„ I m CORNEU UNIVERSITY LIBRARY 3 1924 062 872 67 ALBERT R. MANN LIBRARY New York State Colleges OF Agriculture and Home Economics AT Cornell University EVERETT FRANKLIN PHILLIPS BEEKEEPING LIBRARY SF UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 671 Contribution from tlie Bureau of Entomology L. O. HOWARD, Chief Washington, D. C. PROFESSIONAL PAPER. June 21, 1918 THE DIAGNOSIS OF BEE DISEASES BY ^n^ABORATORY METHODS. By AsTHim H. McOb ay, ApicuUural Assistant, and G. F. White, ^ Expert Engaged in the Investigation of Bee Dj,seq,seg.. Introduction Laboratory methods CONTENTS. Page. Page. 1 Diseases to be diagnosed— Continued. 2 Sanhrnnd in Diseases to be diagnosed 4 European f oulbrood 4 AmerioflTi fmilbrood . 8 Literature 14 INTRODUCTION. Already some of the States have established laboratories for the diagnosis and investigation of the diseases of bees. The number of State laboratories where a rapid and accurate diagnosis of suspected material can be made will no doubt increase in the future in pro- portion as State laws for bee-disease inspection and control are insti- tuted and perfected. Inspectors realize that there will be always some suspected brood and many conditions among adult bees which can not be diagnosed in the field, but which will require laboratory methods for diagnosis. In addition to the demands made by in- ■^pectors for the examination of suspected material, there will be numerous independent requests from beekeepers. During the past several years the writers have examined a large nifmber of specimens of suspected brood and bees sent to the Bureau of Entomology, and have developed and perfected methods and technique in the diagnosis of the known diseases of bees which, it is believed, will prove valuable to others. It is the aim of this paper to present these methods of diagnosis for the benefit of those who may engage in similar work. 47164°— Bui! 671—18 1 mi3 ^ 2 BULLETIN etl, U. S. DEPARTMENT OF AGBICULTUBE. For the understanding and application of the methods herein out- lined, a preliminary training in general bacteriology, supplemented by a special knowledge of the pathogenic bacteria and the methods pursued in the diagnosis of diseases in general, is essential. As effi- ciency depends largely upon a knowledge of pathology, too much emphasis can not be given to it. With such general and special train- ing as a basis, and with a knowledge of the bee diseases, the acquisi- tion of the special methods necessary for a laboratory diagnosis of them becomes a comparatively simple matter. One of the authors (White) began his investigations on the dis- eases of bees in 1902, and from this date to 1909 examined and diagnosed about 500 samples of suspected material and during the period developed the laboratory methods as given in the present paper. Since 1909 the senior author (McCray) has carried on the work of diagnosing the samples received by the Bureau of Ento- mology and up to the present time has examined and diagnosed about 5,000 of them. LABORATORY METHODS. OBTAINING THE SAMPLES. For a satisfactory diagnosis of suspected brood the material at hand should be adequate. The size of the comb containing the af- fected larvae should be ample. A piece about 5 inches square is suit- able, provided it contains a sufficient number of larvae. All samples, no matter how far sent or how long in transit, should be inclosed in wooden boxes to prevent crushing during transportation. Paste- board boxes are apt to allow the sample to be crushed, while samples inclosed in tin boxes are often covered with a rich fungous growth when received. Both the crushing of the sample and the fungous growth obscure the gross features which form an important part of the examination. The sample should not be wrapped in cloth or paper, as this will tend to retain moisture and further encourage fungous growth. If thin paper is used, it frequently adheres so closely that its complete removal is difficult. The sample should con- tain no honey, as a small amount of it in a comb is sometimes suffi- cient to cause an unsightly mass, owing to leakage through the pack- age in transit. As complete a history as can be obtained should accompany each suspected specimen as an aid to diagnosis. The Bureau of Entomology for several years past has sent out a printed question card with blank spaces for answers by the beekeeper sending the suspected brood. Some of the more important of these questions are as follows : 1. Is there any bee disease In your neighborhood? 2. If so, what disease? 3. Have you brought colonies from a distance? If so, give source. DIAGNOSIS OF BEE DISEASES. 3 4. Have you fed honey from other sources than your own apiary? Give source if known. 5. Is the diseased brood mostly capped or is it mostly uncapped? 6. What is the color of the larvse (grubs) soon after death? 7. Later, what is the color of the decaying larvse? 8. Are the dead larval remains ropy? 9. Do you notice any disagreeable odor in the hive? 10. Does there seem to be an unusual number of queenless colonies in the apiary? 11. What disease do you suspect? 