased bees and make increase from healthy colonies. Burning diseased colonies. The only known method at present by which American Foulbrood may be completely stamped out is by burning the bees and combs and sterilizing other parts of the hive by fire. Disease has been entirely cleaned up in restricted areas by this plan and there is every reason to believe it could be completely eradicated from the Province. Immediately the disease is found it should be destroyed. Delay is generally costly. Considerable work has been done in an effort to find some drug or chemical that would effectively cure a colony of American Foulbrood. So far there has been no success along this line. Steps in Eradicating American Foulbrood 1. Inspect the colonies giving each frame a thorough examination. If disease is found, mark the colonies carefully so there will be no possible danger of overlooking them later. 2. Dig a hole in the ground large enough to accommodate all the combs. Unless the hole is large diseased honey will be spilled around the surface of the soil and spread disease. Do not dig the hole too near a tree or the fire will wilt the leaves and injure the tree. Allow for the wind and be sure the fire is situated where there is no danger of spreading. Place some rough chunks of wood in the hole to ensure a good draft and sufficient fuel to thoroughly consume all diseased material. 3. Kill the bees. In the past gasoline was used for this purpose but it is not at satisfactory as cyanogas, which is a commercial preparation of cyanide in powder form. When gasoline is used the bees fly from the hive unless the entrance is closed. Cyanogas kills the bees without their realizing any danger. Field bees will also fly into the hive. When using cyanogas sprinkle a dessert-spoonful on top of the frames and a little In the entrance to catch the incoming bees. Five minutes is long enough to leave the colony after adding the cyanogas, otherwise many of the bees recover as the powder becomes spent and the gas drifts from the hive. Great care should be taken when using this poison as a few good breath.s might easily prove fatal. There is no necessity to wait until evening to kill the colonies. 4. Light the fire. It is advisable to use coal oil and get a good strong fiame so the diseased material may be disposed of as quickly as possible. 5. Carry the hives complete to the fire and throw on the combs. Be sure no dead bees drop from the entrance as most of them exude a drop of honey which sticks to their tongues and may be diseased. If the combs are heavy with honey keep the fire well bolstered up with chunks of wood. 6. Scrape thoroughly and disinfect by scorching all boxes, bottom boards and covers. Covers and bottom boards may be sterilized by 11 holding them over the fire for a few seconds, on a fork or long stick. Boxes may also be treated in this manner or else piled one on top of the other sprinkled inside with coal oil or gasoline and ignited. The flame may be extinguished by placing a cover on the top box, thus excluding the air. 7. All dead bees and other debris that might carry infection should be scraped into the fire. When everything has burned down to an ash, fill the hole in thoroughly. 8. Hive tools should be disinfected with fire and the hands washed with soap and water before examining other colonies. Disease in Super Combs Chemical treatments such as the use of formalin, chlorine, etc., which where once recommended for diseased super combs have been definitely proven unsatisfactory. If a beekeeper is suspicious of his super combs the best practice is to render them into wax and have it made into foundation. The actual cost of this procedure is not great and it is considered sound economy by many successful beekeepers. EUROPEAN FOULBROOD Less than forty years ago this disease caused the beekeeping industry considerable anxiety. It spread with remarkable rapidity, wiping out whole apiaries. Today beekeepers have learned that it can be easily controlled by keeping the colonies strong and using resistant Italian stock. It is no longer a menace to the industry. (a) Symptoms The appearance of larvae dead of European Foulbrood varies consid- erably and the symptoms are more variable than those of American Foulbrood. Always keep in mind the appearance of healthy brood when inspecting for indications of disease. Appearance of comb. The presence of many uncapped cells, contain- ing disease larvae in the early stage of development, gives the comb a spotted irregular appearance. Should the majority of larvae die at a later stage of development after the cells are sealed, the cappings are sunken, perforated and decidedly greasy in appearance. Time of death. All larvae die before the transformation to the pupal period. About ninety percent die one or two days before the cell is capped. Probably ten percent die the first or second day after capping, i.e. when the larva is beginning to straighten out in the cell preparatory to its transformation to the pupa. Occasionally an outbreak of European Foulbrood takes place where colony after colony contains larvae most of which are attacked at this later stage. This type is often confused with American Foulbrood. The variability displayed in time of death makes diagnosis more difficult. In typical European Foulbrood, however, the cell is uncapped. 1^ Plate 3. Characteristic European Faulbrood, death occuring in the early larval stage: 1. Healthy curled larva; 2 to 10 Various cef^Q °L^T^''.^' ^; ■^'^r^ ^''''^^^^' S' 7' Larva twisted in ceU; 9, 10. Dried out scale. —(Reproduced from U.S.D.A. Bulletin £10.) 13 ' i ) '- Plate 4. European Foiillbrood, death occurring in the late larval stage: 1, 2. Capped cells containing larvae dead of European Foulbrood; 4. Dead larvae showin- part removed by the bees; 3, 5, 6, 7. Various stages of decay; 8, 9. Dried out scales.— (iReproduced from U-S.O.A. Bulletin 810.) 14 Position in the cell. Larvae exhibit marked variation in their Dosition according to the age at which they die. Young arvae dying at fhe characteristic^stage while still curled generally ^^.^^^m around as though in pain assuming unnatural positions m the cell. Those aryae that die after capping are larger and lie more uniformly extended in the cell. Colour changes. The pearly white colour of healthy larvae changes to greyish yellow. As putrefaction continues the colour deepens to a dark greyish brown mass. Consistency of dead larva. After approximately three weeks of decay the larva becomes a sticky, somewhat granular mass. The granular appearance at this stage is contrasted to the smooth glue-like appearance of American Foulbrood. Tracheae visible. The tracheae are glistening, silvery air tubes situated below the skin. In many larvae dead of the disease these tracheae may be plainly seen and remain visible during the complete process of putrefaction. The presence of tracheae is an important symp- tom to diiferentiate infection in the advanced larval stage from American Foulbrood. European Foulbrood odour. In advanced cases, especially where many larvae die in the capped over stage, there is a very offensive odour. It is described as resembling that of rotten fish. Scale formation. The dried larval remains are less brittle and more rubber-like than American Foulbrood scales. They are shrivelled, brown in colour and can easily be removed from the cells. The fact that brood dead of European Foulbrood can be removed by the bees makes it possible for strong colonies to clean up the disease. (b) Cause of European Foulbrood Bacillus pluton, a non spore former, is considered to be the organism which actually causes the death of the larva. Invariably, however, there are secondary organisms present, the chief of which is Bacillus alvei. When a laboratory diagnosis is made the presence of Bacillus alvei indicates European Foulbrood. (c) Methods of Spread The organism causing European Foulbrood does not form spores so is unable to live over winter in honey. It is carried over in pieces of dead larvae. From this small beginning in the spring it can spread very rapidly under favourable conditions. If nectar is stored in contaminated cells and fed to larvae they contract the disease. Further spread may be caused by robbing, interchanging combs or drifting nurse bees. (d) Immunity to European Foulbrood Workers, drones and queens are all susceptible to European Foul- brood. Italian bees, due to their vigorous house cleaning habits, stop the spread of disease and eventually eradicate it. Some strains are far more efficient in cleaning out dead larvae than others. Black bees are very susceptible to the disease. 15 (e) Eradication European Foulbrood has not caused very serious trouble since beekeepers have learned the present preventive measures. Most definibe progress in its control has been made through Italianizing all colonies and breeding queens from resistant stock. It must be remembered that European Foulbrood is a disease of weak colonies. The introduction of an Italian queen to a very weak colony is useless if European Foulbrood is present. Weak colonies should be united to stronger ones before requeening. If resistant Italian queens are used and the best methods of beekeeping, which ensure strong colonies, are followed there will be little trouble with European Foulbrood. A colony at the beginning of the honey flow should be strong enough to have eight full combs of Langstroth size filled with brood. Proper wintering of bees is a matter of highest importance in regions where European Foulbrood is found. As very little infection is carried over the winter the first brood of the year usually escapes with little loss. For this reason it is essential to have as much brood as possible get away to a good start early in the spring. The emerging bees are then able to ward off the disease throughout the season. Where the disease has become well established, it is sometimes diflScult for the bees to make headway in cleaning it up. Removal of the queen for a few days causes a period in which no brood is being fed. This gives the bees a chance to make more rapid progress. As soon as the dead larvae are removed, the queen is returned or better still the colony is given a young Italian queen. How the disease spreads is not thoroughly understood. Honey has a definite devitalizing effect upon the organism so is not a serious carrier. IL is never necessary to destroy or disinfect combs, brood, or honey from European Foulbrood colonies. Normally requeening and strengthening the colony will satisfactorily control the disease. SACBROOD Sacbrood is an infectious disease of the brood of bees. Although it is not particularly malignant and rarely, if ever, causes the death of a colony, it is responsible for the loss of much brood. Where the disease is advanced the death of many worker larvae results in the weakening of the colony. Brood rearing space may also be considerably reduced by the presence of dead larvae in the cells. The disease has never proven very serious in Ontario but it is essential that beekeepers be able to differentiate between it and other brood diseases, especially American Foulbrood. (a) Symptoms of Sacbrood Character of comb and cappings. As in other brood diseases the presence of affected brood, interspersed with healthy brood, gives an irregular appearance to the comb. Larvae die after the cells are cappfed. 16 Plate 5. Sac'brood: 1, 2. Healthy brood at the age at which it dies of Sacbrood; 3, 4. Brood recently dead of disease; 5, 6, 7, 8, 9, 10. Brood in various stages of deca'<'. — (Reproduced from U.S.D.A. Bulletin 431.) 17 Cappings Time of Death Position in the Cell Colour Changes Appearance of Pupa Consistency Tracheae Odour Scale COMPARISON OF BROOD DISEASES A. F. B. Many sunken, dark and perforated. During prenupal stage or within two days after transformation to pupa. Straightened out, regular and uniform. Occupies most of lower cell wall. Posterior, or hind end, extending up back wall of cell. From light yellow- brown to dark coffee- brown. Uniform throughout. Same process of decay as larva, tongue extended upwards. Soft mass. Exhibits ropiness at certain stage. Not visible. Odour of heated glue in advanced stages. Hard, dark, uniform scale. Adheres tightly to cell. E. F. B. Usually none. When larvae in advanced stages of development, capping, sunken, perforated and greasy. Before change to pupa. Generally quite early and before capping. Advanced larval stages often cauped. 90% of cases curled in the cell and in unnat- ural positions. 10% in advanced larval stages fairly regular and extended in the cells. Prom greyish-yellow to dark greyish brown. Not uniform. Sacbrood From a light yellowish- grey to dark brown. (Head end darker. Nione die. Somewhat stioky and granular. 'Often plainly visible. Fishy odour in advanced stages. Ruiblber-like, irregular. Black, roughened shrivelled, dark brown curled up at anterior scale. lEasilv removed, or head end. Many uncapped; some perforated. Before change to pupa. After capping. Uniform shape and position in cell with head end curled up. None die. Bodv wall tough, contents watery. Not visible. No odour. A considerable proportion of the cappings are uncapped by the bees, a rule the proportion of brood affected is not large. As Time of death. Affected larvae die after the cells are capped but before the change to the pupal stage is completed. The majority die during the two days prepupal period, i.e. within the two days preceding the transformation. Many uncapped cells are generally observed on diseased combs but they are uncapped by the bees after the death of the larvae. Position in the cell. Dead larvae are extended lengthwise along the floor of the cell. The position is similar to that of larvae affected with American Foulbrood. Sacbrood, however, can be determined by the appearance of the head which is dark, somewhat shrivelled and turns up towards the roof of the cell. Colour changes. Soon after death the larval remains are slightly 18 greyish white in colour. This greyish colour turns to a greyish brown tint which deepens as the process of decay continues. Throughout this period of decay the head end appears somewhat shrunken and much darker than the more posterior, or hind, portion of the body. Consistency of dead larva. The body wall of a larva dead of Sacbrood becomes toughened and may be easily removed mtact from the cell. When removed from the cell the larva is sac-like in appearance. The contents of the sac are watery, containing many fine brown granules. Odour. Sacbrood has no distinctive odour. Scale formation. Scales formed from sacbrood are greyish black, roughened and generally curl up at the anterior or head ends. Scales are not common as the bees generally remove the dead larvae before the scale is formed. When present they are loose in the cell. (b) Cause op Sacbrood No organism has been found present in dead larvae which can be demonstrated to cause the disease. It has therefore been concluded that the disease is caused by a virus which will pass through the finest of filters. A colony may be inoculated with the disease by feeding syrup or honey containing the virus from dead larvae. (c) Spread of Sacbrood Sacbrood virus is readily destroyed. Larvae dead of Sacbrood cease to be infectious after one month. How the disease winters over is not known. Colonies infected in the spring generally recover during the honey flow. The weakening effect of the disease during the spring stays with the colony throughout the honey flow. (d) Control Measures Vigorous colonies rarely suffer to any extent from Sacbrood. Re- queening and strengthening will generally clean up any infection that may occur. It is never necessary to destroy any part of a hive infected with Sacbrood. FUNGOUS DISEASES Fungous diseases, although never particularly serious, cause consid- erable damage in the aggregate loss of bees. There are many types of fungi affecting bees both as brood and adults. Conditions such as the so-called stone brood and chalk brood are typical of the work of fungi. The dead remains become dry and mummified. Molded combs and equip- ment and mouldy fruits, etc., are believed to cause the infection. DISEASES OF ADULT BEES To date there has been no serious trouble from adult bee diseases in any part of Canada or the United States. In England and other parts of Europe they have caused great loss. It is necessary that we know some- thing of the symptoms of these diseases and be ready to stamp out any infestation that might occur. 19 (a) Isle of Wight Disease This disease has been a very serious source of loss to British beekeep- ers. It is caused by a minute mite which crawls into the bee's spiracles choking off its supply of air and possibly secreting a toxine which paralyzes the wing or flight muscles. Most adult bee diseases have quite similar symptoms, making diagnosis from external characteristics diffi- cult. The following is taken from leaflet No. 253, issued by the Board of Agriculture and Fisheries, 4 Whitehall Place, London, England: Sijinptoms: 1. The first symptom noticed is a disinclination to work. They fly about aimlessly and do not gather stores. 2. Later they lose their powers of flight and are unable to travel more than a few yards without alighting. 3. As the disease progresses, the bees are unable to fly more than a few feet, when they drop and crawl. They may be seen crawling up grass stems or other upright objects, but they soon fall down and die. Towards night some may be seen gathered in groups, but they usually die before morning. 4. The abdomen is often swollen. 5. The wings often appear disconnected. Sometimes the legs seem affected, and the bees stagger in their attempt to walk. 6. Finally the whole colony of workers is found clustered in front of the hive, except a few which are found crowded around the queen. 