'h'l' ii'i 3 1924 062 872 233 ALBERT R. MANN LIBRARY New York State Colleges OF Agriculture and Home Economics AT Cornell University EVERETT FRANKLIN PHILLIPS BEEKEEPING LIBRARY UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 431 OMribntlon bom the Bnraau of Entomoloir L.O. HOWABD, Chief ^Bshlngton, D. C. PROFESSIONAL PAPER February 9, 1917 SACBROOD By G. F.^^TTE Expert, Engaged in the Investigation of Bee IMseases CONTENTS Page , 1 Btatoctnl Account ......... 2 NmmeoftheDInase 4 Appearance of Healthy Brood at the Ate atWhtehttDicBofSacbraod .... 6 SymptoiiMof Sacbrood 10 Cause of Sacbtood 24 WeakenlnK Effect of Sacbrood Upon a Colony 30 Amonnt of Tims Beqnired to Produce Hut Dlaease. and the Bapldtty of ita . bwreaae . . .' 31 Methods Oaed in Makint Experimental InaenlatlanB . 32 Means for the DeatmcUoit of the Vlnu of Sacbrood BcatlncBeviited to Destroy Sacbrood Vbua when Suspended In Water ' . . Beatlns BeqnlreC to Destroy Sacbrood Tims Vhen Suspended In Glycerine . Beating Required to Destroy Sacbrood Tints When Suspended in Honey . . Bealatanee of Sacbrood Tbua to Dtyins at Boon TMnpetattura 87 Page BiBstotsBet of Saebmod Tlras to Direct Sunlight When Dry 38 Besistance of Sacbrood Tiros to Direct Sunllcht When Suspended In Water . 39 Besistance of Sacbrood Tims to Direct SunllEht When Suspended In Honey . 40 I.enith of Time that Sacbrood Tims Be> - mains Timlent In Honey 40 Besistance ofSacbroodTlrastothe Pres. once of FermentatlTe Processes ... 41 Besistance of Sacbrood Tims to Fer. mentation in Diluted Honey at Out> door Temperature 42 Besistance of Sacbrood Tims to the Pres< enee of Putrefocllve Processes ... 43 Besistance of Sacbrood Tims to Carbolic Acid 44 Modes of Transmission of Sacbrood . . 46 Diagnosis of Sacbrood 48 ProKnoBla ............. 49 Belatlon of These Studies to the Treat* ment of Sacbrood . 50 Summary and Conclmdons ...... S2 Literature Cited. fiS WASBINOTON GOTEBNMENT PBINTIKO OlflCB 1917 SF .5 O ^ "^ UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 431 Contribution from tlie Bureau of Entomology t. O. HOWARD, Chief Washington, D. C. PROFESSIONAL PAPER February 9, 1917 SACBROOD. By G. F. White, Expert, Engaged in the Investigation of Bee Diseases. CONTENTS. Introdaotion 1 Historical account 2 Name of the disease 4 Appearance of healthy broodattheageat which it dies of sacbrood 6 Symptoms of sacbrood 10 Cause of sacbrood 24 Weakening effect of sacbrood upon a colony. . . 30 Amount oJ virus required to produce the dis- ease, and the rapidity of its increase 31 Methods used in making 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 Resistance of sacbrood virus to drying at room temperature 37 39 40 Resistance of sacbrood virus to direct simlight when dry Resistance of sacbrood virus to direct sunlight when suspended in water Resistance of sacbrood virus to direct sunlight when suspended in honey Iiongth 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 ofsacbrood 46 Diagnosis of sacbrood 48 Prognosis 49 Relation of these studies to the treatment of sacbrood 50 Summary and conplusions 52 Literature cited 53 INTRODUCTION. Sacbrood is an infectious disease of the brood of bees. It is fre- quently encountered and bas often been the cause of fear on the part of beekeepers through a suspicion that one of the more serious maladies — the f oulbroods — ^was present. The disease is more benign than malignant. It is insidious in 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 in which the disease is present, a fact which makes the disease one of great economic importance. 58574°— BuU. 431—17 1 Missing Page SAOBEOOD. , 8 It will be noted that Jones also recognized that there was a disease that resembled somewhat the genuiae 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. Simmins (1887), writing from Eottingdean, England, points out the difference between "deadbrood' ' and foulbrood: That foul brood is often confused with simple dead brood I am well aware. * * * But that every bee 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 dnder, 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," Simmins says that these two diseases are in turn to be differentiated from chiUed brood. He adds the additional fact also that Cheshire had examined 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 in 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, imder 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 larvae 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 grantilar 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 occurred in the older larvae, and showed a gray to blackish colora- 4 BULLETIN 431, U. S. UEPAETMENT OF AGKICULTTJRE. tion, partially dryiBg the lai'vse until mummified. These larvse 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 sacbfood. 