12. Give location of the apiary from which sample was taken by town (or township) and county. While a diagnosis sometimes can be made from larvae which have been removed from their cells, and sent without the comb, such mate- rial is not satisfactory. It is far better to examine the infected larvae in the comb in which they die. The diagnosis of bee diseases should not be based upon the examination of honey alone. > ^ EXAMINATION OF THE SAMPLES. In diagnosis both gross and microscopic examinations are made of the suspected material. GEOSS EXAMINATION. The following points are to be taken into consideration : Character of the caps ; regularity of the brood ; proportion of affected brood ; position of diseased larvae within the cell ; age, color, consistency, and odor of the affected brood ; and kind of larvae affected, whether queen, drone, or worker brood. These factors will be taken up in detail as each brood disease is considered separately. In the gross examination of the comb it should be held in such a manner that a good lighting of the interior of the cells is secured. This is especially important in examining for scales. The best method of examining suspected brood is to hold the comb in a ver- tical position and about level with the eyes; then, by gradually in- clining the top of the comb toward the observer, a point is reached at which the greatest amount of light is thrown upon the floor of the cells. This brings out the scales with great prominence and permits of their close scrutiny within the cells. In examining the affected brood for consistency and adherence to the cell walls in the scale stage, a small pair of curved forceps is convenient. The gross examination of the adult bees will be considered under ITosema disease. In routine diagnostic work unstained water mounts have been found very satisfactory in searching for spores, and stained prepara- tions are made for the vegetative forms. Carbol f uchsin is a suitable stain to use. BULLETIN Q11, V. S. DEPAKTMEITT OF AGKICTJLTUEE. MICEOSCOPIC EXAMINATION. The number of larvae or pupas to be examined in a given sample depends upon various factors. If the case is a typical one, one larva or pupa dead of a disease usually is sufficient. If, on the other hand, the gross appearance is not so definite and the microscopic picture from the first dead remains examined is unsatisfactory, others must be studied. While much might be written concerning the microscopic appearance of smears from larvse or pupse affected with the various brood diseases, as compared each with the others and with smears from healthy brood, such elaborate descriptions are not deemed ad- visable in this paper. Only the more salient features of the micro- scopic picture will be given for each disease considered, as it is believed that such descriptions will best serve those for whose benefit the paper is prepared. It will be understood that when the authors write of the recog- nition of certain organisms by microscopic examination, as, for example, that of Bacillus pluton or Bacillus alvei, either in stained smears made from tissue or in stained smears made from agar plates, they refer to the recognition of the organism under observation only in a general way, meaning rather that the microscopic picture sug- gests the organism. The identification of the organism is complete, naturally, only after a consideration of its cultural characteristics also. CULTURES. In culturing the affected brood agar as ordinarily prepared in the laboratory is used in making plates. Those larvse or pupse are selected which upon microscopic examination have shown evidence of disease. Of course as many additional ones may be cultured as desired. The cultures are incubated for different periods of time, as will be noted in the discussion of the diseases. Dead, not living, larvce are examined. — In the laboratory examina- tion of diseased bee brood, the affected larvse are always received dead, therefore nothing will be said relative to symptoms and appear- ance of affected living larvse. DISEASES TO BE DIAGNOSED. EUEOPEAN FOULBROOD. European foulbrood is an infectious disease of the brood of bees caused by Bacillus pluton (White, 1912). GBOSS CHAEACTEES. {a) The caps and regularity of the Irood. — ^Larvse that die of European foulbrood do so usually before they reach the age at which brood is capped. Brood dead of the disease is therefore usu- ally found in uncapped cells. When the larvse die after capping DIAGNOSIS OF BEE DISEASES. 