7. The queen and the brood are not attacked, though "chilled brood" often appears subsequently, owing to there being insufficient bees to keep the hive warm. (b) NosEMA Disease This disease although fairly widespread is mild in character. It is caused by a protozoan or one celled parasite, Nosema apis, which infests the alimentary canal of adult bees. Bees can be infested with Nosema spores without showing any marked symptoms of disease, and apparently with little effect on their ability to carry on. Death may result from infection and in the case of excessive infection bring about a weakening effect on the colony through the shortening of the life of the individuals. Bees are often observed crawling around outside the hive or climbing up blades of grass. Requeening and strengthening by giving additional capped brood usually brings relief. Cases of this disease have been known in Canada. (c) Dysentery Dysentery is more properly a disorder than a disease of adult bees. Bees are only able to void faeces while in flight. During the winter waste matter accumulates in the lower intestine. If the food is of good quality and the bees are not conflned to the hives for too long a period, they will be healthy in the spring. If the food contains considerable indigestible material and the bees are unable to fly they often die in great 20 numbers. In cases of advanced dysentery faeces are voided within the hive and the disorder becomes evident by the resultant spotting. Material reduction in the adult population is a serious handicap during the spring building-up period. Dysentery can be prevented to ;i great extent by proper precautionary measures. Bees wintered on good honey or sugar syrup are rarely affected. Poor food, such as honey-dew or honey of high water content, nearly always produces the disorder. Proper winter protection and freedom from disturbances during winter are also factors in the control of dysentery. (d) Paralysis Very little is known about bee paralysis. It is possible that there are several conditions which result in the so-called paralysis. At any rate beekeepers frequently report that they have observed worker bees crawl- ing in front of the hive with their abdomens trembling. They keep crawling up the side of the hive and up blades of grass and tumbling to the ground. Occasionally individual colonies become rapidly depleted in bees. There is a possibility that the disease may be caused by certaiji foods. (e) Spring Dwindling Spring dwindling is the term used to describe the condition when adult bees in a colony die off more rapidly than they are replaced by emerging brood. The condition is brought about by poor wintering and by the colony going into winter with too large a percentage of old, worn- out bees. To prevent this colonies should be supplied with vigorous queens that will continue brood rearing as late as possible in the fall. SENDING SAMPLES FOR DIAGNOSIS Brood is frequently found dead in a colony from causes other than infectious disease. We find chilled brood, starved brood and overheated orood. Generally the appearances are characteristic but at times the symptoms may be quite similar to one of the brood diseases. If in doubt a sample of comb, or more preferably a smear consisting of the diseased iarya or pupa, folded in a piece of waxed paper, should be sent to the Apiculture Department, Ontario Agricultural College, for examination. Also If a beekeeper notices adult bees showing symptoms of disease he should send a good number to the Department for examination. Bees that have been dead for for some time are not satisfactory for examination. not bfcrushe™ ^""^ ""^ ^°"^^ "^"""^^ ^^ ^""^^^"^ ^"'^ *^^ ^^"'^ «^°"^^ PESTS OF THE APIARY (a) The Greater or Common Wax Moth This is a pest of weak colonies and of stored combs It is most Th7S marhT,?r '"• ^^l\'"'^ "^^^^^ ^y almost entirely at nfght cracks within thpw" T^f' ''1^^^^ '"P^^« «r i*^ corners Snd cracKs withm the hive. The tmy larvae emerge within a few davs and combs fh/vT^^^'^T 'T ^''^'"'^ through^out th^ hive In brood combs they burrow along the midrib, out of reach of the bees, sphming a 21 tough cocoon as they travel. These larvae feed chiefly on pollen, pupal cases and other impurities in the comb. It is thought that very little, if any, wax is digested. When confined to wax and honey as in comb honey infestations, the larvae are unable to develop into the adult stage. Adult moths do no damage to comb. Combs left exposed act as breeding places for this troublesome pest, and the beekeepers should be careful to keep all combs out of reach of the moth. When weak colonies are attacked the beekeeper should remove as many of the bee moth larvae as possible as well as the webbing and cocoons. The colony should then be united with a strong colony. Italian bees are more resistant than black bees. Strong colonies are rarely infested. Weak or dead colonies in box hives form excellent breeding grounds for moths. Various disinfectants can be recommended for the control of wax moth in stored combs : Sulphur. The amount recommended is 3 pounds per 1000 cu. ft. of storage space, burned for 24 hours in a room made as air-tight as possible. As the sulphur dioxide fumes given off do not destroy the eggs it is necessary to fumigate again two to three weeks later. Its use incurs considerable fire hazard. Fire Insurance under the Ontario Beekeepers' Association is not available to those who disinfect with sulphur. It is most conveniently used in the form of flowers of sulphur. To use, obtain a large flat pan and place some water in it. Stand the pan in which the sulphur is to burn inside this on two bricks. Place live coals in this pan and put the flowers of sulphur on top. Supers are criss-crossed in piles. Carbon bisulphide. Is a yellowish oily liquid which gives off a very disagreeable odour. It may be used quite effectively, although the gas does not kill the eggs, and as is the case with sulphur it is necessary to give a second treatment two to three weeks later. The method used is to place two tablespoonfuls or one ounce of the liquid in a small dish and place this on top of a stack of five supers. For best results, the cracks between the supers should be closed by means of gum paper, or they may be effectively sealed by moistening strips of newspaper and sticking them around the supers. The stack should remain sealed at least 12 hours. At low temperatures this evaporation is very slow, so it is necessary to have the room fairly warm. Every precaution should be taken when using this disinfectant, as the gas given off is highly inflammable. When a dish of carbon bisulphide is used on the top super it is necessary to close off the pile by means of an empty super and lid. Cyanogas. Is a commercial cyanide preparation which is extremely poisonous to humans and animals. Great care should be taken when using it. The gas given off is almost the same weight as air. Cracks in the pile of supers should be closed up and about one tablespoonful of the powder may be placed on a piece of paper somewhere in a pile of five 22 supers. Be careful not to inhale any of the fumes. It is advisable to do tne fumigation outside or in a well ventilated room. Do not tear the supers down for twelve hours. Do not use cyanogas in a dwelling house- it is extremely poisonous. Paradichlorobenzene (P.D.B.) Is one of the most satisfactory in- secticides that can be recommended for wax moth control. It is a white crystalline material, with a not unpleasant odour. It is non-injurious to humans at ordinary concentration. It is non-inflammable and very easy to handle. The gas given off is heavier than air, so a handful of crystals is added on a piece of paper at the top of a stack of five supers. More crystals can be added occasionally throughout the storage period. It is most effective at around 70 degrees F. Storing Combs. Where much trouble is experienced from mice and wax moth, it is advisable to store the supers of comb in stacks of five with a metal lid or queen excluder at the top and bottom of each pile. One of the various disinfectants should be used and cracks sealed up as far as possible. The most suitable disinfectant recommended for general use is Paradichlorobenzene (P.D.B.) . When disinfecting supers, best results are obtained if the stacks are not made more than five supers high. Where the danger of wax moth is not particularly great beekeepers stack the supers up with a sheet of newspaper between each super. Printers' ink apparently acts as a repellant to the wax moth. Comb Honey. Wax moth larvae can very soon spoil a season's crop of comb honey. Moths will lay their eggs in the cracks between the supers; on hatching the young worms crawl to the comb honey and destroy the wax capping. If trouble with wax moth is expected it is advisable to remove the honey from the colonies as soon as possible. Place 8 to 10 shallow frame supers of honey one on top of the other and use similar fumigation methods as used for stored combs. P.D.B. is particularly recommended for use with comb honey. Some beekeepers use sulphur for this purpose, as the fumes tend to bleach wax, thus making the cappings whiter. (b) Other Moths The larvae of several moths destroy comb. The Mediterranean Flour Moth and Indian Meal Moth larvae sometimes damage comb when burrowmg for pollen. The larvae spin silken threads similar to those of the Common Wax Worm, but do not cause as much damage to the comb. The Lesser Wax Worm does damage similar to the Common Wax Worm, except that the webs are finer and more generally on the surface =r^ uf "^ ^^'^ '^ "°* ^''. important pest in Canada, but causes con- siderable damage m warm climates. These moths can be controlled by fumigation in the same mannpr a: S ^^SP^P^^MM ^^^^^^Qmi^^b ^^^M^||BMIj I^^E^^^^^^^^ w^p^ *^^^^i^^^SHB^^^S^^^^!§tKirffMf^^^S&s^ ^^^^iP^*^^ ■ ''''^^^^i^^iflH^B|EH|HHH wyw%xf%i^f--^' ipJ'cC:^ %MM:;0i ^^Si 3^wi ^^M ^MHii^^ P^HmH^^W ^"^^Sh lyiffllj^ PiGunE 1. — Webs and tunnels made by larvae of the wax moth in a comb. extracting, and the expense of storage room and treatment for such combs, many beekeepers store supers of these empty combs on the colonies during the winter. This gives added room for the bees to occupy during a warm spell, and a sudden change in temperature may chill or even kill them before they can return to the cluster. It also gives an opportunity for the dissipation of colony heat and thereby increases the quantity of food consumed by the bees, and, during long periods of cold or inclement weather, weak colonies or colonies short of stores may starve. In many such cases of starva- tion the wax moth destroys the combs before the beekeeper becomes aware of the death of the colony. THE WAX MOTH AND ITS CONTROL 6 In any study of the economic importance of an insect, not only the loss but the benefit from the insect must be weighed. The wax moth is not an unmitigated evil. In the first place, the destruction of combs by the wax moth has not only tended to prevent the keep- ing of bees in box hives but has also tended to improve general bee- keeping practices. The wax moth has also been an ally of the bee- keeper by helping to destroy combs in bee trees or other inaccessible places, which might harbor germs of some of the brood diseases. Since bees in box hives cannot be examined, requeened, or other- wise controlled, the colonies are likely to become weak, and under such conditions an invasion of the wax moth destroys the colony and the combs. Many States have laws to prevent the keeping of bees in box hives, and the wax moth has furthered the aim of this legislation by destroying such colonies in its spread. So thorough is the destruction of colony and combs in most box hives that, unless there are large stores of honey in the hives, bees are no longer attracted to them. Particularly in the Southern States, where the honey flow is slow and extends over a long period, it has become a practice to give more super room at the beginning of the flow than the bees actually need. This is not the best practice if a large crop of honey is desired, and the destruction of such unprotected combs by the wax moth has been of direct benefit to beekeepers in forcing a change of method. The destruction by wax moth larvae of combs in bee trees is prob- ably a great aid in preventing the spread of bee disease through the robbing of honey by other bees and, in those areas where queen breeders and package shippers are located, the destruction of stray colonies has also been of real value. Since the germs of American foulbrood have been found in the excrement of wax moth larvae there is a theoretical possibility that the disease might be spread by this means, but actually there is no evidence to warrant pinning additional guilt on this pest. The benefits of the wax moth are small, however, when compared with the losses of entire colonies and of stored combs, or the extra care and manipulation necessary to combat the insect. HISTORY AND DISTRIBUTION The earliest works on beekeeping contain references to the wax moth. Aristotle (384-322 B. C), Virgil (70-19 B. 0.), and Colu- mella (middle of the first century, A. D.), all mention the wax moth as an enemy of the honeybee. The range of foods that can be eaten by the larvae of the wax moth would suggest that it might at one time have had other foods than those obtainable in the hive, but at present wax comb in some form is practically its only food. F. B. Paddock, who has made a study of the present-day distribu- tion of the wax moth, was unable to determine the date of its intro- duction into the United States. From his distribution records some interesting inferences may be drawn: (1) The wax moth has been spread by man more than by the natural activity of the moth. The introduction of the moth into Sweden with beehives from Germany prior to 1750 and its introduction into Australia, New Zealand, and other island regions all point to the conclusion that the wax moth 4 CIECULAK 3 8 6, U. S. DEPABTMENT OF AGEICTJLTUEE must have been aided in its distribution by man and by poor bee- keeping methods. (2) The insect finds its most favorable conditions in the Temperate Zone. According to Paddock, the wax moth is present in Ontario, Canada, but has been unable to establish itself in Manitoba and British Columbia. The high altitudes of the Rocky Mountains are also free, but the wax moth can be found almost any- where else in the United States where there are bees. In the Southern States the wax moth does damage practically throughout the year, with the possible exception of December, Jan- uary, and February; and during mild winters wax moths may ap- pear even in January. It is probable that colonies are infested, at least with eggs, throughout the whole season of bee activity and that only in active colonies is wholesalei damage prevented. In supers and hive bodies brought from the apiary and stored, larvae of all stages will be found, ordinarily within a week, unless the combs are treated. Under storage conditions, the lengths of the egg, larval, and pupal stages vary considerably, and the number of broods per year is largely determined by temperature and humidity. Distribution, under such conditions, is rapid because of the movement of combs and bee equipment, even without the active flight and dispersion of the adult moths. LIFE HISTORY THE EGG The egg of the wax moth is small, white, somewhat elliptical, and rather inconspicuous (fig. 2). It measures about one fifty-fourth of an inch in greatest length and about one-sixtieth of an inch in great- est width. The size and shape vary somewhat, depending on the number of eggs laid in one spot and the character of the site in which they are laid. At 75° to 80° F. the eggs hatch in from 5 to 8 days, but with low temperatures (50° to 60°) the period may extend to 35 days. Under apiary conditions the incubation period is probably almost entirely dependent on temperature. The eggs of the wax moth are probably laid most frequently in the cracks between hive parts; that is, between supers, between hive body and bottom board, or between the super and cover. Egg masses have been found in cracks between the inner cover and top super of the hive, where they had beeji deposited by the female, apparently from the outside of the hive. Eggs are also laid inside the hive in more or less unprotected places. Under controlled conditions, when females were allowed access to combs, the eggs were found on the comb (fig. 2) along the edges of the frames and almost always in the portions of the hive farthest from the light. Egg masses in the hive are difficult to see and may often be overlooked. THE LARVA The young larvae, upon hatching, are very active and do not look like the familiar wax worms. Beekeepers have called them wood lice and have not connected the appearance of these forms with the dam- age from the worms, which they noticed later. They are often seen upon the inner covers of hives and in the cracks between supers and THE WAX MOTH AND ITS CONTROL 5 hive parts. They are less often observed within the hive, especially those with strong colonies, partly because they are very small and vei-y active, and partly because they resemble the wax in color. The young larviic attempt to burrow into the wax almost imme- diately after emergence from the egg. The first burrows are often incomplete and may be mere roughenings of the surface of the wax. After the first day, however, they make small tunnels between the cells and toward the midrib of the comb, in which the typical silken strands of the web may be found. The growth of the larvae depends upon several factors, of which the quantity and quality of food and the temperature are most FIG0EE 2. — Efigs of the wax moth laid on a comb. Greatly enlarged. important. The length of the larval period, from the time of the hatching of the egg until pupation, has been found to range from 28 days to 4 months or even as long as 140 days or nearly 5 months. During this period the large larvae have grown from about one twenty-fifth of an inch to seven-eighths of an inch in length. The food of the larvae is not confined to beeswax, and it is even probable that little pure wax is digested but rather that the larvae derive most of their nourishment from the impurities in the wax. Foundation, especially in frames, is seldom attacked and then usually only by the small larvae. In some cases newly emerged larvae have been seen chewing, or attempting to chew, other