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 examined by Burri and himself 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, Kursteiner has continued to find this disease in the examination of suspected samples. The foregoing references to the hterature show that beekeepers in different counties had been observing dead brood in their apiaries which was imlike 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 nataxe, the writer (1913) used the name "sacbrood" to designate it. The name was coined to suggest the saclike appearance of the dead larvro 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 probability 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 unawares 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 knowing of it hope may be entertained that the losses resulting from it may be reduced. PICKLED BROOD. The term "pickled brood" was introduced into beekeeping Htera- ture 20 years ago (1896), by WOliam R. Howard of Texas. The condition which he described under this term he declared was caused SACBEOOD. 5 by a fungus to which he gave the name Aspergillus poUini. In a second article (1898) he writes that pupae and adult bees, as well as the larvBB, are attacked by the disease, stating his beUef that the disease in adult bees had been diagnosed as paralysis. Technically, therefore, the term "pickled brood" refers to an infectious disorder of bees affecting 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 beekeepers have been led to the mistaken behef 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 likely included tmder the term "pickled brood" as it is popularly used. Brood dead of starvation and that found dead before capping and not dead of an infectious disease seem tQ 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 ?" In the replies received more than one disease was sometimes suggested as being suspected. Out of 189 replies received from beekeepers sending samples of sacbrood, European foulbrood was suggested in 55 rephes, piclded brood in 39, foulbrood in 19, blackbrood in 15, poisoned brood iu 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 natural to suppose that sacbrood would have been one of the conditions occasionally referred to under the term "piclded brood." BULLETIN 431, V. S. DEPABTMENT OP AGBICXTLTUKE. As sacbrood has been proved, however, to be a distinct disease aud different from all other disorders, naturally it is incorrect to use the terms "sacbrood" and "pickled brood" synonymously, either in the popular or in the technical sense.^ APPEARANCE OP 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 will 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 all cells in the un- capped areas will be without caps while practically all in the capped areas will 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 cefls. 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 (Aj) is formed by two sides which together may be termed the roof of the cell. The angle which is lowermost (figs. 1 and 2, Aj) is formed by two sides which with equal propriety may together be termed the floor of the ceU (fig. 2, F). When a cell is cut along its long axis i For the purpose of an explanation lor those wbo may have learned to refer to sacbrood by the term "pickled hrood," it might he 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." i Fig. 1.— Looking into an empty worker cell uncapped by bees. The uppermost angle (Ai), the lowermost angle (Aj), 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.) SAOBKOOD. the cut surface of the older ones shows the presence of a varying num- ber of old cocoons (fig. I, C). Near the mouth of the cell on the side walls (figs. 1 and 2, C) wiU often be noted a wrinkling of the surface. This wrinkling is caused by the presence of old cocoons. The tw;o remaining walls are parallel and will be referred to as the lateral walls (fig. 1, L). The bottom is conc«,ve on the inside. The cap FiQ. 3.— End view of cell capped. The 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. II, I). The long axis of the cell is nearly horizontal, the bottom of the cell being normally only sUghtly lower than the mouth. The long axis measures approximately one-half inch, while the perpendictilar 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 r'ies of sacbrood is found. Fig. 2. — Empty worker cell cut in halt along the long axis of the cell, showing cocoons (C) at the base and near the mouth of the cell, and the lowermost angle (A^) farmed by the two walls which constitute the floor (F) of the cell. Enlarged about 8 diameters. (Original.) 8 BULLETIN 431, U. S. 0EPABTMKNT OF AGBICULTTJBE. APPEARANCE OF A HEALTHY LAKVA AT THE AGE AT WHICH IT DIES OF SACBKOOD. The symptoms which differentiate sacbrood from the other brood diseases are to be found primarily in the post-mortem appearances of the larvse dead of the disease. As an aid in interpreting the description of these appearances a description of the healthy larvae is first made. , Larvse ' that die of sacbrood do so almost invariably after capping and at some time during the four days just preceding the change in form of the maturing bee to that of a true pupa. During the first 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- FiQ. ■!.