5 the caps usually are entire, but may be punctured. The caps may be slightly sunken, but usually are not. Owing to the fact that such a small percentage of the affected brood becomes sealed, a comb of brood affected with European foulbrood with its few sealed cells and large amount of young uncapped brood presents a distinctive appear- ance (PI. I, fig. 1) and shows a marked contrast to the solid areas of brood of uniform age in healthy combs. (&) Proportion of affected Irood. — European foulbrood usually has made rather extensive ravages by the time the beekeeper detects it; hence in many samples received for diagnosis a very large pro- portion of the larvae in the comb are affected. Toward autumn, however, it is not unusual to receive samples containing a small num- ber of affected larvse. (c) Position of larvce within the cell. — The usual position for larvae affected with European foulbrood is that of lying curled at the bottom of the cell. Other affected larVae lie extended in the cell, but these are few in number. {d) Age of the dead larvce. — ^In most of the specimens received for diagnosis by far the larger proportion of the affected larvae are young, lying curled at the bottom of the cell as just stated. Besides this comparatively young brood, older larvae, including a few sealed ones, may be found affected. {e) Color. — ^A change in color is one of the first abnormalities noted in brood dead of European foulbrood. Yellow or gray and combinations of these two colors are among the first to be noted. Later the yellow and gray gradually deepen, until quite a dark brown is attained. Larvae dead of this disease often present a peculiar appearance, as though they were melting away under the influence of heat. The transverse tracheal branches stand out prominently. This melting appearance of the larvse, the yellow, gray, and brown colora- tion, prominent tracheal branches, and large amount of uncapped affected brood are characteristics not easily confused, in the majority of cases, with those of other diseased conditions of the brood. (/) Consistency. — ^Larvae dead of European foulbrood are com- paratively friable. However, larvae which are somewhat viscid usu- ally may be found. Perhaps slimy, rather than viscid, expresses better the consistency of some of these larvae. {g) Odor. — A slight, inoffensive odor is frequently to be noted in European foulbrood. The yeastlike odor which has been described is not constant in brood affected with European foulbrood. A simi- lar odor may be detected in samples other than those which contain European foulbrood. (A) Kind of hrood affected. — Sometimes samples are received which contain only affected drone-brood. Most cases, however, con- sist only of worker-brood. Queen larvae also may be attacked. 6 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTUajii. (i) Scales. — Scales are formed by the drying of the affected larvae, and from the foregoing description of the dead brood some conception may be gained as to their form and appearance. Scales of European foulbrood, like most of the affected larvae, are small and lie at the bottom of the cell, from which they can be separated with ease. The color of the scale is in general yellow, gray, or brown, and the cross markings formed by the transverse tracheal branches usually are still in evidence. This is the usual type of scale found in European foulbrood. Occasionally there will be received, how- ever, a sample containing only a few scales, or perhaps a single scale, in marked contrast to the scale just described. These scales are always few in number in a given comb area, are usually dark brown in color, are less easily removed than the small ones, and are not brittle but rubberlike in consistency. MICROSCOPIC FINDINGS. The appearance of Bacillus pluton, the etiological factor in Eu- ropean foulbrood, in stained preparations usually is sufficiently char- acteristic to render its microscopic identification comparatively cer- tain. Besides Bacillus pluton, the following secondary invaders may be found : Bacillus cdvei, Streptococcus apis, Bacillus vulgatus, Bacillus mesentericus, Bacillus orpheus, and Bacterium eurydice. Without careful observation Bacillus pluton and Streptococcus apis might be confused. Upon careful examination it is found that Bacillus pluton presents considerable variation in size and morphol- ogy in the individual organisms. Some of them occur in the form of cocci, yet the general picture is that of an organism with more or less pointed ends. Thin smears should be made in order to obtain details of morphology. It is important to have a true conception of the microscopic appear- ance of BaclUus pluton. The essential facts are the typical mor- phology and the manner of grouping of the individual organisms. The general shape of the group is often more or less circular, although nmnerous groups of more or less irregular form may be observed. Groups of varying shapes and sizes will be noted as successive fields are brought into view. It is the presence of these groups, containing a sufficient number of organisms with the pointed ends described that serves to differentiate Bacillus pluton from Streptococcus apis. Streptococcus apis usually occurs in forms which are sufficiently coccuslike to lead to little or no hesitancy in differentiating it from Bacillus pluton. Forms which are sufficiently pointed to resemble B. pluton do occur, however, and if only a few are present in a field the differentiation of these species is not possible. By making a sufficient number of smears from a sufficient number of larvae, forms DIAGNOSIS OP BEE DISEASES. 7 in abundance typical of either B. pluton or Strep, apis usually can be found. In stained smears of Strep. t. of Agriculture. ME II. FiQ. 1.— Comb Containing Brood Dead of Sacbrood. Natural size. (White.) Fig. 2.