larvae which had been injured. The larvae prefer the darker brood combs to the white extracting combs. In the brood combs the larvae confine their work mostly to the midrib and bases of the cells, and combs are often found with perhaps the outer one-fourth of the length of the cells untouched and the central portion, including the midrib, completely destroyed and replaced with a mass of web and refuse. Under such conditions 6 CIRCULAft 3 8 6, XJ. S. DJii'AllTMJiJSrT OF AGRICUL.XUKJ!; the cells containing pollen are mostly avoided, although cells con- taining honey may be riddled. It is known, however, that larvae will eat pollen and develop on it. Wax moth larvae sometimes chew off the cappings of the cells containing sealed brood, and, while the bees may repair some of the damage, many cells will be left only partially closed. Although larvae can develop on foundation, the mortality of such larvae is high, and the developmental period of those which survive ip much longer than that of normally fed larvae, and the resulting adults are small and almost white. It is almost certain that damage reported by beekeepers in Louisiana as caused by the lesser wax moth {Achroia grisella Fab.) is caused by such poorly fed larvae of Galleria meUoneUa, since no specimens of the true lesser wax moth were observed during the author's studies. When the larvae are forced to exist on the lighter comb and the outer portions of the cells which have been left untouched by the previous broods, the damage done by them, such as the webbing and external feeding, and their later appearance greatly resemble the work and appearance of the lesser wax moth. The optimum temperature for the development of the larvae is between 85° and 95° F., about that normally found in a beehive during the active season. At lower temperatures development is slower, but, unless the temperature falls below 60°, no other influence on the larva has been noted. At temperatures of 40° to 45° the larvae seem to become dormant, and no feeding or growth takes place. THE PBHPUPA Before pupation the full-grown larvae spin a dense, tough, silken cocoon. IJsually this cocoon is firmly attached to the side of the hive, to the frame, or other solid support, but in some cases the cocoons are found in the mass of tunnels and refuse of the wax of the frames or on the bottom of the hive (fig. 3). In many cases a hollow is chewed out of the wood of the hive or frame, and the cocoon is placed in this for added protection. Frames may be found in which holes have been bored completely through the end or top bars, and the cocoon and pupal case will be found inside these holes. This habit of the wax worm is responsible for a considerable part of the damage caused by the insect, since in heavily infested colonies not only the wax but also the frames are destroyed. In such cases particles of the wood borings are incorporated in the cocoon, which is then well disguised. The fully grown larvae migrate to considerable distances before the cocoons are spun, and pupal cases may be found beneath the hive and even on the more protected parts of the hive stand. THE PUPA Within the cocoon the larva changes to the pupa. The duration of the pupal stage within the cocoon ranges from 8 to 62 days, depending on temperature. As with many other insects, the pupal period allows the wax worm to pass through the fall and winter protected against climatic influence to a large extent. In the South, especially during warm winters, the adults may emerge at any time during the winter. THJi WAX MOTH AND ITS CONTKOL THE ADULT The adult wax moths are about three-fourths of an inch in length and have a wing spread of about 1 to I14 inches in well-developed specimens. _ They are commonly seen in the resting position with their grayish-brown wings folded, rooflike, closely about them (fig. 4, A and B). The moths are not easily disturbed, but when molested they run rapidly before they take wing. The males are slightly smaller than the females and may be distinguished from them by the shapo of the outer margin of the fore wmg, which is smooth in the female but roundly notched in the male. The sexes FiGUEE 3. — Pupal cases, or cocoons, ot the ^vax moth. may also be distinguished easily by the palpi of the mouth parts, which are prominent in the female but absent in the male. The moths vary widely in size and color, according to the type of food consumed by the larvae and to the length of time of develop- ment. Small, poorly nourished larvae, or those which, because of low temperatures or other factors, develop slowly, transform into small adults, sometimes less than half the normal size. Such small adults might easily be confused with the lesser wax moth. Larvae fed on dark brood combs transform into adults which may be dark gray to almost black, while larvae which survive on pure wax, or on foundation, transform into moths that are silvery white and smaller than those reared on brood comb. The female starts depositing eggs from 4 to 10 days after emergence and continues depositing them as long as her bodily vitality lasts. Egg laying may be rapid at times, and as many as 102 eggs have been deposited by a femaJe in 1 minute. The total number of eggs laid by 8 CIECULAB. 3 8 6, V. S. DEPAETMENT OF AGEICULTUEE a female varies to a considerable extent under laboratory conditions, but it is usually less than 300. The adults may live as long as 3 weeks. NrMBESt OP BROODS It seems doubtful whether there are definite generations of the wax moth during different periods of the year in the Southern States. Kather it is probable that the moth is always present, that larvae in all stages, pupae, and adults may be found at any time, and that devel- opment goes on except during periods of low temperature. -4 ^ B Figure 4. — Adults of the wax moth : A and B, With wings folded ; C and D, with wings spread ; A and C, females ; B and O, males. Note the deep clefts in the tips of the fore wings of the male, OTHER MOTHS CAUSING DAMAGE TO STORED COMBS Mention has been made of the lesser wax moth, but this moth does not cause so much damage to stored combs as does the wax moth. The work of the lesser wax moth is similar to that of the wax moth, but the tunnels are smaller, the webs finer, and feeding and webbing are more confined to the outer surf a,cc of the combs. The Mediterranean flour moth {Ephestia huehnieTl 11. V. 1 ^. 11 A 1 ■».ll 4 / / 't ^', Figure 5. — Supers loaded with comLi ready (or fumigatiou. Tlie joiiils are sealed with gummed paper tape, and the crystals of paradichlorobenzene have been sprinkled heavily over the top bars. of paper laid on the top bars. Since the gas is nonpoisonous and not disagreeable, treatment may be made in ordinary storage without taking the infected material out of doors. At intervals during the storage season the covers of the stacks should be raised, and unless some are still present, more crystals added. Paradichlorobenzene is at present as cheap as any of the materials mentioned in this circular, with the exception of sulphur, and is by far the easiest and least dangerous to use. The crystals last for some time, since they volatilize slowly, and not only kill the larvae and adults first present and the larvae as they hatch from the eggs, but repel moths from outside which might otherwise enter and start a fresh infestation. Paradichlorobenzene is most effective at tempera- tures above 70° F. and volatilizes more rapidly as the temperature rises. Inspections of stored materials should be made at intervals of 2 or 3 weeks, depending on the temperature of the storehouse and the prevalence of adult moths. THE WAX MOTH AND ITS CONTROL 11 CARBON MSULPIIIDB Carbon disulphide has been a standard fumigant for wax moths and similar insects until recently, and with proper precautions is still satisfactory. As commonly sold conmiercially, it is a more or less yellowish, somewhat oily liquid that changes readily at ordinary temperatures into an ill-ynielling gas. The liquid is about one-fourth heavier than water, and the gas is heavier than air. It is MgMy infaminahh\ atid the vapor is explos-ive v^heii: fnixed. with air in certain- propmiions, and therefore this chem'ical nrnst not ie hcmdled around -fire of any hind. Preferably it should be used out of doors or in a well ^-entilated or open shed. In using carbon disulphide the supers should be sealed in the same manner as for paradichlorobenzene. One ounce of liquid is sufficient for five supers, and more than this number of supers should not be placed in a single stack, since the weight of the gas carries it quickly to the bottom of the stack, and the top super may not be adequately fumigated. The stack should remain sealed for not less than 12 hours. Carbon disulphide is effective against larvae and adults but not against eggs; consequently, it may be necessary to repeat the treatment after any eggs have had time to hatch. FUMIQANTS THAT ARE LESS EFFICIENT FOR WAX MOTH CONTEOL Other substances may be used for the control of the wax moth in stored equipment, but, as explained in the following paragraphs, they are not so efficient for this purpose as either paradichloro- benzene or carbon disulphide and are therefore not recommended. The fumes from burning sulphur effectively control the larvae and adults of the wax moth but are ineffective against the eggs. Sulphur was one of the earliest of the substances used to control the wax moth in stored combs. The early method was to stack the supers over a pan of live coals over which was sprinkled powdered sulphur. About 2 ounces of powdered sulphur (flowers of sulphur) is suffi- cient for a stack of five supers. At least one empty super should be placed at the bottoon of the stack so that the heat will not melt the combs. Present-day practice is to put the sulphur in a dish, wet it with denatured or wood alcohol, and ignite it directly. The work should be done in a well-ventilated room or out of doors, and precautions must be taken against ignition or overheating of the combs. Calcium cyanide is effective against the larvae, pupae, and adults of the wax moth, but cannot be depended on to destroy the eggs. It is obtainable either as dust or as fine or coarse crystals. For use in fumigating bee equipment the crystals are preferable to the dust. In the presence of moisture (such as that found in the a,ir) the crystals form a deadly gas, noninflammable and nonexplosive, hwb extreTtiely poisonous to people and ammals. Care WMst he taken when vmng the substance, and the ffas must not ie breathed. For use put one full tablespoonful of crystals on a sheet of paper and place the paper on the top of the frames in a super. Quickly place the other supers on top, using not more than five supers per stack, and tape the joints between supers with gummed paper tape. The fumigation should be done out of doors, or in a well-ventilated room. 12 CIECULAR 3 8 6, U. S. DEPAETMENT OP AGRICULTURE Leave the stack for at least 12 hours before disturbing it, and air the supers well before storing them. Carbon tetrachloride is effective against wax moths, but does not. have enough penetrating power to kill larvae in cocoons or in thickly webbed refuse. It is a colorless liquid with a sweetish, disagreeable odor. The gas formed is heavier than that of carbon disulphide, and it is used in the same way. The gas is noninflammable and non- explosive, but poisonous. GENEEAL DIBECTIONS ANB SUMMAEIZED INFORMATION ON THE USE OP FTJMIGANTR Use not more than five supers in a stack and seal the joints with gummed-paper tape to make the stack gas tight. With gases heavier than air, make sure that the base of the stack is tightly closed, since the gases sink to the bottom of the stack and may escape. A pad of newspapers placed beneath the stack will help to confine the gas. Fumigate out of doors, if possible, or at least in a well-ventilated room. Read carefully the directions for using the selected fumigant and have everything in readiness before fumigation is begun, espe- cially if cyanide is to be used. Caution. — Carbon disulphide gas is KigKkf eo^losive, and any chance of ignition must he carefully guarded against. Both carbon disulphide and. calciwrn cyanide and their gases are poisonous to people and to animKds and must be stored and Kcmdled vAth extreme care. When using paradichlorobenzene, put the crystals directly on the top bars of frames of the top super, as shown in figure 6, or on a paper laid on the top bars, and renew them throughout the season whenever the crystals have disappeared. As th.e other fumigants mentioned are not effective against rein- festation from hatching eggs, examinations must be made at intervals to see if any eggs have hatched, especially if the storage room is warm. If the temperature is above 70° F., repeat the treatment after 2 or 3 weeks. The stacks being fumigated must be kept sealed for at least 12 hours, and preferably for 24 hours. Air the combs thoroughly before placing them on the hives. Table 1 gives an outline for reference in fumigating against the wax moth. CONTROL MEASURES IN STORED COMB HONEY The control for wax moth damage to stored comb honey is the same as for other stored comb products. The supers should be removed from the colonies as soon as possible after the flow ceases and piled in tiers of not more than 8 to 10. All joints between supers should be covered with paper, and the bottom of the stack should be sealed to prevent leakage of gas. Paradichlorobenzene crystals should be sprinkled over the sections of each super as it is placed in the tier as well as on the sections of the top super, since circulation of air is poor in such stacks. The treatment should be continued until the honey is graded and marketed. Carbon disulphide may be used according to the directions given, if desired. THE WAX MOTH AND ITS CONTROL 13 s e S e S g 'a III on « OS a 13 ■S a a a a S 2 ^ fl 2.S o fl o §1 9 &£ P.go « H >< ^ o " am ph .a t .a S a Ba a ■3 o 5.3 2 ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE WHEN THIS PUBLICATION WAS LAST PRINTED Secretary of Agriculture Henry A. Waixace. Under Secretary Rexford G. Tugwell. Assistant Secretary M. L. Wilson. Director of Extension WorJc C. W. Warbtjeton. Director of Personnel W. W. Stookberger. Director of Information M. S. Eisbnhoweei. Director of Finance W. A. Jump. Solicitor Masiin G. White. Agricultural Adjustment Administration Chester C. Davis, Administrator. Bureau of Agricultural Economics A. G. Black, Chief. Bureau of Ayrioultural Engineering S. H. MoCeory, Chief. Bureau of Animal Industry John R. Mohler, Chief. Bureau of Biological Surrey Iea N. Gabejeilson, Chief. Bureau, of Chemistry and Soils H. G. Knight, Chief. Bureau of Dairy Industry 0. E. Reed, Chief. Bureau of Entomology and Plant Quarantine. Lbjb A. Strong, Chief. Office of E-i-periment Stations - James T. Jardinb, Chief. Food and Drug Administration Walter G. Campbell, Chief. Forest Service Ferdinand A. Silcox, Chief. Grain Futures Administration J. W. T. Duvel, Chief. Bureau of Home Economics Louise Stanley, Chief. Library Claribbl R. Barnett, Librarian. Bureau of Plant Industry Frbdemck D. Richby, Chief. Bureau of PuMic Roads Thomas H. MaoDonald, Chief. Soil Conservation Service H. H. Bennett, Chief. Weather Bureau Willis R. Gregg, Chief. This circular is a contribution from Bureau of Entomology and Plant Quarantine... Lee A. Strong, Chief. Division, of Bee Culture Jas. I. Hamblbton, Principal Api- cuUurist, in Chai-ge. 14 U. S. GOVERNMENT PRINTING OFFICE: 1936 For sale by the Superintendent of Documents, Washington, D. C. - - - Price 5 cents A300-718-10m TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 231 jUNE, 1918 DIVISION OF ENTOMOLOGY THE BEEMOTH OR WAXWORM B. YOUNGBLOOD, Director COLLEGE STATION, BRAZOS CX)UNTY, TEXAS. AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS W. B. BIZ7EIX, A. M.. D. C. L., President TEXAS AGRICULTURAL EXPERIMENT STATION BOAKD OF DIRECTORS John I. Guion, Ballinger. President Term expircfl 1919 L J Hart; San Antonfo. Vice-President Term expires 1919 E; H. Astin. Bryan • Term expires 1919 J R. KuBENA. Fayetteville • Term expires 1921 A B. Davidson. Cuero Term expires 1921 Will A. Miller. Jb.. Amanllo Term expires 1^1 John T. Dickson. Paris Term expires 1923 H. A. Bbeihan. Bartlett Term expires 1923 F M Law, Houston Term expires 1923 MAIN STATION COMMITTEE L J Hart, Chairman Will A. Miller, Jr. GOVERNING BOARD, STATE SUBSTATIONS P L Downs, Temple. President Term expires 1919 Charles Rogan. Austin. Vice-President Term expires 1923 J E BooG-ScoTT. Coleman Term expires 1921 R M Johnston. Houston Term expires 1918 ♦STATION STAFF ADMINISTRATION B. Youngblood, M. S., Director A. B. CoNNEB, B. S., Vice Director Chas. a. Felkeb. chief Clerk A. S. Wabe. Secretaru W. T. Bbink. B S., Executive Assistant in Charge Library and Publication Edith H. Phillips, M. S., Technical DIVISION OF VETERINARY SCIENCE •*M. Francis, D V. S., Veterinarian in Charge H. Schmidt, D. V. M.. Veterinarian D. H. Bennett, V. M. D.. Assistant Veterinarian DIVISION OF CHEMISTRY G. S. FRAPS, Ph. D., Chemist in Charge; State Chemist T. B Leith. B. A.. Assistant Chemist Frances Summerell, B. S., Assistant Chemist ■ — t Assistant Chemist DIVISION OF HORTICULTURE H. Ness, M. S., Horticulturist in Charge W. S. Hotchkiss, Horticulturist DIVISION O? ANIMAL HUSBANDRY J. C. BuBNS, B. S., Animal Husbandman, Feeding Investigations J. M Jones, A. M., Animal Husbandman, Breeding Investigations P. V. EwiNG, M S., Animal Husbandman, Sivine Investigaiions DIVISION OF ENTOMOLOGY F. B. Paddock, M. S., Entomologist in Charge; State Entomologist H, J. Reinhard, B. S., Assistant Ento- mohgisi : . Assistant Ento- mologist County Apiary Inspector§ R. C. Abernalhy. Ladonia; William Atcb- ley, Mathis; J, W. E. Basham, Barstow; T. W.Burleson. Waxahachie.W. C. Col- lier. Goliad; E. W. Cothran. Roxton; G. F- Davidson. Pleasanton; John H-Tb 'd, Seguin; S.T. GrahanuMilano; J. B.King, Batesville; N. G. LeGear, War.o; R. A. Little, Pearsall; S. U Stephens. Uvalde; M. B. Tally. Victoria. R. E. Watson, Heidenheimer: M.E. Van Every, Fabens; R. A Nestor, Buffalo; J E Bush, San Antonio; H A, Jones, Oakdale; T. A. Bowdon, Palestine; E. R, Jones. Bee- viile. DIVISION OF AGRONOMY A. B. Conner. H. S.. Agronomist in Charge A. H. Leidigh. R. S., Agronomist , Agronomist Louis Wermelskibchfn, B S., Agronomist DIVISION OF PLANT PATHOLOGY AND PHYSIOLOGY T. J. Taubenhaus, Ph. D., Plant Patholo- gist and Phiisiologtst in Charge DIVISION OF POULTRY HUSBANDRY H. N. Harvey, B. S., PouUryman in Chargt DIVISION OF FORESTRY E, O. SiECKE, M. F., Forester in Charge, State Forester DIVISION OF PLANT BREEDING E. P. Humbert, Ph. D., Plant Breeder in Charge DIVISION OF DAIRYING W A Doubt. Dairyman ♦••SOIL SURVEY , Soil Surveyor 3. F. Stroud, Soil Surveyor DIVISION OF FGED CONTKOL SERVICE F D Fuller. M. S., Chi^ James Sullivan, Executive Seeretarg J. H. Rogers, Inspector W. H. Wood, Inspector S. D. Pearce, Inspector W. M. WiCKEs, Inspector W F. Christian. Inspector J. W. Snell. Inspector J. J Kelly. Inspector SUBSTATION NO. 1: Beevllle, Bee Connty 1. E. CowABT. M S., Superintendent SUBSTATION NO. 2: Troup, Smith Coamy W. S. Hotchkiss, Superintendent SUBSTATION NO. 3: Ani^leton, Brazoria County E. A. Miller, B. S., Superintendent SUBSTATION NO 4: Beaumont, JeffersoD County H. H. Laude, B. S., Superintendent G Purvis, Scientific Assistiini ' SUBSTATION NO. 5: Temple, Bell County D. T. Killough, B. S.. Superintendent SUBSTATION NO. 6- Denton, Denton Coanty C. H McDowell, B. S.. Superintendent SUBSTATION NO. 7: Spur, Dickens County R. E. Dickson. B S . Superintendent E M Smeltzer, Scientific Assistant SUBSTATION NO. 8: Lubbock, Lubbock County R. E. Karfer, B. S., Superintendent D. L Jones Scientific Assistant SUBSTATION NO. » Pecos. Reerea Coanty J W. Jackson, B. S., Superintendent SUBSTATION NO. 10: (Feeding and Breeding Substation), College Station, Braaofl County N. E Winters, M. S., Superintendent L. G. Wilkinson. Scientific Assistant SUBSTATION NO. 11: Nacogdocbes, Nacog- doches County G. T. McNess, Superintendent SUBSTATION NO. 12: Chiilicothe, Harde- man County > •••A. B. Cron, B. S., Superintendent V. E. Hafner. B. S., Scientific Assistant SUBSTATION NO. 14, Sonera, Sutton County E. M. Peters, B. S., Superintendent CLERICAL ASSISTANTS Daisy Lee. Begistration Clerk Ruth Lord. Stenographer H. F. Stasney, Mailing Clerk B. C. Franks, Stenographer Emma Campbell, Stenographer Ethelwyn Frazier, Steno- Mabgabet Sheldon, Stenographer Ruth Gilliam, Stenographer grapher •As of July 1. 