— Lateral view of healthy worker larva showiug the nornial position within the cell. For oonTen- ience of description the length is divided into thirds— anterior third (AT), middle third (MT) and posterior tliird (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 cr 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 ia 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 poiating toward the mouth of the cell. Its posterior or caudal end lies upon the bottom of the cell, ' As beekeepers usually refer to the brood at this age as "larvse," the term is used here to designate the developing bee at this stage of its growth. SACBEOOD. 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 thirds (PT). Anterior third. — On removing the cap from a cell the anterior cone-shaped ^ „ , . ... . ,n _ T,i Iio. 5.— End View of healthy worker larva portion 01 the larva is seen (ng. 5; Jrl. in normal position in the cell. Cap II, a). The apex of this cone-shaped tom and tumed aside with forceps. En- , . V . ,. 1 • 1 , , larged about 8 diameters. (Original.) 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- pointy 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 10 BULLETIN 431, V. 8. DEPABTMENT OF AGKICULTUEE. ceptible, wMte line. Sometimes there may be seen a narrow area along the median line of the ventral sm^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 similar in appearance to the one on the dorsal side, but less distinct. A cross section of this third is slightly elliptical in outline. The middle third passes more or less gradually into the posterior third. The juncture 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 surface 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 oflF into segments by ridges and furrows similar to, but less regular than, those of the middle third. TISSUES OF A HEALTHY LAHVA AT THE AGE AT WHICH IT DIES OF SACBROOD. Upon removing a larva in the late larval stage and puncturing its body wall lightly, a clear fluid almost water-hke in 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 soHd 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 primarily in the FiQ. 6. — Healthy larva and cell riewed from above and at an angle. (Original.) SACBEOOD. 11 colony as a whole, and not in individual Kees. Therefore, in describ- ing the synaptoms of a bee disease, the colony as a whole should be considered as the unit for description, and not the individual bee. A symptom of disease 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 larvaj dead of the disease. With sacbrood thus produced in ex- perimental colonies the symptoms of the disease have been studied, and the description 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 OBSERVED FHOM A CASUAL EXAMINATION. Fig. 7.— Larva dead of sacbrood lying in the 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 (P\. 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 r^oticeably diminished. Should a large amount of the brood become affected, however, the colony naturally becomes weakened thereby, the loss in strength soon becoming appreciable. Brood that dies of the disease does so almost invariably in capped ceUs, but before the pupal 12 BULLETIN 431, U. S. DEPAKTMENT OF AGBICULTUBE. Fig. 8.— End view of capped cell whioh con- tains a larva dead of sacbrood, being simi- lar to the one shown in figure 9. The cap here is not different 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 larvae 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 in cells which have been tmcapped (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 found to have been punctured by the bees. Usually only one puncture is found in a cap (PI. II, d), but there may be two (fig. 10) or even more (PI. 11,/). 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, l) has a hole through it which suggests by its position and imiform circumference that it has never been completed. Through such an opening (fig. 11; PL II, e) or through one of the larger punctures 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, until 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 granular 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 Fio. 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. 43 r, U. S. Dept. of Agriculture. Plate I. Fig. 1.— Marked Saobrood Infection. Size Slightly Less than Naturau (Original.) l^Sim^i^tB'^^d g^aJH^CjSli s ^^9L.-^c>BS FiQ. 2.— Heavy Sacbrood Infection, Showing a Number of Different Stages OF Decay of Lahv/e. Eqqs, Young Larv/e 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. Comparison of a Healthy Larva and the Remains of Larv/e Dead of Sacbrood. "■< :^i??P "' * healthy larva; 6, c, d, e, and /, caps over larviB in first, second, third, fourth and fifth stages of decay, respectively; g, a healthy larva, end vievr; h, i, j, k, and I an end view of the five stages of decay; m, a healthy larva viewed from above; nova and r cor- responding view of the five stages of decay; s and y, healthy larva removed from the cell; i, u, v, w, and z, larval remains in different stages of decay removed from the cell- mo, a larva recently dead of sacbrood vrtth 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: j/?/, almost a pupa; 2s, a pupa dead of sacbrood which had only recently transformed. (Original.) ■ """.I '•'=^<="i"j' SACBROOD. 