— Stained Smear Preparation Showing Spores of Nosema apis. Highly magnifled. (Original.) SACBROOD AND NOSEMA SPORES. DIAGNOSIS OF BEE DISEASES. 9 that on thrusting the forceps into the brown larval remains and withdrawing them a portion of the decaying mass adheres and is drawn out, often to a distance of 3 or 4 inches. The viscidity is often referred to by the term " ropiness " in beekeeping literature. In the rare instances in which young uncapped affected larvae are en- countered the ropiness is less pronounced. (g) Odor. — The odor is characteristic and may be described as unpleasant. Often it is feeble or absent altogether, probably having disappeared after the removal of the diseased brood from the hive. Affected brood-comb will absorb other odors if given the oppor- tunity, thus masking the original characteristic odor. Such dis- appearance and masking of the odor has been observed where speci- mens of diseased brood in combs from various sources had been thrown together and allowed to lie about preparatory to being de- stroyed (McCray, 1916). (h) Kimd of hrood. — It is affected worker-brood that is most often encountered in American foulbrood samples, although drone-brood is sometimes affected. (J) Scales. — The scales of American foulbrood are distinctive and are characteristic of the disease. If they are present in guiScient numbers the disease can be diagnosed from the gross appearance alone. The lower cell walls can be easily illumined by tilting the upper portion of the comb toward the observer, as previously de- scribed. The scales appear extended along the lower cell wall, are quite dark in color, and adhere closely to the floor and base of the cell. Sometimes they adhere so closely as to break when an attempt is made to remove them from the cell. Often a semblance of the form of the pupse is evident in the dried-down mass forming the scale. Some of the mouth parts of the pupae sometimes protrude sufficiently to adhere to the roof of the cell. MICROSCOPIC FINDINGS. In considering the microscopic appearance of stained smears from infected brood in this disease, there usually is only BaoUlus larvae in the spore form to engage the attention of the observer. In some instances, however, samples containing the disease in its earlier stages are received and then BacUlus larvae in the rod or vegetative form may be encountered. Such a sample usually is more difficult to diagnose. To receive a sample in which some older larvae con- taining spores of BacHMm larvae can not be found, however, is a very rare occurrence. In a stained smear made from an infected larva and mounted in water, the microscopic picture is rather characteristic. The most striking feature of the mount is the large number of spores adher- ing to the cover glass, floating with the current, or dancing free in 10 BULLETIN 671, U. S. DEPAETMENT 03? AGRICULTTXBJi. the water medium. Many of the spores stain slightly about the periphery, which aids somewhat in the observation. Good results may be obtained from an unstained water mount. The vegetative forms of BociUms larvae, when they are present, are observed to be slender rods, which tend to occur in chains. CULTURES. In culturing affected larvae it is the absence of growth on the agar plates that is important in the diagnosis of American foulbrood. This is because the spores of Bacillus larvae will not germinate and grow on the ordinary media of the laboratory, and other growth is absent because there are seldom secondary invaders present. Occa- sionally there will be a spreading growth of B. vulgatvs, or B. mesen- tericus, and very rarely of B. alvei. On quite rare occasions a con- siderable number of colonies of B. vulgatus or B. mesenteneus have been found. As both of these species form spores, as a rule, within 24 hours, their differentiation from B. alvei usually can be made in this way. A report on a sample of American foulbrood, therefore, nearly always can be made within a day. SACBROOD. Sacbrood is an infectious brood disease of bees caused by a filter- able virus (White, 1913 and 1917). GROSS CHAEACTBRS. (a) Character of caps and regularity of the hrood. — ^Larvae usually die after capping in this disease, some of the dead brood being un- capped by the bees later. Occasionally the caps are punctured. An area of comb affected with sacbrood therefore presents an irregularity. So far as the age of the affected larvae themselves is concerned, there is considerable uniformity owing to the fact that death in this disease occurs after sealing during the two-day period of rest just preceding pupation. The affected brood, however, is interspersed among healthy brood of varying age, which adds to the irregular appearance of the affected comb (PI. II, fig. 1). (&) Proportion of affected hrood. — ^As a rule there is not a large proportion of affected brood in a given comb area. Often there will be an affected larva only here and there. (c) Position within the cell. — The position of the affected larvse is that of extension lengthwise along the floor of the cell, against which the dorsal portion of the larva lies. The head is turned upward, to- ward the roof of the cell. {d) Age. — The brood dies after it has been sealed. {€,) 6'