1918. ••In cooperation with A. & M. College of Texat. •••In cooperation with United States iJepartment of Agriculture CONTENTS PAGE Introduction ^^ History 6 Dispersion 6 Distribution 6 Systematic position 7 Economic importance 7 Hosts 8 Methods of study 8 Life history 9 The egg 9 Incubation period 10 The larva 11 Description 11 Feeding habits 11 Length of period 13 Description 14 Pupation 14 Construction of cocoon 15 The pupa 16 Transformation 16 Description ,1'^ Duration of pupal stage ,■ 1'? Adults 18 Description 18 Emergence 19 Duration of life 19 Proportion of sex 19 Habits 21 Period between emergence and copulation 31 Life of unmated females 22 Mating , ^3 Age at beginning of oviposition 33 Process of oviposition *'^ 24- Time of oviposition . ., Habits during oviposition "* • • 3-i Period of oviposition Adults — continued. page Effect of age of sex 35 Eate of oviposition 35 Effect of humidity on development 35 Seasonal history 30 Natural control 33 Predaceous enemies 33 Parasites 33 Climate 33 Artificial control 34 Trap lights 34 Decoy boxes 34 Fumigation 34 Sulphur 35 Carbon bisulphide 35 Bulletin No. 231 June, 1918 THE BEEMOTH OR WAXWORM F. B. Paddock, M. B., State Entomologist, Entomologist in Charge INTRODUCTION This paper is a revision of Bulletin 158 of ' this Station, now out of print. The continued interest manifested in the beemoth makes this revision and reprint necessary. Additional information has been derived from experiments in the practical control of this pest since the first treatise appeared in the aforementioned bulletin in 1913. In modern vrorks on apiculture the opinion is expressed that in the present age of modern beekeeping the beemoth cannot be judged a pest of the apiary. It is not only true that box hives favor this insect in its work of destruction, but that warm climates also favor it, even where movable frame hives are used. The long open winters in the southern states allows this insect to feed freely on stored combs. No doubt as the practice of producing extracted honey grows ' in the northern states, that section Y^ill experience more trouble with the work of this insect in the stored combs. At the present time the beemoth or waxworm is a serious hindrance to the beekeeping industry in Texas, as well as in all of the southern states. Many beekeepers no longer dread the beemoth but keep every colony provided with a vigorous queen. Under such conditions it is difficult for the beemoth to enter the hive to deposit its eggs. The waxworm has become very largely an enemy of the box hive and a destroyer of stored combs and honey and is .found usually around the bee hive and in piles of used comb. In large apiaries, the wax and comb that is carelessly left lying around affords ample food for the insect to breed in. In this way the pest is maintained in a yard, ready to infect any weak colony. With many beekeepers the beemoth is a source of constant trouble, for if the bees are not closely watched and become queenless the colony is certain to become infested in a short time. If the beemoth once becomes established, it is hard to extermi- nate. At present the beekeepers are not able more than to keep this pest in check. It is hoped that a more thorough knowledge of the habits and life history of the insect will result in a better control of this enemy and a reduction of the loss now suffered from its ravages. Almost every beekeeper is acquainted with the work of the insect, generally known as the "web worm" or "miller," but it is not com- monly known that the worm, following maturity, develops into a moth or miller. The worms or larvae feed in protected places, within the comb, which makes them difficult to fight successfully. 6 Texas AGBicuLTtTEAL Expekimen"t Station HISTOEY This species was known to observers and beekeepers in very early times. Perhaps the first mention was that made by Aristotle, who lived in 384-322 B. C. Later Virgil (70-19 B. C.) made mention of the beemoth in his writings. In the first century, A. D. Columella, a Eoman writer on agricultural subjects, mentioned the beemoth as an enemy to the honey bee. Eeamur (1685-1757) in France told of the damage done by the beemoth. In Holland, Swammerdam (1637-1680) refers to species of the beemoth, commonly called at that time the '^bee wolf." Linnaeus (1707-1778) in Sweden tells of the presence of this pest among the beekeepers of that country. The introduction of the beemoth into America occurred about the beginning of the nine- teenth century. It is said that the pest was found in Australia prior to 1878 and in .ISTew Zealand it was not noticed until 1904. When this pest was introduced into Texas is not known. DISPEESION It has been said that man is the spreading agency of this pest. It is undoubtedly true in the larger sense, for it is very doubtful if the pest spreads fast through its own flight. The carelessness of the bee- keeper is almost wholly responsible for the maintenance and spread of the waxworm. As beekeeping has progressed from Asia through Europe and Northern Africa to the North American continent and to the islands of the Pacific, so has been the spread of the beemoth. Wherever beekeeping has been introduced, a few years later the presence of the pest is recorded. Within small areas the spread" of the pest is largely through the exchange of infested combs. In Texas it has not been traced, nor is the first location known. The dispersion seems to- be restricted by the climatic conditions of Colorado and other western states. In these localities it has been introduced often but had failed to establish itself. DISTEIBUTION The beemoth is now found in much of northern Asia and Africa north of the desert, throughout Europe, Great Britain, North America, Australia, New Zealand, Ceylon and India. The distribution of this pest in Texas includes the following counties: Anderson, Atascosa, Bandera, Bastrop, Bee, Bell, Bexar, Blanco, Bosque, Bowie, Brazoria^ Brazos, Brooks, Brown, Burleson, Burnet, Caldwell, Callahan, Cass,. Cherokee, Coleman, Collin, Colorado, Comanche, Concho, Cook, Coryell,. Crockett, Dallas, Delta, Ellis. Erath, Falls, Eannin, Payette, Franklin,' Freestone, Gonzales, Greg,?, Grimes, Guadalupe, Hamilton, Harrison', Hays, Henderson, Hill, Houston, Hunt, Jasper, JefPerson, Karnes' Kaufman, Kendall, Kerr, Kimball, Lamar, Lampasas, Lavaca, Lee, Leon, Liberty, Limestone, Llano, Madison, McCullough, McLennan' jMason, McMuUen, Medina, Milam, Mills, ]\rorris, Navarro Nolan Nueces, Panola, Parker, Polk. Eains, Bed Eiver, Eobertson, Eockwalf The Beemoth or Waxworm 7 Eunnels, Rusk, Sabine, San Jacinto, Schleicher, Shackelford, Smith, Stephens, Taylor, Travis, Trinity, Tyler, Uvalde, Val Verde, Waller, Ward, Washington, Wood, Wilson, and Williamson. The dissemination of the Ijeemoth in Texas has been very complete, for there are few counties in the State where bees are kept that are free from the pest today. The counties shown to be infested as reported by beekeepers include all of the important beekeeping counties of the State. There is no doubt that further inquiry will show the presence of this pest in still other counties. SYSTEarATIC POSITION The very early writers refer to the beemoth as Tinea mellonella. Later it was known as Galleria cereana Fabr, which genus was erected by Fabricius. A more careful search of the early records revealed that Linnaeus described two species of beemoths known as Tinea mellonella and T. cereana. Writers were then very confused and we find the beemoth called G. cereana; G. alveria, T. cerella. In early American literature this species is referred to as G. oHiquella Walker. All recent publications on this insect refer to the species as G. mellonella L. The name early applied to this species would indicate that it was placed in the family Tineidae, which contains many small fringe-winged moths, the larvae of which are often case-bearing. It can easily be seen how this might have been done, as the waxworms construct very substantial tunnels that might have been classed as cases. Today the beemoth is placed in the family, Galleriidae, which is now included with the Tineidae as the micro-lepidoptera. The lesser beemoth, a closely associated species, is now known as Achroia grisella Pabr. The two species of moths have often been con- fused by writers on beekeeping subjects. This species is not so widely distributed as the larger beemoth. ECONOMIC IMPOETANCE What this pest is costing the beekeepers of the State is hard to de- termine. The price of bees, honey and wax varies in the different, sec- tions of the State. Often the loss of colonies is attributed to other causes and frequently the presence of the beemoth is not detected. In the reports which have been received from beekeepers, no mention has been made of the loss of stored comb, but this must certainly be con- siderable. The loss in some cases is very heavy. In the year 1911, 136 bee- keepers reported losses varying from five per cent, to their colonies to as high as ninety-five per cent. Many more beekeepers reported the presence of. the beemoth as "general," indicating that they suffered no small loss. In one very well kept apiary that has come under the observation of the writer, there has been an annual loss of three per cent, due to the beemoth. It is safe to say that in many of the larger ■8 Texas Ageicultural Espeeiment Station apiaries throughout the State this loss is not uncommon, while in the smaller apiaries and in boxhive apiaries the loss is much greater, as was indicated by the reports referred to above. The census of 1910 shows approximately 295,000 colonies of bees in the State, and it is generally conceded that these figures are below the actual number. Assuming that five per cent, is the average annual loss of colonies due to the waxworm, including the large losses in the poorly kept apiaries, it is seen that the annual loss amounts to at least 14,000 colonies. At an average valuation of $4.00 per colony, this amounts to $56,000 a year, a very considerable annual tax on the beekeeping industry of the State. There is no way to arrive at the loss of combs and honey. HOSTS In India, according to T. B. Fletcher, the beemoth attacks wild bees, as well as domesticated. He says, "In India, in districts where bees are not domesticated, it attacks the combs of the wild honey bees to such an extent that the bees often desert their nests in disgust and swarm oflE and found a new one, while it is very rare to find a deserted comb which does not bear traces of the ravages of this pest." This is the only reference to the attacks of the beemoth on wild bees. Observations of the writer have never disclosed the presence of the beemoth in the •domiciles of wild bees. In the hives of domesticated bees, the midrib of the comb seems to be the preferred food. The old brood-rearing combs are preferred to the new combs. This is probably due to the taste of the beemoth for the cast skins of the bee larvae. The larvae, however, are never eaten hy the waxworm, although the bee larvae may die in a badly infested comb. This may be due to a lack of nourishment, as the waxworm will devour pollen wherever it is found, stored in cells or as food for the bee larvae. Small quantities of honey are consumed by the wax- worms. Propolis is eaten in small quantities. Although the frames and hive are eaten out for pupation it is doubtful if the wood is a food, but probably it is used slightly in the construction of the cocoon. In stored empty combs the waxworm shows a decided preference to the combs in which brood has been reared. Of course, any comb is readily attacked and destroyed. In the case of stored honey, the comb is preferred as food and the honey is only eaten in small quantities. Honey cappings are readily devoured whenever found and often serve as the only food of the waxworm. It has been said that if fed on pure wax the worms will die. The writer observed one instance in which two full sheets of medium brood foundation were riddled by the wax- worm. METHODS OE STUDY For the purpose of observing the details of oviposition, small pieces of old brood comb were used. For the observations on the larvae larger pieces of old brood comb were used, the small pieces of comb being The Beemoth or Waxworm placed in lantern globes having checso cloth over the top. The habits of the moth were observed in the large rearing cages. Sometimes fresh cappings were supplied the moths for oviposition and for food for the larvae. There was no apparent difference in the activity of the adults with these two foods. LIFE HISTOEY The larva ("webworni") upon reaching maturity, constructs a cocoon by means of silken threads which it is able to spin. After the cocoon is completed, the larva changes to the pupal stage. This is the stage in which the form of the larva is reconstructed to make the moth which will emerge later from the cocoon. The moths mate and the females deposit the eggs which hatch into the larva. This is called the "life cycle." The life history of the moth has been assumed by almost every writer on the subject of beekeeping. No definite experiments, however, have been recorded of observations on the details of the life cycle of this insect in this country. The paper by Fletcher in 1911 gave data col- lected in India. In the United States it is certain that the details of the life cycle will vary much for there is evidence that even in this State there is a variation within the broods as well as a seasonal vari- ation. THE EGG The egg is elliptical and pearly white in color. The shell is slightly roughened by wavy lines running across it diagonally at regular inter- vals. If the egg is not deposited on dark comb, it is very difficult to see and even then experience is necessary to detect all eggs present. There is a slight variation in the size of the egg, as is shown by the measurements given in the following table: Table I. — Measurements of eggs. Date Jan. I.'i. 7913 Mar, 14, 1913 Mar. •2.'i. 1913 Apri 2. 1913 Number. 9 eggs 3 eggs 9 eggs 10 eggs I Length. .485 mm. .473 mm. .520 mm, ,433 mm. Width. ,440 mm. .360 mm. .400 mm. .378 mm From the foregoing table the average length was .478 mm. and the average width was .394 mm. for the thirty-one eggs taken in the lab- oratory over a period of three months. The embryonic development of the egg has not been studied, but a few observations have been made upon the incubation period. Through- out this period the egg gradually changes from a white to a yellow color. About four days before hatching, the developing larva becomes visible as a dark ring inside the shell. The perfectly formed larva can be distinctly seen for at least twelve hours before the shell bursts. During this time the larva is engaged in cutting an opening in the 10 Texas Agricultural Experiment Station shell and its final emergence from the egg is made through a ragged hole in the top. After the larva is out of the shell it appears white and clear. INCUBATION period The period of incubation varies greatly with the brood and in the cooler portions of the year this period is irregular within the brood. The low temperature at which the vitality of the eggs is destroyed has not been determined. In table 2 the eggs were from moths placed in an unheated room and during this period the temperature averaged 65° P. Table 2. — Duration of egg stage, spring I9I2. Laid. Hatch ed. Period. Mar. 8, 1912 April 4, 1912 27 days Mar. 9. 1912 April 4, 1912 2S days Mar. 10, 1912 April 4, 1912 2.5 days Mar. 13. 1912 April 4. 1912 23 days Mar. 13. 1912 April 4, 1912 23 days Mar. 14, 1912 April 4, 1912 22 days Mar. 21, 1912 April 4, 1912 14 days Mar. 27, 1912 April 7. 1912 11 days The average period of incubation of this brood was 31.8 days. Table 3. — Duration of egg stage, fal'. 