13 "scale" (figs. 22\ 23; PI. II, I, r, and x). This scale is not adherent to the cell waU. In sacbrood the brood combs may be said to have no odor. Larvae imdergoing 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 larvse dead of the disease, however, will make it possible to avoid any such confusion. A more careful study of the dead larvse is therefore justified. Fio. 10. — Cap of cell containing the remains of a larva dead of sacbrood. The cap is slightly sunken 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 diu-ing the time when ' they are motionless, lack of movement can not be used as an early sign of death. In 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- scription that would be correct for a dead larva on one day, there- fore, may and probably would be incorrect for the same larva on the following day. Moreover, all larvse dead of the disease do not imdergo the same change in appear- ance, causing another considerable range of variation. For con- venience of description, this gradual and continual change in appear- ance is here considered in five more or less arbitrary stages. As the Fig. 11.— End view of cell containing alarva dead of sacbrood, with a cap which has the appearance of never having been com- pleted. (Original.) 14 BULLETIN 431, TJ. S. DEPARTMENT OF AGRICULTURE. same plan will be followed and similar terms will be used iu describing these stages as were employed in the description of a healthy larva of the same age, the interpretation of the description will be aided if the appearance of a healthy larva as described above is borne in mind. PiHST Stage. Uncapping a larva showing the first symptoms of the disease, it will 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 tlie 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 cephalic end of the an- terior third (fig. 12; PI, II, &, h) may have assumed, 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-Uke 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 ol all three thirds. Cap torn across. (Original.) SAOBROOD. 15 verse tracheae 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 still 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 Fio. 16. — 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 appea,rance 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 in evidence. By microscopic examination the granular appearance is found to be due chiefly to fat ceUs. 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. li— 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 BTJLLETIlSr 431, U. S. DEPARTMENT OP AGRICULTURE. amount of sacbrood is readiJy 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 ot an- terior tliird of larra dead o! saobrood. (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 little 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 slightly from that observed in the preceding one. The apex is farther from the roof of the cell (PI. II, i). The deflection is more marked and is seen in a greater number of larvse. 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. (Original.) SACBEOOD. 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 juncture 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 resiilts of inociilations 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. Thibd Stage. Fig. 18.— Tlurd stage: Larvadead ofsaobrood, 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 brdwn. This third stiU preserves its conelike form (figs. 17 and 9; PL II, j), the distance of the apex from the roof of the cell being stiU 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°— Bull. 431—17 3 18 BULLETIN 431, U. S. DEPARTMENT OF AGKICULTUEE. 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 lighter. The ventral surface of this third (figs. 18 and 7) is less convex from side to side than in the preceding stage, and the segmental markings, while still 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 marldngs are still pronounced and much resemible those of the normal larva. That the watery content of the sac is being lessened through evapo- ration is evidenced by the dinainution of the quantity of the watery- Fia. 19.— Third stage: Larva dead of saobrood, lateral view. (Original.) appearing substance seen at the lateral marguis 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 stiU maintaiQS, in a general way, the form and markiags of the normal larva (fig. 4), The turgidity is gone, although the position in the cell 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 noicroscopic 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 SACBKOOD. 19 are not infectious. The status of the virus in this stage is not defi- nitely kncwn, 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 third 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 veiy deep brown, often appearing almost black. As a result of drying, the apex of this conehke third Fig. 20.— Fourth stage: Eemains of larva dead of sacbrood. (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, k) of decay almost obhter- ated through the wrinkling of the smiace, 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 shghtly 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.— Fourth stage: Eemains of .larva dead of sacbrood, ventral view. ( Original. ) 20 BULLETIK 431, V. S. DEPAETMENT OF AGEIGULTUB13. third. The effect of the drying on this third is quite perceptible also. The surface markings and notched majgin of the normal larva are stiU indicated in the decaying remains, but are much less pronoimced. The subcuticular fluid is no longer in evidence. ConsisUncy of the cordents of the sac— Upon tearing the sac, the contents are found to be less fluid than in preceding stages. The decaying tissue mass is still granular in appearance. As the drying Fia. 22.— Fifth stage: Scale, or larval re- mains, in saobrood as seen on looking into tlie 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 fourth stage do not seem to be infectious. Fifth Stage. Fig. 23.— Fifth 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 moistiire, leaving the dry, mummylike remains known as the "scale." Anterior third. — The anterior third (fig. 22 ; PL 11, T) through dry- ing is retracted from the mouth of the cell, with the apex drawn stiU deeper into the cell 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.^ThB middle third (fig. 23; PI. II, r), is deeply concave from side to side and may show remnants of the segmental markings of the larva. The surface is often roughened through drying. Sometimes both longitudinal and transverse tracheae are SACBEOOD. 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 ceU, 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 roughened^ and directed somewhat forward. This margin, like 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, X.) 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 cut lengthwise, lateral view. COrigiual.) 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 boatlike appearance and cotild be styled "^ gondola- shaped." This general fom^ 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, U. S. DEPAETMENT OP AGRICULTUEE. through the stage described above as the third stage (figs. 7, 9, 17, and 18; PI. II j, p). That the dead larvse are allowed to remain ia the cells often for weeks is in part the cause of the irregularity ob- served in the appearance of the brood combs (p. 11). (Pis. I, IV.) APPEAEANCE OF THE TISSUES OF A LAEVA DEAD OF SACBKOOD. The gross appearance of a larva during its decay after death from sacbrood has just been described. The saclike appearance of 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 cells (FC) are coraparatively 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 larvse 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 (C^), which is at a con- siderable distance from the hypodermis (Hyp). Another cuticula (Ci) is already quite well formed and lies near the hypodermis. Be- tween these two cuticulse (C2 and CJ during the earlier stages of decay there is a considerable space ("intercuticular space") (IS). This space is filled with a watery-lookuig fluid. That the fluid is not water, but that it is of such a nature that a coagulum is formed in it during the preparation of the tissues for study, is shown by the presence of a coagulum in the sections. The body (B, A) wall of the larva is composed of the cuticula (Ci), 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 fotmd among the fat cells, especially in the ventral half of the body. The oenocytes in the prepared tissues are irregular in outline, having a nucleus regular in outUne. The cytoplasm is uniformly graniilar and does not contain the black staiaing bodies found ia the fat cells (C). SACBEOOD. 23 Fig. 25.— The tissues of a worker larva after being dead of saobrood about one week. A, cross section, semldiagrammatlc, of the abdomen in the region of the ovaries, showing a recently cast cuticula, or molt skin (Cs), a vswly formed oatioala (CO, the hypoder^iis (Hyp), the stomach (St), the ovaries ( Ov), the heart (Ht), the ventral nerve cord (VNC), the dorsal diaphragm (DDph), trachese (Tra), oeno- cytes (Oe), and fat cells (FC). Between the cuticula Ca and the cuticula Ci is a considerable intercu- ticular space (IS). B represents the body wall in this pathological condition, showing the cuticula Ca and the cuticula Gi, both bearing spines (SC2 and SCi), and theinterouticular 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 irregtdar outline, irregular nucleus (Nu), and deep staining bodies- (DSB). D, oenocyte with uniformly staining cytoplasm, and with a nucleus (Nu) having 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, U. S. DEPARTMENT OF AGBICtTLTUBE. The molt skin (Cj) is probably the one that is shed normally about three days after the larva is capped. The cuticula (Cj), already quite weU 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 saebrood, the cuticula (Cj) probably assisting somewhat at times. The presence of the subcuticular fluid is made more intelli- gible by these facts. Larvae dying of saebrood at an earlier or later period in their development wiU present an appearance varying somewhat from that just described. Contrasted with the