1912. r.aid. Hatelnd. Period. Aug. 27, 1912 Sept, 3, 1912 7 days Aug. 27, 1912 Sept, 3. 1912 7 days Aug. 28, 1912 Sept, 3, 1912 " 6 days Aug. 30. 1912 Sept. 5. 1912 6 days bept. I, 1912 S?pt. 7, 1912 6 days S'^pt. 3, 1912 SL-pt. 10. 1912 7 days S!>pt. 3. 1912 S=pt. 11, 1912 8 days Sjpt. 4, 1912 Sept. 12, 1912 8 days bspt. 5, 1912 Sept. 14, 1912 9 days These eggs were kept in the laboratory and the temperature averaged about 95° F. The average period of incubation of this brood was 7.1 days. Table 4. — Duration of egg stage, winter 1912. laid. Hatched. Period. Dec. 6, 1912 Dec, 11, 1912 5 days Dec. 6, 1912 Dec. 12, 1912 fi days Dec. 4, 1912 Dec. 13, 1912 9 days Dec. 7, 1912 Dec. 14, 1912 7 days Dec, 7, 1912 Dec. ir,. 1912 8 days Dec, 7, 1912 Dec, 16, 1912 9 days Dec, 6, 1912 Dec, 18, 1912 12 days Dec, 7, 1912 Dec, 20, 1912 Dec, 10. 1912 Dec. 23, 1912 13 days Dec, 14, 1912 Dec. 26, 1912 12 days These eggs were kept in the laboratory with artificial heat which The Beemoth or Waxworm 11 averaged 80° F., although it was quite irregular. The average period of incubation oi' this brood was 9.5 days. THE LAEVA DESCRIPTION When first hatched the young larva (worm) is only one to three millimeters (1/25 to 1/8 of an inch) in length. They have a dirty white, waxy color. The head is slightly yellow and smaller than 'the prothoracic segment, which is decidedly prominent. The true or thor- acic legs are especially well developed and the pro-legs or abdominal legs are not apparent when the larva is first hatched, and only appear normal when the larva is about three days old. FEEDING HABITS After emerging from the shell through a ragged hole, the larvae are quiet for a short time while they are apparently drying in preparation for their work of destruction. Soon they become active, but only upon close examination is it possible to detect them hurrying over the comb in their attempt to gain an entrance. Within a short time after hatch- ing, the first meal is taken by the larvae. This consists of scales of wax which they can loosen from the comb in their attempts to gain an entrance. The larvae do not enter the comb near the eggs from which they hatched. In fact, several entrances are attempted before one is finished. This may be due to the extra hard comb in such areas or it may be that they are frightened and never return to continue. It was not observed whether entrances started are taken up by other larvae. The entrance is made at the top of the comb between the walls of adjoining cells. It is during this short period of an hour- or more that the larvae are at the mercy of the bees but no doubt few if any are killed at this time in the dark hives. The entrance is extended by the larvae into tunnels directed toward the center of the comb or the bottom of the cells. The presence of the larvae is readily noticeable as soon as the tunnels are well started. In making these 'tunnels the larvae push back of them and out of the tunnel bits of chewed wax not used for food. This makes the surface of the comb appear rough and poorly kept. These bits of chewed wax contain strands of the web of the larvae. It is evident that the web is secreted continually by the larvae. Some larvae leave their tunnels considerably and may even work two or three tunnels down to the mid- rib of the comb. During this period of reaching the center of the comb the growth of the larvae is very slow. During this time only a small proportion of the wax is consumed for food. These tunnels ex- tending to the bottom of the cell are increased in size to accommodate the growth of the larvae, in which case only the thinnest wall protects them. The time consumed in extending the tunnels to the center of the comb varies greatly, from four to eight days. 12 • Texas Agricultural Experiment Station When the center of the empty comb is reached, holes are cut through the bottoms of the cell walls and the larvae leave their tunnels and wander along, the mid-rib from cell to cell. At this time the old tunnel serves as a shelter and it is enlarged. The material thrown out falls- on the bottom of the cells. This refuse is about four-fifths chewed and webbed wax and one-fifth excrement. When the comb is disturbed^ the larvae may run through three or four cells to their tunnels. At first only holes are eaten through the cells, but in a few days lines of web can be seen outlining the passageway from cell to cell. The holes are enlarged to admit free passage of the growing larvae. The silk spun by the larvae is numerous enough in the course of a few days ta form a gallery which gives very good protection. If the larvae are- shaken from the comb they seem lost for a time but shortly proceed in the direction of the comb. What causes them invariably to seek the- comb it is impossible to say. If they encounter refuse before reaching the comb, they will avail themselves of such shelter. At all times the larvae avoid light as much as possible. When disturbed, the larvae will often drop by means of a thread. In this way they return to the- exact place of feeding. Prom this central gallery the feeding is extended out along the bot- toms of the cells or the middle of the comb. The silk is spun wherever the larvae go, so that very soon the bottoms of the cells are replaced by a layer of silk thread covered with excrement of the larvae and particles- of chewed wax. The time required for this varies, of course, with the temperature. After the mid-rib has been eaten, the larvae start on the walls of the cells, the ones farthest away from the light being the first destroyed. As this feeding continues along the cell walls, the threads of silk are extended to cover the new feeding ground, and not only serve to protect the larvae but also act as a scaffold to support the damaged cells. Soon the center of the. comb appears as a mass of tangled refuse and dis- carded wax. The feeding is at times that of a colony all working com- paratively close together. At various places through the comb there are constructed false cocoons that serve as a protection to the larvae while they are resting during the day. When small pieces of comb were used, the larvae would leave the comb during the day and remain in well constructed galleries in the refuse under the comb. After the larvae were three-fourths grown, they worked practically none during the day although the cages were darkened. At night the gnawing of the larvae was distinctly audible. Dark comb is preferred for feeding to light comb. When small pieces of comb were used and additional food was necessary, another piece of empty comb was placed under the old comb. The larvae would immediately attack the new comb, going to the bottom of the cells, eating the mid-rib, lower cells, then upper cells, exactly as the first piece of comb was eaten. The feeding continued until the walls were entirely eaten, but the top of the cells was never eaten, perhaps because this would expose them The Beemoth or AVaxworm 13 to outside influences and enemies. The area of feeding was gradually extended from the point of infestation finally to include the entire comb. If the comb does not furnish sufficient food the larvae begin to feed on the refuse under the comb in which there is considerable wax in small pieces. In this they construct such a large amount of web that they are absolutely protected from enemies. In a few cages, balls of fresh cappings, containing normal amounts of honey, were supplied as food for the larvae. The entrance was usually made on the top side of the cappings. A tunnel was made aa in the ease of empty comb, directed toward the center of the ball. This tunnel, however, was not extended to the center but was extended around the mass, keeping a uniform distance from the surface. The refuse was thrown back and out of the tunnel as was done when feeding on empty comb. When the tunnel was extended around to a point just above the point of contact with the bottom of the cage, an exit was made. Here much refuse was accumulated and the larvae seemed to prefer to eat here. The growth of larvae feedin,g on cappings was slower than when feeding on comb and there was a greater variation in the growth. '\ATien feeding on stored combs of honey, the worms apparently fed only on the comb, which allowed the honey to drip. The amount of chewed wax was not so great as when feeding on empty comb, nor were there so many webs visible. LENGTH OF PEEIOD There was a great variation in the length of the larval period even within the brood when food and climatic conditions were apparently the same in all cages. Throughout the observations made on the feed- ing habits this fact was readily noticeable. In some cages this vari- ation in the size of the larvae was apparent as early as seven days after hatching. This variation is much more apparent during the later part of the period, and during the colder portion of this year the variation is still more pronounced. As indicated above, the food has an effect upon the length of this period, it being greater when cappings were supplied than when empty comb was used. Table 5.— Length of larval period, spring 1912. Hatclied. Matured. Period. April 8, 1912 April 4, 1912 April 4, 1912 April 4, 1912 April 3, 1912 April 12, 1912 April 3, 1912 April 5, 1912 May 25, 1912 May 29, 1912 .May 27, 1912 May 29, 1912 May 25, 1912 May 25, 1912 May 21. 1912 May 27, 1912 47 .days 52 days 43 days 55 days 52 days 43 days 48 days 52 days These larvae were kept in the laboratory at normal temperature with 14 Texas AGBicQLTUiLi.L Experiment Station empty combs for food. The average length of the larval period of this brood was forty-nine days. Table 6. — Length of larval period, fall 1912. Hatched. Matured. Period. Aug. 26. 1912 Sept. 25, 1912 30 days Aug. 25, 1912 Se.nt. 25. 1912 31 days Aug. 25, 1912 .Sent. 27. 1912 33 days Aug. 26, 1912 .<:ept. 28, 1912 33 days Aug. 26, 1912 Sept. 30. 1912 35 days Aug. 26, 1912 Oct. 2, 1912 37 days Aug. 26, 1912 Oct. 4, 1912 39 days Aug. 24, 1912 0.-I. 5. 1912 ■42 days These larvae vs^ere kept in the laboratory at normal temperature, with empty comb for food. The average length of the 'larval period of this brood was thirty-five days. In the fall brood many of the larvae do not mature and pupate until cold weather occurs and a few larvae feed throughout the winter. At any time a comb was inspected there was a vast variation in the size of the larvae from eggs deposited at the same time. Table 7. — Length of larval period, winter 1912. 13. Hatched. Matured. Period. .Sept. 4. 1912 Dec. 10. 1912 97 days Sept. 9. 1912 Dec. 15, 1912 101 days ■Sept. 7, 1912 Dec. 17, 1912 101 days Sept. 5, 1912 Dec. 17. 1912 103 days Sept. 3, 1912 Dec. 10. 1912 98 days Sept. 6. 1912 Dec. 20. 1912 105 days -Sept. 14, 1912 Jan. 27. 1913 .Sept. 13, 1912 Jan. 31. 1913 140 days These larvae were kept in the laboratory with normal artificial heat, with empty comb as food. The average length of the larval period of these was 110 days. Fo observations were made on the number of moults of the larvae. DESCKIPTION On September 15, 1913, ten specimens of mature larvae were meas- ured and the average length was twenty millimeters. The head is small and pointed, reddish brown in color, with a light v-shaped line on top, this "v" opening towards the front of the head. The body is larger than the head, long, cylindrical, smooth except for a few short hairs. The general color is a dirty gray with the prothoracic shield brown and having a broad band across it. PUPATION Having completed its growth, the larva seeks a place in which to pupate, though sometimes the end of the feeding gallery may be en- larged and closed to serve as a cocoon. The cocoon may also be spun The Beemo'l'h or AVa'xwoem 15 in the refuse under the comb and this mass of webs afEords an excellent protection' to the p\ipa. The most common places are cracks or corners about the hive, or between the frames and the hive or in the "hee space" at the end of tlie top bars. The larva prefers to get into a place which' it can chew in order that a cavity may be constructed and the cocoon thus be better protected. Having found the location for the cocoon, the larva begins the spin- ning of the silk thread about itself, starting just above the head and working backward more than the length of the body. A thin layer of silk is spun in the general shape of the cocoon and this frame work is covered with fine silk from the inside. The larva is able to reverse itself within the cocoon, which it does many times during its con- struction. The outer layer, upon hardening, becomes very tough, and even like parchment, while the inner layer remains soft and flufEy. CONSTRUCTION OF COCOON The time consumed in the spinning varies with the season and varies most in the cooler portions of the year. Within the brood there is some variation. Table 8. — Period of rocoon construrlion. summer 1912. Started. Aug. 10. Aug. 10, Aug. 10. Aug. 10, Aug. 10, Aug. 10, Aug. 10, Aug. 11, Aug. 11, Aug. n, Aug. 11, Aug. n, Aug. 12, Aug. 12, Aug. 12. Aug. 12, Aug. 12, Atig. 13, Aug. 13, Aug. 13. 1912 1912 1912 1912 1912 1912 1912 1912 1912 I9I2 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 Completed. Period. Aug. 11, Aug. 12, Aug. 12, Aug. 13, Aug. 13, Aug. 14, Aug. II, Aug. 15, Aug. 15, Aug. 16, Aug. 13, Aug. 13, Aug. 14, Aug. 14, Aug. 13. Aug. 13, Aug. 13, Aug. 15, Aug. 15, Aug. 15, 1912 1 day 1912 2 days 1912 2 days 1912 3 days 1912 3 days 1912 4 days 1912 1 day 1912 4 days 1912 4 days 1912 5 days 1912 2 days 1912 2 days 1912 2 days 1912 2 days 1912 1 day 1912 1 day 1912 1 day 1912 2 days 1912 2 days 1912 2 days The average period of construction of the cocoon of this brood was 2.25 days. The cocoons were' spun in cages in the laboratory with normal temperatures. fable 9. Period of rocoon construction, fal! 1912. started. Oct. 25, 1912 Oct. 24, 1912 Oct. 25. 1912 Oct. 25, 1912 Oct. 27, 1912 Oct. 29, 1912 Oct. 26, 1912 Oct. 26, 1912 Oct. 26, 1912 Oct. 26, 1912 Completed. Oct. 30, 1912 Oct. 30, 1912 Oct. 30, 1912 Oct. 30, 1912 Oct. 30. 1912 Oct. 30, 1912 Oct. 31, 1912 Oct. 31, 1912 Oct. 31, 1912 Oct. 31, 1912 Period - 5 days 6 days 5 days 5 days 3 days 3 days 5 days 5 days 5 days 5 days 16 Texas Agricultural Esperiment Station" These cocoons were spun in cages in the laboratory Tvith normal artificial heat. The average period of construction of this brood was 4.73 days. THE PUPA TRANSFORMATION ' As the cocoon nears completion, the larva becomes very sluggish, and the body shortens. The last act of the larva is to make an incision in the cocoon near the head end which provides for the easy emergence of the moth at maturity. The time required in the transformation from the larva to the pupa varies with the broods and somewhat within the broods. Table 10. — TransformaLion period, summer 1912. Larva. Aug. 13, Aug. 7, Aug. 10, Aug. 13, Aug. 14, Aug. 13, Aug. 11, Aug. 17, Aug. 14, Aug. 16, Aug. 19. Aug. 18, Aug. 18, Aug. 19, Aug. 19, Aug. 19, Aug. 19, Aug. 19, Aug. 21, Aug. 21 , 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 Pupa, Period. Aug. 15, Aug. 15, Aug. 15, Aug. 15, Aug. 16, Aug. 16, Aug. 20, Aug. 20, Aug. 20, Aug. 21, Aug. 21. Aug. 21, Aug. 22, Aug. 22, Aug. 22, Aug. 22, Aug. 23, Aug. 23, Aug. 23, Aug. 23, 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 2 days 8 days 5 days 2 days 2 days 3 days 9 days 3 days 6 days 5 days 2 day.s 3 days 4 days 3 days 3 days 3 days 4 days 4 days 2 days 2 day.s This transformation took place in the cages in the laboratory at normal temperature. The average period of transformation of this brood was 3.75 davs. Table 11. — Transformation period, fall 1912. Larva. Pupa. Period. Oct. 23, 1912 Oct. 29, 1912 6 days Ucl. 23, 1912 Nov. 3, 1912 Oct. 23, 1912 Oct. 29, 1912 6 days Oct. 22. 1912 Oct. 28, 1912 6 days Oct. 22, 1912 Oct. 27, 1912 5 days Oct. 21, 1912 Oct. 27, 1912 Oct. 24, 1912 Nov. 3, 1912 10 days Oct. 20, 1912 Oct. 25, 1912 Oct. 20, 1912 Oct. 25, 1912 5 days Oct. 23, 1912 Oct. 27, 1912 4 days This transtormation took place in the laboratory with normal arti- ficial heat. The average period was 6.40 days. The Bbemoth ok AVaxvyorm 17 DESCRIPTION The newly formed pupa is white. At the end of the first twenty- four liours It turns to :i straw color, very light at first, deepening slowly. By the end of the fourtli day the pupa is light brown and this color gradually deci^ns, so that by the end of the pupal period the insect is dark brown. The male pupae average fourteen millimeters (about two- thirds of an inch) in length and the female pupae are fully sixteen millimeters in length. A row of spines arises just back of the head and extends to the fifth abdominal segment; the body line is somewhat curved downward. DUHATION OE PUPAL STAGE The pupal stage varies greatly with the seasons, being especially long during the fall and winter. Table 12. — Duration of pupal stage. 1912. Pupated. Jan. 17, 1912 Jan. 18, 1912 Jan. 18, 1912 Jan. 21, 1912 Jan. 20, 1912 Jan. 20., 1912 Emerged. Mar. 9, 1912 Mar. 3, 1912 Mar. 7, 1912 Mar. 11. 1912 Mar. 9, 1912 Mar. 15, 1912 Period. 52 days 45 days 49 days 51 days 49 days 55 days These pupae were in cages in the laboratory with no artificial heat. The average length of the period was fifty days. Table 13. — Duration of pupal stage, 1912. • Pupated. Emerged. Period. Aug. 12, 1912 Aug. 20, 1912 8 days Aug. 13. 1912 Aug 19, 1912 6 days AuB. 12, 1912 Aug. 19, 1912 7 days Aug. 12. 1912 Aug. 18, 1912 6 days Aug. 11, 1912 Aug. 20, 1912 9 days Aug 14, 1912 Aug. 21, 1912 7 days Aug. 13. 1912 Aug. 21, 1912 S days Aug. 13, 1912 Aug. 24, I9I2 9 days Aug. 14, 1912 Aug. 23, 1912 8 days Aug. 15, 1912 Aug. 23, 1912 9 days Aug. 16, 1912 Aug. 24, 1912 8 days Aug. 16, 1912 Aug. 24, 1912 8 days Aug. 17, 1912 Aug 25, 1912 8 days Aug. 17, 1912 Aug. 25, 1912 8 days Aug. 18, 1912 Aug. 24, 19)2 6 days Aug. 18, 1912 Aug. 26, I9I2 8 days Aug. 19, 1912 Aug. 29, 1912 10 days Aug, 18, 1912 Aug. 26, 1912 8 days Aug. 20, 1912 Aug. 27, 1912 7 days Aug. 20, 1912 Aug. 27, 1912 7 days These pupae were in cages in the laboratory with normal temperat^ure. The average length of the period was 7.85 days. 18 Texas AGKicuLTUR-iL Experiment Station Table 14. — Duration of pupal stage, 1912. Pupated. Emerged. Period. Oct. 3T 1912 Nov. 20, 1912 21 days Nov. 1 1912 Nov. 20. 