stomach (midintestine or midgut) of a feeding lairva, 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 larvae, 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 saebrood, 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 saebrood is, therefore, a composite affair. Upon examining the mass microscopically, it wiU be foimd that the grauTilar appearance is due for the most part to fat cells suspended in a liquid. The Uquid 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 liquefaction of some of the tissues present. The granular mass suspended in a watery fluid, as a symptom of saebrood, is by these facts rendered more easily understood. CAUSE OF SACBROOD. DooHttle (1881), Jones (1883), Sunmins (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 saebrood. There has been no consensus of opinion, however, as to the infectiousness of the disease. On this point Dadant (1906) writes: Whatever 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. SACBEOOD. 25 In the quotation Dadant expresses the behef 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 thefoulbroods, 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. Kecent studies have added others relative to sex, age, race, cHmatic 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. Pupge are rarely affected (PL II, zz). If one succumbs to the disease, it is quite soon after trans- formation from -the larval stage. Primarily it is the larvse that are susceptible. When a larva dies of the disease, it does so almost invariably after capping, and usually diu-ing the 2-day period immedi- ately preceding the time for the change to a pupa. Sex. — Worker and drone larvae may become infected. Queen larv^i 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 estabhshed. The question : "What race of bees is there in the diseased colony ? " was asked beekeepers sending samples of diseased brood. Out of 140 replies received from those sending sacbrood samples,' 53 reported hybrids, 49 reported Italians, 21 reported blacks, and 17 reported Italian hybrids. These replies show that the bees commonly kept by American beekeepers are susceptible, although their relative suscepti- bility is not shown. The bees which have been inoculated in the experimental work on sacbrood have been largely Italians or mixed with Italian 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 stat'e- pients. Climate. — Historial evidence strongly suggests that sacbrood is found in Germany (Langstroth, 1857), England (Simmins, 1887), 58574°— Bull. 431—17 4 26 BULLETIN 431, U. S. DEPARTMENT OF AGBICULTUEE. and Switzerland (Burri, 1906). Beuhne (1913) reports its presence in Australia, and Bahr (1915) has encountered a brood disorder among bees in Denmark wbicli lie 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 climates or the coldest climates in which bees are kept has not yet been completely established. 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 coimtry 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 nature that it can appear under widely different cHmatic 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 climate 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 climatic 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 quality 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 6f localities, wherein the food and water used by the bees vai-y as greatly almost as is possible in the United States, the conclusion may be drawn that its occmrence 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 r6le 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 SACBROOD. That sacbrood is an infectious disease was demonstrated by the writer (1913) through experiments performed during the summer of 1912. This was done by feeding to healthy colonies the crushed tissues of larvae 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 fiilter 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 fi[lter* (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 found 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. Having filtered the aqueous suspension of crushed sacbrood larvse through paper, the filtrate is then filtered by allowing it to pass through the walls 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 filtering ia this instance gravity is the only force used. 28 BULLETIN 431, tr. S. DEPAETMENT OF AGEICULTUEE. nies. WTaen all tMs is properly done, sacbrood will appear in the inoculated colonies. This shows that the virus ^ of this disease, to a Tig. 26.— Berkefeld filter (B) -with 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 Inreferrii^ to the inlecttng agent in sacbrood, the term "rirus" 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 larvae dead of the disease is prepared as before. This is filtered by the aid of pressure obtained Via. 27.— A convenient apparatus which can be employed in using the Pasteur-Chamberland, Berkefeld, and other filters. Pasteur-Chamberland filter (b) with a glass mantle (a), a rubber stopper (c) through which passes the filter, a oonnectirg 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 (j), and a vacuum pump