1912 19 days Nov. 7, 1912 Nov. 22, 1912 19 days Nov. 3 1912 Nov. 24, 1912 21 days Nov. 1 1912 !Mov. 22, 1912 21 days fi 1912 Nov. 24, 1912 18 days Nov. fi 1912 Nov. 21, 1912 18 days 1 1912 Nov. 15, 1912 15 days 7 1912 Nov. 25. 1912 IS days Nov. 7, 1912 Nov. 25, 1912 19 days These pupae were in cages in the laboratory with normal artificial heat. The average length of the pupal period was 18.9 days. Tabic 15. — Duration or pupal stage, 1912. Pupated. Emerged. Period. Nov. 3, 1912 Nov. 1. 1912 Oct. 26. 1912 Oct. 28, 1912 Nov. 10, 1912 Nov. 1. 1912 Nov. 1. 1912 Nov. 30. 1912 Nov. 30. 1912 Dec. 1, 1912 Dec. 5, 1912 Dec. 20, 1912 Dec. 15, 1912 Dec. 15. 1912 27 days 30 days 35 days 38 days 40 days 45 days 45 days These pupae were kept under the same conditions as those recorded in table 14. From this it is obvious that a portion of the pupae have a prolonged period. For those recorded in table 15 the average length of the period was 35.5 days. ADULTS DESCRIPTION Although very familiar to many beekeepers, the beemoth is yet not definitely known to many who should be acquainted with it in order that they might more readily combat it. Having been a pest for such a long time, it is remarkable that more beekeepers are not acquainted with this pest of the apiary. Perhaps the reason that these moths are not more commonly known is due to the fact that they are seldom to be seen on the wing, except at dusk, unless frightened from their hiding places. The adult beemoth is about five-eighths of an inch (fifteen milli- meters) in length, with a wing expanse of about one and one-quarter inches (thirty to thirty-two millimeters). The moth with its wings folded appears ashy gray in color but the back third of each front wing is bronze colored. This wing is thickly covered with fine scales which Tub off easily when the moth is touched. On the outer and rear mar- gins of the forewing is a scanty row of short hairs. The hind wings are uniform in color, usually gray, with traces of a few black lines The Beemoth or Waxworm 19 extending from the outer margin inward toward the base; on the outer and rear margins is a thick fringe of hairs on which is a dark line running parallel with the border of the wing. The body is brown, the shade varying with a covering of scales. These scales rub oS easily and are not always present on the older moths. The male is slightly smaller than the female. A difference between the sexes is noticed in the fore- wing, which in the ease of the male is deeply scalloped on the outer mar- gin. This scallop carrier a heavy fringe of hairs, almost black in color. Another difference is in the mouth parts, the palpi of the male being rudimentary. EMERGENCE The moths emerge during the first part of the evening. In the cages emergence started just before sundown (6:30 p. m.) and no moths emerged after 9 p. m. They at once sought some protected place in which to expand their wings and dry, and by the next morning they. were able to fly. The first and lasit emerging individuals of the brood are smaller in size than the average, regardless of sex. The quantity of the food has a great deal to do with the size of the adults. The last larvae of the brood are always undersized, but are almost always able to pupate and reach maturity. DURATION OF LIFE In no instance were the moths of either sex seen to feed during their existence. In many cages fresh cappings were supplied for oviposition but the honey in those cappings did not serve as food for the moths. jSTo other attempts were made to supply food to the moths. The dura- tion of the life of the adults varies greatly, apparently depending upon several conditions, retarded fertilization and oviposition, brood and tem- perature conditions. The males usually live longer than the females, as shown in table 16. The average length of the life of a female was twelve days and the average length of the life of the male was twenty- one days. The average time that the male lived longer than the female was nine days. Table 16. — Length of adult life. Date. Female lived. Male, lived. Jan 5, 1912 21 days G days 11 days 12 days 12 days 21 days .30 days Sept. 5, 1912 Sept 9, 1912 IS days 21 days Sept 9, 1912 18 days Sept 10 1912 . . . 21 days Sept. 19. 1912 21 days PROPORTION OF SEX On October 25, 1912, 182 larvae were placed in separate vials to de- termine sex and rate of emergence. The results are shown in table 17. 20 Texas Ageicultueal Experimext Station Table 17. — Proportion of sex. Date. Males. Females Nov. 20, Nov. 21 , Nov. 22, Nov. 23, Nov. 24, Nov 25, 1912 1912 1912 1912 1912 1912 . . . 3 5 5 4 3 1 2 2 1 5 13 1 2 4 3 2 1 7 11 4 .5 4 3 1 Nov. 26. 1912 3 Nov. 27. Nov. 29. Nov 30, 1912 1912 1912 1 Dec. 1 , Dec. 2, Dec 3. 1912 1912 1912 .... 2 14 9 Dec. 4. Dec .") . 1912 1912 4 2 Dec. 6. 1912 3 Dec. 7. Dec 8. 1912 1912 . . . 6 9 Dec. 9. Dec. 10. 1912 1912 Dec. 11, Dec. 12, 1912 1912 6 16 78 104 The results that are given would seem to indicate a preponderance of females and a tendency of the males to emerge somewhat ahead of the females. Another collection of eightj'-five larvae was made on November 29, 1912, and each larva was placed in a separate vial. The emergence record of this collection is shown in table 18. Table 18.- -Proportion of sex. Date. Males. Females. Dec 1, 11, 12, 19, 26. 18. 19, 1912 2 7 14 3 3 2 6 13 19 3 2 1 De- 1912. .. Dp" 1912 . , Dec. 1912 Dec. 1912 1913 Tnn 1913 31 44 In this case there were more females than males and the early emer- gence of the males is more pronounced than in table 16. On November 30, 1912, another collection of sixty-one larvae was made. These larvae were placed in separate vials to observe the emer- gence of the adults. The results are shown in table 19. Table 19. — Proportion of sex. Date. Males. Females. Dec. 26, 1912 7 6 10 2 6 Dec. 30. 1912 Jan. 3, 1913 Jan. 6, 1913 Jan. 18, 1913 7 10 12 1 25 36 Tjie Bekmoth or Wax worm 21 1 The females predominate in this collection, as in those of the first two shown in tables IG and 17. Of the 318 larvae observed. 184 de- veloped into females and 134 into males. HABITS During the day the moths seek a sheltered place away from light and enemies, where they apparently settle down and draw their wings around them, remaining very quiet. Usually they are well protected by their color-, which resembles weatherbeaten wood. If disturbed during the day, the moths will make a dart or short flight, acting as though blinded by light. When an object is met, the moth quickly settles down and seems very anxious to a\oid flight. That the moths are hard to disturb in the daytime is shown by the fact that in several of the cages used in the experiments small ants attacked the moths and killed them without any apparent struggle on the part of the latter. Only by close examination could it be detected that the moths were dead and not resting in the usual manner. It is only during the later part of the oviposition period that the females are active during the daytime. The male moths are very active throughout their existence. PERIOD BETWEEN EMERGENCE AND COPULATION The moths under normal conditions probably mate soon after emer- gence. No cage observations were recorded but a series of unmated females were killed and examined to determine the condition of the eggs. The first moth was killed one hour after emergence. Many of the eggs were full size but were not close to the ovipositor. The second moth was killed fourteen hours after emergence. In this moth fully one-third of the eggs were fully developed and a very few were close to the ovipositor. The third moth was thirty-eight hours old when killed. Four eggs were extruded before death. The eggs were crowded close to the oviduct and were well rounded, the immature eggs being somewhat flattened on the ends. A count was made of 1128 eggs, 700 of which were full sized. The fourth moth was sixty-two hours old when killed. Fully two-thirds of the eggs were full sized, the remainder varying in size. The fifth moth was eighty-six hours old when killed. In this moth fully three-fourths of the eggs were full sized and they were closely packed in the lower portion of the repro- ductive orgam The next female was 110 hours old when killed. The eggs were of the same comparative size and condition as in the previous moth. Those eggs nearest the ovipositor, however, assumed a yellowish color. The next moth killed was 134 hours old. The proportion of full sized eggs was the same as in the two previous cases noted but the smaller eggs were increased in size. 22 Texas Agricultural Experiment Station LIEB OF UNMATED FEMALES Several observations were made on the length of life of the unmated females, which period is very irregular. The results of these observa- tions are shovra in table 30. Table 20. — Life of unmated females. Emerged. Died. Period. Mar, 19, 1912 Mar. 27, 1912 8 days Mar. 18, 1912 Ma-. 26. 1912 8 days Aug. 14, 1912 Aug. 22, 1912 8 days Aug 25, 1912 Aug. 31, 1912 fl days Aug. 20. 1912 Sept. 9, 1912 19 days Aug. 30, 1912 Sept. 9, 1912 10 days Aug. .30, 1912 Sept. 9, 1912 10 days Aug. 30, 1912 Sept, 9, 1912 10 days Sept. 1. 1912 Sept. 16, 1912 15 days Aug. 31, 1912 Sept. 19, 1912 19 days Nov. 20, 1912 -Oe-. 11, 1912 20 days MATING During the mating period the males are more active than the females, and at this time can be noticed "drumming" with their wings, the vibrations of which are at times sufficient to produce a low hum. Mating takes place at night, as would naturally be expected from the nocturnal habits of the species. In one case a pair of moths were observed in coitu early in the morning, but this was no doubt an abnormal condition and the female died in a short time. Another case was observed when the moths were in coitu from 7 p. m. till 10 :30 p. m. The next morning no eggs were deposited, but the following night the female began ovipositing. This was an exceptional case, as the female had been confined for a week after emergence before having the opportunity to mate. AGE AT BEGINNING OF OVIPOSITION The females begin to oviposit in a comparatively short time after .emergence. This period, of course, varies with the brood and within the brood. The comparative age of the male and of the female un- doubtedly has an influence on the length of this period. The results of the observations on the age of the female when the first eggs are deposited are shovm in table 21. The Beemoth or Waxworm Table 21. — Age of female. 33 Emorsed. Mar. 8, Mar. 6, Autf. 29, Aug. 26, AuH. 31, AuH. 29. Au^. 29, Au«. 2S, Aug. 30, Auo. 28, Sept 7. Sept. 4 , Sept. 3, Sept. 3, Nov. 23-, Nov. 26, Nov. 2'( . Dec. 2, Dec. 2, Dec. 2, Nov. 27, Nov. 25, Nov. 22, Dec. 3, Nov. 30, Nov. 30, Dec. 7, Dec. 10, Dec. 9, 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 First eggs. Mar. 14, Mar. 21 , Sept. I, Sept. 2. Sept. 2, Sept. 3, Sept. 3, .Sept. 3, Sept. 3, Sept. 5. Sept. 6. Sept. 10. Sept. 11. Sept. 11, Dec. 1 . Dec. 1 . Dec. 4, Dec. 4, Dec. 5, Dec. 5, Dec. 5, Dec. 5, Dec. 5, Dec. 7, Dec. 7, Dec. 9. Dec. 11, Dec. 14, Dec. 14, 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 1912 Age. 6 days 15 days 3 days 7 days 7 days 3 days 5 days 5 days 6 days 6 days 9 days 3 days 7 days 8 days 8 days 8 days 8 days 10 days 3 days 3 days 8 days 10 days 13 days 4 days 7 days 9 days 4 days 4 days 5 days PROCESS OF OVIPOSITION While depositing 'eggs the female seems mindful only of the task she is performing and is not easily disturbed. Some of the females appear nervous while ovipositing but work steadily. In the cages rela- tively small pieces of comb were supplied for oviposition. The female usually went over the top and down the sides of the comb, repeating this course continuously. In going over the top of the comb the ovi- positor is extended and appears to be dragged along. Apparently the interior of every cell was inspected by the ovipositor but never were any eggs deposited in these empty cells. Prom the top of the comb the female went to- the sides, where suitable places for oviposition were readily found. A very thorough inspection was made of the crevice before the eggs were deposited, sometimes a situation would not be' accepted one time but an egg would later be placed there. When a suitable location was found, the moth exerted a tremendous force back- ward, such as to bend the abdomen, perhaps to force the ovipositor into the crevice as far as possible. Then there was a moment of quiet when the body was rigid, then a quick jerk and the female was on her journey again. During the inspection work the antennae were vibrating con- tinuously but they were motionless while the egg was passing down the oviduct. The eggs are always securely fastened to whatever object they are laid upon. The eggs are always laid in cavities. In the cage experi- ments these were on the side of the comb, -often where the walls of cells had been turned in. Only one egg is deposited at a time, although in 24 Texas Agkicultueal Experiment Station working over the comb, the female often places the eggs close together. On the smaller pieces of comb, furnished to moths confined in cages, as many as seven eggs were found in a single cavity. The number of eggs actually deposited by one female has not been determined. In the cages, under artificial conditions, if the comb was not supplied for the female she would deposit her eggs in any rough place detected by her ovipositor. _In many instances the female would refuse to oviposit on cappings which were furnished in some of the cages, but would go around the base of the lamp globe in which they were confined and fill every crevice with eggs. Sometimes these eggs would be fastened on the outside of the glass, and in such cases the globe would be fastened to its resting place. TIME OE OVIPOSITION Oviposition usually takes place at night, beginning at early dusk. In every cage the most of the ovipositing was completed by 9 :30 p. m. On the last day the female may oviposit during the afternoon, especiallj^ if the day is cloudy or the cage is not directly exposed to light. HABITS DURING OVIPOSITION Whenever freed from the cages, the females always started immediately for the windows of the laboratory, but the males, when turned out, sought protection in darkened places. The female was active as soon as darkness started, but upon turning on the electric lights in the room, sought the darker places. If a bright light, such as a candle or read- ing lamp was placed close to the cage, however, the female at once attempted to reach the light. The male was not so readily affected by light, seeming to prefer quiet and protection. During the cool evenings of early fall, the moths are active only on those nights when no breeze is blowing. At this latitude the usual breeze stops during the later part of the evening,- and the moths may become active for a short period. The male is never found on the food during the oviposition period, and rarely is the male found on food preceding the period. The female is not found around the food before the oviposition period, but may often be found in the better protected places during the period. PERIOD OF OVIPOSITION The period of oviposition of the beemoth varies considerably within the brood as well as with broods. During the last part of the egg laying period the female appears to be in a great hurry, and during the last few days she deposits during the day, as well as during the night, at times stopping to rest. If disturbed during the resting period, she vigorously resumes her egg laying. The females usually die while ovipositing and the last three or four eggs are barely extruded from The Bkiiimoth ou AVaxwoem the ovipositor. If a female is being killed or injured, she will attempt to oviposit even al'ter she is unable to walk. The cage records on the period of oviposition are shown in table 23. Tnblo 22. — Period of oviposilion. Kirs I ogRs. Last eggs. Pe riud. M:ir, 9, 1912 Mar. 13. 1912 4 days Milr. 12. 1912 Mar. 17. 1912 ,1 day.s Mar. 20, 1912 Mar. 26, 1912 6 day.s Mill-. 21, 1912 Mar. 28 , 1912 8 days M.n. 27, 1912 April 7, 1912 13 days S,-pl 3. 1912 Sept. 6, 1912 .'i days Si'lU 5, 1912 Sept. 8, 1912 .i days Srpl ."i. 1912 Sept. 8, 1912 3 days •Srpl .">. 1912 Sept. 9, 1912 4 days .S,.pl i;. 1912 •Sept. 9, 1912 3 days .S,.pl <)' 1912 Sept. 18. 1912 9 days Nipt 12, 1912 Sept. 20. 1912 8 days IX',- 11. 1912 Dec. 19. 1912 8 days >>cv. II. 1912 Dee. 17, 1912 3 days r>cv 14, 1912 Dee. 18, 1912 4 days Dcr. 14, 1912 Dec. 20, 1912 6 day; Do". 16. 1912 Dec. 23. 1912 7 days EFFECT OF AGE OF SEX The age of the sex apparently does not have any constant effect on the fertility of the eggs. Females of excessive age were mated with freshly emerged males. Eggs were deposited which hatched. Females that had just emerged were mated with males otf excessive age and the eggs that were deposited hatched. In one cage a female was mated with a male that had mated in another cage the previous night. Mating took place and all eggs deposited hatched. The females will deposit their eggs even when they have not had the opportunity to mate. In all cases when the sexes were not properly paired the females would finally oviposit, the period of oviposition being, however, much shorter than the natural one. Although many females which did not mate were confined in cages, and although they deposited eggs, none of these unfertilized eggs ever hatched. It seems a fairly safe conclusion that parthenogensis does not occur with this species. RATE OF OVIPOSITION In many instances females have been observed depositing their eggs at the rate of one -every minute for a period of thirty minutes, and then after a short rest have continued again at the same rate. EFFECT OF HUMIDITY ON DEVELOPMENT Experiments were planned to determine the effect of a low humidity on the hatching of the eggs and development of the larvae and pupae. For this purpose a large egg incubator of the hot water type was secured. The temperature of ninety degrees F. was decided upon and a humiditv of approximately thirty-five per cent. A recording hygro- 26 Tesas Ageicultueal Experiment Station thermograph was placed inside the incubator and the records of the- temperature and the humidity maintained in the experiments are shown in table 24. The eggs were deposited by the moths in cages in the laboratory and. the following morning they were placed in the incubator. The larvae were supplied with ample food in the form of fresh eappings. The larvae that hatched in a twenty-four hour period were kept together. The results of this experiment are shown in table 23. The eggs of one batch were placed in the incubator over a period of one day. After the first twenty-four hour period, the hatching was very slow, only four to six larvae from a batch. The prolonged exposure in the in- cubator dried the eggs badly and after a short time the eggs would not hatch. The larvae started their work in the normal manner but this condition was soon changed. Table 23 — Effect ol humidity on development. Eggs deposited. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. .5 Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dor-. Dec. Dec. Dec. Dec. Dec. 11 Dec. 11 Dec. 11 Dec. 11 Dec. 11 1912.. 1912... 1912.. 1912... 1912... 1912... 1912.., 1912... 1912.. 1912... 1912.. 1912.., 1912... 1912... 1912.. 1912... 1912... 1912... 1912... 1912... 1912... 1912.. 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912.. 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... 1912... Eges hatched. Larvae died Dec. Dec. Dec. 13, 1912 15. 1912 16, 1912 Dec. Dec. Dec, Dec. Dec. Dec. Dec. Dec. 16, 1912 16, 1912 17, 1912 18, 1912 19, 1912 20, 1912 22, 1912 23, 1912 Dec. Dec. Dec. Dec. 17, 1912 16, 1912 17, 1912 IS, 1912 Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. 2R, 1912 24, 1912 24, 1912 26. 1912 12, 1912 16, 1912 17, 1912 IS, 1912 Dec. Dec. Dec. Dec. 17. 1912 18, 1912 20, 1912 24, 1912 Dec. Dec. Dec. Dec. 20, 1912 21, 1912 22, 1912 23, 1912 Dec. Dec. Dec. Jan. 20. 1912 2). 1912 27, 1912 2, 1913 Dec. Dec. i >ec. Dec. Jan. 22. 1912 23. 1912 26, J912 28. 1912 1, 1913 Dec. Dec. Dec. Dec. Dec. 23, 1912 24, 1912 25, 1912 26, 1912 28, 1912 Mar. 24, 1913 April 12, 1913 Mar. ,5, 1913 April 5 , Mar. 24, April 12. Mar. 24, April 5, Mar. 24, April 12, Mar. IS, 1913 1913 1913 1913 1913 1913 1913 1913 Mar. 24, April 12, Mar. 24 , Mar. 24, 1913 1913 1913 1913 Mar. 24. Mar. 24, April 5, April 5, April 12, Dec. 31. Mar. 24, April 12, 1913 1913 1913 1913 1913 1912 1913 1913 Mar. 24, Mar. 12, April 12, Mar. 5 , 1913 1913 1913 1913 Mar. 24, Feb. 26, April 5 . April 5, 1913 1913' 1913 1912 April 5 , April 5 . April 5 , Feb. 21, 1913 1913 1913 1913 Mar. 12, An.ril .'>, Mar. 12, April o . Mar. 24, 1913 1913 1913 1913 1913 Feb. 21, Feb. 26, Mar. 24, Mar. 24. Mar. 24, 1913 1913 1913 1913 1913 Remarks. Work very unnatural. Variation in size. Very little growth. No egg,« hatched. Larva died. Larva one-third ?;rown. Larva one-fourth grown. Larva one-fourth grown. Larva one-half grown Very little work. Larva one-fourth grown, verv little work. No eggs hatched. Larva one-third .grown . Very httle growth. Larva one-half grown. Very little growth. No eggs hatched. Larva one-half grown. Verj' little growth. Larva two-thirds grown.. Larva two-thirds grown. Larva one-third grown Very little work. Larva one-third grown. Larva one-half grown. No eggs hatched. Larva one-third grown. Larva one-third grown. Larva one-third grown. Larva one-third grown No eggs hatched. Very little work. Very little work. Lgrva one-fourth crown.. Larva one-fourth grown- -Nc eggs hatched. Ver"/ little work. Larva dried. Larva onc-tliird grown. Very little work. No egg,s hatched. I-arva one-fourth grown- Larva one-fourth grown. Very little work. Very little work. Larva two-thirds grown. No eg.ps hatched. Larva one-third grown. Very little work. Very little work. Very little work. Very little work. The BicK.MdTii on Waxwokh Table 23. — Effect of humidity on development— conlinued. 2r Eggs de|)u:.iti'd. I'lgv's 1 inldied. Doc. 29. li)12 ■ Ian. 2. 1913 Jan. 9, 191,) ■Dec'.'23;'i9i2 Dec. 21. 1912 Dec. 26. 1912 Dec. 2(i, 1912 'bee. is; 1912 Dec. 17, 1912 Dec. 19. 1912 Dec. 22, 1912 Dec. 26, 1912 Dec. 31, 1912 Jan. 2, 1913 Larvae died. Remarks. Dec. 11, 1912 , Dec. 11, 1912 Dec. 11, 1912 Dec. 11, 1912, Dec. 11, 1912 . . . Dec. 11, 1912 . , Dec. 11, 1912. Dec. U. 1912 Der. 11, 1912 Dec. 3, 1912. Dec. 3, 1912 D,c. 3, 1912. .. . Dec. 3, 1912 Doc. 3, 1912 Dec. 3, 1912 Dec. 3, 1912 Dec. 3, 1912 Mar. 21, 1913 Mar. 2 1, 1913 I'Oh. 21. 1913 ' April' 12' 'igi.'i Mar. 21, 1913 Mar. 21. 1913 April 12, 1913 Mar.',3ii '1912 ' Mar. 31. 1913 Mar. 31. 1913 Mar. 31, 1913 Mar. 31, 1913 Mar. 31, 1913 Mar. 31, 1913 Larva two-thirds grown. Larva two-lhirds grown. Very little growth. No eggs hatched. Larva two-thirds grown. Very little work. Very little work. Larva one-half crown. No eggs hatched. Larva two-thirds grown. Very little work. Very little work. Very little work. Very little work. Very little work. Very little work. No egos hatched. Very little work. Very little work. Very little work. No eggs hatched. Very little work. Very little work. Very little work. ■Very little work. Very little work. Very little work. Very little work. Very little work. No eggs hatched. Dec. 21, 1912 Dec. 22, 1912 Dec. 26. 1912 Mar. 31, 1913 Mnr. 31, 1913 Mar. 31, 1913 Dec. 5, 1912 Dec. 5. 1912 Deo. 5. 1912 Dec. M, 1912 Dec. 14, 1912 Dec. 14. 1912 Dec. 14, 1912 Dee. 30, 1912 Dec. 31. 1912 Jan. 1. 1913 Jan. 2. 1913 •Jan. 3, 1913 Jan. 4, 1913 Jan. 5. 1913 Jan. 7, 1913 Mar. 3i, mh Mar. 31. 1913 Mar. 31, 1913 Mar. 31. 1913 Mar. 31, 1913 Mar. 31, 1913 Mar. 31, 1913 Mar. 31, 1913 Dec. 14. 1912 Dec. 14, 1912 Dec. 14. 1912 Dec. 14, 1912 Dec. 14. 1912 ' for the temperature maintained, the work of the larvae was much prolonged. The table shows that in no case did a larva ever reach more than two-thirds normal size when it died. Very quickly after death the larva would become dry and hard. IJsually the larva grew but little in the long period of exposure and a point was reached where growth was not possible and death ensued. In table 24 is shown the mean atmospheric humidity prevailing at College Station over the period of months when the experiment was conducted. Compared with this is the mean humidity of the incubator where the experiments were conducted. Table 24. — Mean atmo spheric humidity prevailing at time of experiment. In'-iibator. Room. Hunnidity. Temperature. Temperature. Dale. Max. Min, Mean. Max. Min. Mean. Max. Min. Mean. Dec. 1, 1912 43 37 4D.C 92 90 91.0 Der. 2, 1912 41 38 39.5 92 91 91.5 Dec. 3, 19-12 41 40 40.5 92 91 91.5 Dec. 4 1912 45 44 44.5 93 92 92.5 Dec 5 1912 44 37 40.5 93 91 92.0 Dec 6 1912 37 36 36.5 91 89 90.0 Dec. 7. 8, 9, 10 1912 I9I2 1912 1912 38 38 35 39 35 35 31 35 .36.5 36.5 33.0 37.0 93 91 93 92 91 90 90 90 92.0 90.5 91.5 91.0 Dec Dec. Dec. Dec. 11 1912 39 33 36.0 92 88 90.0 Dec. 12, 1912 36 31 33.5 92 89 90.5 Dec. 13, 1912 32 30 31.0 92 90 91.0 Dec. 14, 1912 34 32 33.0 93 90 91.5 38 Tksas Agricultural Explrimkxt Statiok" Table 24. — Mean atmDspheric humidity prevailing at time of experiment — continued. Incubator. Date. Humidity. Temperature. Max. , 1912 34 . 1912 47 , 1912 41 , 1912 43 , 1912 42 , 1912 43 . 1912 42 , 1912 40 , 1912 35 , 1912 34 , 1912 35 . 1912 35 , 1912 33 , 1912 36 . 1912 37 , 1912 34 , 1912 35 , 1913 37 , 1913 34 , 1913 33 . 1913 39 , 1913 41 . 1912 33 . 1913 32 , 1913 31 , 1913 31 , 1913 41 , 1912 3S , 1913 33 . 1913 34 , 1913 37 . 1913 42 , 1913 44 , 1913 45 . 1913 45 , 1913 45 , 1913 42 , 1913 33 , 1913 35 , 1913 35 , 1913 34 , 1913 34 , 1913 33 , 1913 30 . 1913 30 . 1913 31 , 1913 31 , 1913 26 , 1913 27 , 1913 27 , 1913 33 , 1913 39 , 1913 39 , 1913 39 , 1913 37 , 1913 35 , 1913 37 , 1913 41 , 1913 41 , 1913 37 , 1913 36 . 1913 35 , 1913 35 . 1913 37 , 1913 41 , 1913 47 , 1913 47 1913 45 1913 43 1913 38 1913 37 1913 32 1913 38 1913 40 Min. Mean. Max. Min. Mean. Temperature. Max. Min. Mean. Dec. 15 Dec. 16 Dec. 17 Dec. 18 Dec. 19 Dec. 20 Dec. 21 Dec. 22 Dec. 23 Dec. 24 Dec. 25 Dec. 26 Dec. 27 Dec. 28 Dec, 29 Dec. 30 Dec. 31 Jan. 1 Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan Jan. Jan. Jan. Jan. Jan. Jan. Jan.' 12 Jan. 13 Jan. 14 Jan. 15 Jan. 20 Jan. 21 Jan. 22 Jan. 23 Jan. 24 Jan. 25 Der. 28 Jan. 29 Jan. 30 Jan. 31 Feb. Feb. Feb. Feb. Feb. Feb. Feb. . Feb. S Fib. 9 Peb. 10 Feb. 11 Feb. 12 Feb. 13 F,-b. 14 Feb. 15 Feb. 16 Feb. 17 Feb. 18 Feb. 19 Feb. 20 Feb. 21 Feb. 22 Feb. 23 Feb. 24, Feb. 25 Feb. 26 33 42 36 35 38 42 41 38 3 32 33 30 31 32 31 33 33 31 31 30 3: 33 30 30 30 29 31 32 32 33 33 37 42 42 42 44 33 33 34 33 32 32 31 28 28 27 28 24 23 26 32 33 38 36 33 33 33 33 3B 35 35 35 34 35. 38 41 45 42 36 35 32 28 30 31 .33.5 44.5 38.5 39.0 40.0 42 41.5 39.0 31.0 33.0 34.0 32.5 32.0 .34.0 35.2 33.5 34.0 31.0 32.5 31.5 35 37.0 31.5 31.0 30.5 30.0 36.0 35.0 32.0 38.5 35.0 39.5 43.0 43.5 43.5 44 5 37.5 33.0 34.5 ,34.0 33 33.0 32.0 39.0 29.0 29.0 2.8.5 25.0 25.0 26.5 .32.5 36.0 38 37.5 35.0 34.0 35.0 39.5 38 36 35 35 34 36 39 44.0 48.0 43.5 39.5 36.5 34.5 30.0 .34.0 35.5 91 93 91 90 91 91 90 89 91 92 92 94 93 93 93 90 90 91 92 92 1 931 92l 93 92 92 93 92 93 90 91 92 92 93 92 92 90 93 92 92 91 93 92 91 91 91 91 91 92 90 92 92 91 91 92 90 90 89 92 92 93 91 90 91 91 92 92 92 91 91 92 91 92 92 92 89 89 90 88 90 89 89 88 89 9u 90 91 90 91 90 86 89 89 89 89 90 85 89 89 88 89 90 89 88 88 90 90 90 90 90 89 89 89 89 90 90 90 89 87 89 89 89 89 87 88 90 89 90 89 88 87 88 91 91 90 89 89 88 89 90 90 90 90 89 89 89 88 92 90 .-0 91.0 90.5 89.0 90.5 90.0 89.5 88.5 90.0 91.0 91.0 92.5 91.5 92.0 91.5 88.0 89.5 91.5 90.5 90.5 91.5 88 . 5 91.0 90.5 90.0 91.0 91.0 91.0 89 89.5 91.0 91.0 91.5 91.0 91.0 89.5 91.0 90.5 90.5 90.5 91.5 91.0 90.0 89.0 90.0 90.0 90.0 90.5 89.5 90.0 91.0 90.0 90.5 90.5 89.5 88.5 88.5 91.5 91.5 91.0 90.0 '89.5 89.5 90.0 91.0 91.0 91.0 90.5 90.0 90.5 90.0 90.0 92.0 90.5 73 72 69 73 77 68 72 75 73 78 74 74 72 75 80 73 75 80 75 80 69 80 80 79 80 80 83 77 82 81 SO 78 79 79 73 77 73 75 73 80 75 75 72 74 74 78 71 74 67 73 77 74 70 67 72 71 76 75 73 71 75 85 70 83 83 74 58 60 62 59 60 62 60 56 61 64 59 60 71 55 62 60 59 64 67 62 60 66 69 72 74 73 73 73 67 67 67 69 69 69 65 61 67 66 66 65 65 60 62 66 68 65 59 68 60 66 6: 63 61 60 63 64 68 68 70 70 65 65 62 64 70 67 The Bbemoth or Waxworm 29 Tabic 24.— Mean atmospheric humidity prevailing at time of experiment— oontinued. ito. Incu balor. Room. Di Humidity. Temperature. Temperature. Max. Min. Mean, Max. Min. Mean. Max. Min. Mean. Feb. 27 Feb. 28 Mar. 1 Mar. 2 1913 , 1913 . 1913 . 1913 33 32 30 26 30 27 23 23 31.5 29.5 26.5 24.5 90 91 91 92 90 88 90 90 90.0 89.5 90.5 91.0 72 69 82 69 66 59 61 60 69.0 64,0 71.5 64 5 , 1913 32 29 30.5 91 90 90.5 80 72 76 Mar. 4 1913 32 28 30.0 93 91 92 78 65 71 5 Mar. 5 1913 30 25 27.5 92 91 91.5 71 66 68 5 1913 32 25 28.5 94 85 89.5 80 67 73 5 Mar. 7 1913 28 24 26.0 93 91 92.0 83 74 78 5 Mar. 8 1913 28 25 26.5 91 91 91.0 79 73 76 Mar. 9 1913 30 28 29.0 91 91 91.0 77 65 71 Mar. 10 1913 33 30 31.5 92 91 91.5 82 67 74 5 Mar. n 1913 36 31 33.5 92 91 91.5 85 68 76 5 Mar. 12 1913 39 32 35.5 92 91 91.5 80 70 75 Mar. 13 1913 32 22 27.0 93 91 92.0 90 66 78 Mar. 14 1913 24 21 22.5 92 89 90.5 90 60 75 Mar. 15 1913 21 19 20.0 93 90 92.0 80 60 70 Mar. 16 1913 19 19 19.0 91 ' 90 90.5 76 58 67 Mar. 17 1913 24 20 22.0 92 88 90.5 74 62 68 Mar. 18 1913 28 24 26.0 93 89 91.0 70 64 67 Mar. 19 1913 33 28 30.5 94 92 93.0 69 68 68 5 Mar. 20 1913 33 25 29.0 94 89 91.5 80 ' 63 71 5 Mar. 21 1913 25 25 25.0 92 89 90.5 80 63 71.5 Mar. 22 1913 30 26 28.0 90 90 90.0 71 63 67 Mar. 23 1913 33 30 31.5 92 90 91.0 74 71 72 5 Mar. 24 1913 46 44 45.0 93 90 91.5 77 73 75.0 Mar. 25 1913 1913 1913 1913 46 40 36 34 40 32 28 30 43.0 36.0 32.0 32.0 92 92 92 93 91 90 88 90 91.5 91,0 90.0 91.5 Mar. 26 Mar. 27 Mar. 28 75 65 70.0 Mar. 29 1913 30 33 36.0 91 89 90.0 74 68 71,0 Mar. 30 1913 38 37 37.5 91 90 90.5 70 64 -67.0 Mar. 31 1913 40 38 39.0 93 90 91.5 82 71 76:5 April 1 1913 48 38 43.0 93 91 92.0 78 72 75.0 April 2 1913 50 45 47.5 95 86 90.5 76 74 75.0 April 3 1913 47 38 42.5 91 86 88.5 74 69 71.5 April 4 1913 40 34 37.0 92 88 90.0 72 65 68.5 April 5- 1913 34 30 32.0 92 90 91.0 75 68 71.5 April 6 1913 38 32 35.0 90 88 89,0 74 70 72,0 April 7 1913 42 38 40.0 92 90 91.0 ■ 77 70 73.5 April 8 1913 45 42 43.5 92 91 91,5 74 70 72.0 April 9 1913 43 39 41.0 91 88 89,5 72 65 68.5 April 10 1913 41 31 36.0 91 89 90.0 67 63 65.0 April 11 1913 33 30 31.5 92 90 91.0 67 62 64.5 April 12 1913 32 22 27.0 93 90 91 5 67 61 64.0 Apiil 13 1913 22 i.8 20.0 91 89 90.0 68 62 65.0 Table 25 — Comparison of humidity. Year. Atmosphere. December. January. February. March, 1914 78.1 74,2 64,5 62,8 64,6 72.8 78.9 70.8 72.0 69.6 66.7 .•58.7 70,5 1915 72.6 1916 61,8 1917 63.0 Average . . 69.9 71.8 66.8 66.7 • Year. Incubalor. December. January. February. March. 1912 36.7 1913 34.2 35.7 .30.0 30 Texas Ageicultural Experiment Station SEASONAL HISTORY From the work which was done in trying to identify the different broods or generations of this insect, it appears that there are three broods in the extreme southern part of the United States. The third brood is not nearly so large as the first two, due to the fact that some of the second brood of larvae do not pupate until late fall. There is a decided overlapping of the generations, which makes it difficult to determine the exact number of broods a year. At almost any time, from early spring until December, examination of a colony of bees is likely to reveal this insect in all stages. It is often assumed from this that the life history is short and there are several generations each year. In well protected hives the development may continue throughout the year' without interruption. Usually the winter is passed with about one-third of the insects in the pupal stage and the remainder in the larval stage. Warm spells during the winter cause some of the moths to emerge from their cocoons; in the laboratory many moths emerged when the temperature was maintained constantly at sixty degrees F. It is not unusual to see moths on the windows of the honey house, try- ing to escape during the warm spells in December and January. Their presence may be accounted for on the supposition that they have just emerged from their cocoons or that they may have been in hibernation as adults and become active with the rise in temperature. Such moths do not reproduce in localities where freezing temperatures are fre- quent. Even the most vigorous moths cannot withstand a freezing temperature for more than three days. Moths in well protected places can survive an outside temperature as low as twenty-six degrees P. for as long as five days. The moths are never active during the day when the temperature is below fifty degrees P., so at such times reproduction does not take place. For College Station, Texas, the following life history and duration of broods has been carefully determined: The maximum number of moths which mature from the over-winter- ing larvae and pupae appear about the first of April. These moths are active for some time before any eggs are deposited and it is the middle of April before the eggs _ are laid for the first brood of larvae. Usually twelve days are required for the eggs of this brood to hatch, so that by the first of May most of the first brood of larvae are out. The larval period of this brood is quite long, most of the larvae feeding at least forty-five days before completing their growth. A majority of the larvae of the generation are ready to pupate by the middle of June, but there is a considerable variation in the rate of growth, for some of these larvae feed for six weeks longer before attaining full size. The pupa- tion of the first brood takes place during the last two weeks in June, and by July 1 some of the moths of the second generation a]:e to be seen. The moths of this generation emerge at about the same time and give the impression of constituting a very large brood. Most of the eggs are laid very soon after emergence of the moths and by the middle The Beemoth or Waxworm ' 31 of July all of the eggs of the second generation are deposited. The high temperature at this time of the year shortens the egg period, only ten days being required for these eggs to hatch. There is a considerable variation in the maturing of this brood of larvae. Normally the larval period is shorter than for the first brood and by the first of September many of the larvae are full grown. Some of the larvae may continue to feed for four weeks longer and then pupate. Some of the larvae which mature early in September may pass through a short pupal stage and soon emerge as moths. This accounts for the appearance of the number of moths about the first of October. This brood is usually small and scattered and few of the larvae which result from the eggs of these moths reach full size. Some of the larvae of the second generation do not pupate during the fall, but live over the winter in the larval stage and pupate the following spring. The following summary shows the stages which normally occur dur- ing each month of the year at College Station, Texas: April : itoths reach maturity from the over-wintering larvae and pupae. Eggs are deposited. May: Eggs hatch. Larvae are about three-fourths grown. June: Larvae reach maturity. Some pupae. July: Pupae. Adults of the second generation. Eggs deposited by the second generation of moths. August: Larvae of the first generation. Pupae of the first generation. Moths of the second generation. Eggs of the second generation. Larvae of the second generation. September: Pupae of the first generation. Moths of the second generation. Eggs of the second generation. Larvae of the second generation. Moths of the third generation. Eggs of the third generation. October: Larvae of the second generation. Pupae of the second generation. Moths of the third generation. Eggs of the third generation. November: Larvae of the second generation. Pupae of the second generation. Larvae of the third generation. December: Same stages as during November. January: Same stages as during November. February: Same stages as during November. March: Pupae. 32 TexIs Agricultural Experiment Station NATURAL CONTEOL PREDACEOUS ENEMIES Of the natural enemies of the beemoth, the most important is the honey bee itself. It is a well established fact that if the colony be kept strong, healthy, and with a vigorous queen, it will defend itself against the beemoth. This is particularly true in the case of "Italian" bees. In the Ohio Cultivaior for 1849, page 185, Micajah T. Johnson says, "One thing is certain: if the bees, from any cause, should lose their queen and not have the means in their power of raising another, the miller and the worms soon take possession. I believe no hive is destroyed by worms while an efficient queen remains in it." This seems to be the earliest published notice of this important fact by an American observer. This fact is of vital importance in the fight against the beemoth, for if the pest can be kept from its favorite food control measures are made much easier. The fact that the bees under natural conditions are able to defend themselves should leave the problem of control to such means as will destroy the pest in places other than the hives. Recently it has been found advantageous to introduce Italian blood into the colony, as the workers of this race seem to be more efficient fighters of the beemoth. In most cases this is sufficient for the control of the pest in the colonies, but it must be remembered that the colony cannot be kept under close observation and maintained at full strength unless domiciled in a frame hive. A small red ant, Solenopsis geminata Pab. was found to be an enemy of the beemoth, as many of the cage experiments were destroyed by these ants killing the moths and larvae. The attack is made on the moths during the day or when they are at rest. Usually the ant crawls under the wings of the moth, and begin the attack on the abdomen. There is no apparent struggle on the part of the moth, for close exam- ination was necessary to determine that the moth was dead and not resting. The abdomen seems to be all that is desired, and this is car- ried away in small pieces to the nest of the ants. The same species of ant also destroyed moths which had recently been prepared for exhibits. At such times only the abdomen was taken by the ants. In their attacks on the larvae the ants entered the cages and crawled over the comb and wax ia search of their prey and if any larvae were exposed, they were attacked. The larger larvae are more frequently attacked, as they are less active and usually feed in more exposed places than do the smaller ones. Unless the larvae were well protected by webs in the refuse, they were destroyed by the ants. Apparently there are days and even parts of days when the ants are most active in their destruction. Never were the ants present in sufficient numbers to attempt tracing them to their nests. No observations have been made upon this ant in or about the apiary and while it proved very destructive under arti- ficial conditions, the moths and larvae might be better able to protect themselves under natural conditions. The Bekmoth or Waxworm 33 PARASITES Three hymenopterous parasites have been recorded from the beemoth. One is chaleid, Eupelmus ccreanus, found by Eoudani in Italy; another is Bracon bravicoriiis, which was found by Marshall in France, and a tliird species, Apentoles lateralis, was recently found by A. Conte in France. The last species was found near Lyons, where it spread very rapidly. It is apparently of considerable importance since it has also been reported to attack the larvae of several other moths in England and Germany. The adult parasite is about one-sixtTi of an inch (4 mil- limeters) in length, very lively, and avoids light. The body is black and the wings are transparent, with black specks. The larvae of the beemoth are attacked while quite young and never attain a large size. A single parasite develops in each larva. The bees are said to pay no attention to the presence of the parasite, so that it can easily enter the hive in search of the beemoth larvae. It was artificially introduced into hives by Conte with very satisfactory results. CLIMATE Cold is perhaps the greatest climatic factor in the control of the beemoth. Young and vigorous moths when exposed were able to with- stand a temperature of a few degrees below freezing. When a temper- ature of twenty-eight degrees F. occurred, all exposed moths were killed. Even with some protection all moths did not survive this temperature. The young moths may withstand this temperature for one night, but not for a longer period of time. The larvae are greatly susceptible to cold. Larvae of all ages even up to two-thirds grown, were placed out-of-doors, and with only the meager protection of light tunnels and small pieces of comb. When- ever a temperature of thirty-two degrees F. was reached, all larvae were killed. Under natural conditions the larvae are better protected to pass through the winter by better built feeding galleries. The nat- ural mortality was observed in an infested hive that was allowed to remain untouched throughout the winter. The results are shown in table 26. It will be seen that the high mortality was among the larvae. Of the 208 larvae examined, 146 or seventy per cent, were dead, and of the 158 pupae examined, eight or five per cent, were dead. TablR 26. — Mortality uT waxworms. Date. Alive. Larvae. Pupae. April 9, 191.3 ai6 66 150 Di-ad. Larvae. Pupae. 150 142 8 34: Texas AGErcuLTUR.4.L Experiment Station AETIFICIAL CONTEOL Unfortunately, the only natural enemy of the beemoth that is present to any great extent in Texas is the honey bee itself. In the absence of any natural enemies of importance, the measure of artificial control must be made all the more effective if the beekeeper is to free his apiary of the pest. If the moths are driven from the hives by strong colonies of Italianized bees, they will surely seek scraps of comb and wax about the ground and stored comb and honey in the honey house. It seems quite likely that in such cases the eggs are deposited as near to the comb as possible, as along the cracks between the supers, and the larvae, after hatching, find their way to the comb through crevices much smaller than the moth can enter. TRAP LIGHTS Trap lights were employed to learn if the moths were attracted to them. On September 10, 1913, a large lantern and an acetylene lamp were placed in an apiary where the beemoth had been present con- stantly. The lights were so placed as to throw the rays across the apiary over a great number of hives. The night was warm, clear, and still. The lights were run from 7:30 to 10 p. m. Not a single bee- moth was ever present at either light. Again on September 27, 1913, the trap lights were put in operation in the same apiary. The day had been rainy and it was still misty in the evening, which was also very dark. No moths were ever at the lights. DECOY BOXES Decoy boxes containing pieces of comb were placed in and around the apiary. These were put out during September, 1913, and remained under observation until December. None of the twelve boxes was ever infested during this period. FUillGATION One of the methods of artificial control, and one upon which many beekeepers depend, is fumigation of combs and honey. Gas is able to penetrate material that it is not possible to treat in any other manner. The fumigation process is not difiicult, for when once started no fur- ther attention is necessary until the treatment is complete. It is not necessary to watch the entire process. Stored material, such as comb honey and empty combs, should be examined from time to time and at the first evidence of the waxworm they should be fumigated. Stored material of this kind should be examined at least once every week dur- ing the summer and once every month during the winter season, so as to ■ detect the infestation at the start. In the present investigation two materials have been used in the fumigating experiments. These were selected because almost every bee- keeper is acquainted with them and they can be obtained in practically The Beemoth or Waxwoem 35 every locality at a reasonable price. They are sulphur and carbon bisulphide, or "high-life." Sulphur Dry powdered sulphur, or "flowers of sulphur," is a light yellowish powder with which everyone is familiar. AVhen sulphur is burned, it unites with the oxygen of the air and forms a poisonous gas known as "sulphur dioxide."' This gas is effective in kilUng some kinds of in- sects, including the waxworm. A common method of burning the sulphur is to place it on a pan of red hot coals and immediately tier up the infested supers over the burning sulphur. The bottom super should not contain any infested material and the pile should be covered as quickly as possible. A number of experiments were made with sul- pher fumigating combs containing waxworms. The result of these experiments are given in table 36. Table. 26. — Results of fumigaling infested combs with sulphur dioxide. Stagp of beemoth. Amount of sulphur used par cubic foot. Time the combs were confined to fumes. Effect. Larvae. . . . One-fourth ounce One hour Killed One hr;ur Killed Two-lhirds ounce Killed The larvae which were used for these experiments were from ten to twentj' days old and in every case they were well protected by the webs and refuse. The larvae which were used in the experim.ents were of different ages and some better protected than others. When the larvae are not very well protected, they are quite susceptible to the gas, but the larger larvae, which are often enclosed in a iuass of webs, were not killed except when extremely large doses of sulphur were used. From the experiment with sulphur dioxide, it is_ evident that only extremely large doses will effect the eggs of the beemoth; so large, in fact, that such fumigation would not be practical. These results seem to indicate that the sulphur fumes are not ordi- narily penetrating enough to effect the eggs, and only when the larvae are young and not well protected will the gas 'effect them. While the method is simple, there are minor details upon which the success of the operation depends. The sulphur must be burned at a high tem- perature in order to generate the most effective gas. While the method is generally effective under proper conditions, it cannot be recom- mended in preference to fumigation with carbon bisulphide. Carlon Bisulphide {"Eigh Life") The commercial bisulphide is an oily liquid, very volatile and exceed- ingly foul smelling. It is cold to the touch and because of its rapid evap"oration it produces a freezing sensation when dropped on the skin. When exposed to air at ordinary temperatures the bisulphide rapidly changes to a gas or vapor which is a little more than two and one-half 36 Texas Ageicultue.4l Experiment Station times as heavy as air. This is a point to be remembered in its use, since it goes first to the bottom of whatever it is confined in. When mixed with air, it becomes highly inflammable and sometimes explosive. Such a mixture of air and bisulphide gas may be exploded by even a spark such as might be made by hitting a nail with a hammer. The liquid, upon evaporation, leaves a residue of impurities. Its rate of evaporation is in proportion to the temperature and the area of the exposed surface. Its efficiency is the greatest with the rapid evapora- tion, and this is secured in relatively warm weather, but artificial heat must never be used to hasten its changes into gas. Carbon bisulphide is obtainable from practically every druggist. When carbon bisulphide is to be used for fumigation of infested ma- terial, the greatest precaution should be used to keep all fire, such as lights, cigarettes, etc., away from the liquid and where it is being used. For this reason it is well to take the material that is to be fumigated out-of-doors and at least 100 feet away from any building. The in- fested material should be placed iu supers or hive bodies if possible. These are piled as high as is convenient and all cracks between the containers made as nearly gas-proof as possible. Especially should the bottom be tight. A good plan is to -place an inverted hive cover on the ground, lay a piece of canvas over it and then tier up the supers on this. After the pile has been completed, an empty super should be put on top, in which should be placed a large shallow pan. Into the latter the bisulphide is to be poured. When all is in readiness, pour the bisulphide into the pan, and immediately put a hive cover on the top of the tier to confine the gas. This operation is best performed in the evening, and the pile of supers should be left intact the following morning. When the supers are taken down the confined gas will escape immediately, even before they can be carried separately into a building. The results of fumigating infested material with carbon bisulphide is shown iu table ??. The Bebmoth or Waxwokm Table 27.— Results of fumifiating infested combs with carbon' bisulphide. 37 Stage of bcemotti. Moth. Moth. Moth. Moth. . Pupae. Pupae. Pupae. Pupae. Larvae (in cocoons) Larvae (in cocoons) Larvae . (in cocoons) Larvae . (exposed)* Larvae (exposed)* Larvae (exposed)* Larvae (exposed)* Larvae (exposed)* Larvae (exposed) * Larvae . (exposed) * Larvae (exposed) * Larvae (exposed)* Larvae (exposed)* Larvae Larvae (exposed) ♦ Amount of liquid carbon bisulphide used per cu. ft. One-hnif ounce. Two-thirds oune Thiec-oisliths ,ounoo One-fourth ounne. . . One-sixth ounce. . . . One-fourth ounce. . . Three-eighth.s ounce One-half ounce One-eighth ounce. . . One-fourth ounce. . . Five-eighths ounce. . One-sixth ounce. . . . One-eighth ounce. . . One-eighth ounce. . . One-foiirth ounce. , . One-fourth ounce. . . One-fourth ounce. . . One-half ounce Three-fourths ounce Three-fourths ounce One ounce One ounce One ounce rime of ■niilint'mcnL. 15 iTiiniites. , 20 minutes. 20 minutes. . '20 minutes.. 24 hours. . . . 24 hours ... , 24 hours. . . . 24 hours. . . , 24 hours 24 hours 24 hours ... 24 hours. . . 24 hours . . . 24 hours . . . 24 hours . . . 4 hours. . . 24 hours .. . 24 hours . . . 24 hours . . . 24 hours. . . 24 hours. . . 24 hours... 24 hours . . . EffocI Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Remarks. The moth was unable to walk with- in 10 minutes after being con- fined. The moth was unable to walk with- in 10 minutes. Not all the bi- sulphide evaporated. The moth was dead before all the bisulphide evaporated. Several larvae in cocoons were also killed. Several larvae in cocoons were also killed. Several larvae in cocoons were also killed. Several larvae in cocoons were also killed. Some died in one hour in cocoon. Some died in one and one-half hours in cocoon. Larvae 10 days old an(J well pro- tected. These larvae were live days old and well protected in webs. Larvae were 25 days old, and pro- tected. These were 20 days old and fairly well protected. These were 20 days old and ex- posed. These were 12 days old and ex- posed. These were 15 days old and fairly well protected. Eggs were present which hatched afterward. Eggs were present; hatched after- ward. Eggs were present, hatched after- ward. Eggs were present; hatched after- ward. ♦These larvae were feeding in enipty combs. In all the experiments conducted, the eggs of the beemoth were nn- injured by the fumes of carbon bisulphide. It is possible that in cases of extremely large doses the eggs may be injured. A number of experiments -were conducted to determine the efEect of the fumes of carbon bisulphide upon the larvae. Comb containing larvae of various ages and different degrees of protection was fumi- gated. Many experiments were made with the larvae in cocoons, and these showed that the carbon bisulphide was very effective. The larvae which are hardest to kill are those about three-fourths grown and well protected in a mass of webs and refuse. Ordinarily the larvae succumb to the average dose of carbon bisulphide in a comparatively short time. The outcome of the experiments demonstrated the effectiveness of carbon bisulphide for the destruction of the larvae. Several experiments were conducted to determine the effect of carbon 38 Texas Ageicultueal Experimbnt Station bisulphide upon the pupae. Jt was found that they are quite sus- ceptible, buf a long exposure to the fumes is necessary as the pupae do not consume air very fast. From the experiments conducted with the moths, it was found that they are very susceptible to the fumes of carbon bisulphide. With the average dose the moths are overcome in from ten to fifteen minutes and are killed in from fifteen to twenty minutes after being confined. All fumigation should be allowed to continue for at least twelve hours, for those larvae which are best protected by webs and refuse will not be killed unless plenty of time is given for the gas to penetrate the material. The liquid will evaporate in a few hours, but the resulting gas will be eft'ective for several hours. The following table has been prepared to show at a glance how much liquid carbon bisulphide is required for effective fumigation of ten frame supers and hive bodies containing infested material. Table 28. — Amount of carbon bisulphide to use in fumi- gating ten frame supers for the waxworm. Number of supers in the tier. Cu. ft. contained in tier. Amount of liquid bisulphide re- cjuired. 2 1.74 2.61 3.48 4.35 5.22 R.09 6.9fi 7.83 8.70 9.57 10.44 f 1 1* n 2 21 2i 2? ounce 3 4 5 ounce 6 ounce 7 8 ounce 9. . . . 10 n ounces 12 ounces Table 29. — Amount of carbon bisulphide to use in fumi- gating ten frame hive bodies for the waxworm. Number of bodies in the tier. Cu. ft. contained in tier. Amnunt of liguid bisulphide re- uuired. o 2.90 4.35 5.80 7.25 8.70 10.15 11.60 § ounce 1 ounce IJ ounce ' If ounce 3 4 5 6 7 8 For eight-frame supers and hive bodies, use eighty per cent, as much bisulphide as is given in the foregoing table for the corresponding num- ber of supers or bodies. Example: Suppose that the beekeeper has six ten-frame shallow ex- tracting supers containing combs which he wishes to fumigate. All are tiered up as previously directed and an empty super is placed on top. This makes seven supers in all. Eeference to the preceding table shows that this tier of seven supers contains 6.09 cubic foet of space and that for the destruction of all of the waxworms in it one and one- half ounces of the liquid bisulphide are required.