PAS et Ke we AN Dts: % toa) ru, xe ye Soma ey Vacate ; Wi 3s on Sas ae : oe ; Sees : : sas Sr . : Saphiolees A ey) . " rns, ae; Sate atic Y meal Ros : 2 < ey Ate te ae a - - 4 ‘ om iM . Aetna eed An i aaah os, nee AS ry ‘ . ‘ ~ iY vane’ . fyi jig , a, wif ’ teh. 8 Uitte Ss say Ua i s ~ J d y tb << ee i? i p : >> = rh, V/ a4 4 ‘ i re w/\, <4 Ps ay 4 Li a >> M¢ — EA 4 Oe SS 2 he a : Vo " Cee i j > ‘ tS WAAAY VARA < LAD ARAL eee “Ww A VAS Issued May 10, 1912. USS VOr rr wEVENT OF AGRICUBPURE, BUREAU OF ENTOMOLOGY—CIRCULAR Nos 15 L. O. HOWARD, Entomologist and Chief of Bureau. . THE CAUSE OF EUROPEAN FOUL BROOD. ‘ BY G. F. WHITE, M. D., Pa. D., Expert in Bacteriology. WASHINGTON ! GOVERNMENT PRINTING OFFICE : 1912 38171°—Cir. 157—12——1 ee BUREAU OF ENTOMOLOGY. L. O. Howarp, Entomologist and Chief of Bureau. C. L. MARLATT, Entomologist and Acting Chief in Absence of Chief. R. S. Ciirton, Executive Assistant. W. FEF. Tastet, Chief Clerk. FE. H. CHITTENDEN, in charge of truck crop and stored product insect investigations. A. D. HopkKINs, in charge of forest insect investigations. W. D. HUNTER, in charge of southern field crop insect investigations. F. M. WEBSTER, in charge of cereal and forage insect investigations. A. L. QUAINTANCE, in charge of deciduous fruit insect investigations. E. F. Puixyies, in charge of bce culture. D. M. Rocers, in charge of preventing spread of moths, field work. Roiia P. CurRIg£, in charge of editorial work. MABEL COLCcorRD, in charge of library. INVESTIGATIONS IN BEE CULTURE. HK. F. PHILLIPS, in charge. G. F. WHITE, J. A. NELSON, experts. G. S. DemutH, A. H. McCray, N. E. McInpboo, apicultural assistants. PEARLE H. GARRISON, preparator. H. A. SurFrace, D. B. CASTEEL, collaborators. JI CIRCULAR No. 157. Issued May 10, 1912. United States Department of Agriculture, BUREAU OF ENTOMOLOGY. L. O. HOWARD, Entomologist and Chief of Bureau. THE CAUSE OF EUROPEAN FOUL BROOD. Bye Gack, With. Me) Phe :: Expert in Bacteriology. HISTORICAL. The purpose of this preliminary paper is to discuss briefly the ex- citing cause of European foul brood. With diseased material furnished by Cheshire, Cheyne made a bacteriological study of foul brood. The latter isolated and described a bacterium from the brood dead of the disease and identified it as Bacillus alveit. Cheshire agreed that the identification was correct. A joint paper by these men appeared in 1885, and for about a decade and a half thereafter Bacillus alvet was generally considered to be the cause of foul brood. The disease which, it is believed, Cheyne studied is the one that has received the designation European foul brood. This disease is also believed to be the one which William R. Howard worked upon and named “black brood.” In 1900 he described as its cause a bacterium to which he gave the name Bacillus milii. It is probable that Burri in 1906 was studying the same disease in Switzerland when he referred to “ sour brood.” In the disease he dis- covered a bacterium to which he referred as the “ guntheri—forms.” Maassen in 1907, working probably with the same disease, appar- ently encountered the “ guntheri-forms” reported by Burri and named the new species Streptococcus apis. SOME EARLIER WORK BY THE WRITER ON EUROPEAN FOUL BROOD. In 1907 the writer observed in European foul brood the bacterium which had been named Streptococcus apis. It was observed at the same time that there was present also another microorganism quite al 2 THE CAUSE OF EUROPEAN FOUL BROOD. similar in appearance, but clearly different from the one which Burri had observed and Maassen had named. AI] attempts, however, to cultivate this new species were unsuccessful. Until more was known about this organism it was referred to in 1908 as “ Bacillus Y.” In 1907 the writer demonstrated that American foul brood, an infectious brood disease, could be produced by feeding to healthy colonies pure cultures of Bacillus larve. ‘This fact emphasized the probability that if European foul brood is also caused by a bac- terium this disease, too, could be produced by feeding pure cuitures of the bacterium causing it. To demonstrate this, it was desirable in the first place to determine whether or not the virus of European foul brood was present and active in the diseased brood. Healthy bees were fed sirup which contained a suspension of the diseased brood, and European foul brood was produced. This showed that the diseased brood did con- tain the virus and that the disease could be produced by feeding. This being done, pure cultures of Bacillus alvei isolated from the dis- eased material were subscituted for the diseased brood in the inocu- lation experiment. Pure cultures of Streptococcus apis were isolated — and used likewise. Then cultures of Bacillus alvei and cultures of Streptococcus apis were used simultaneously in making the inocula- tions. European foul brood was not produced in any of the experi- ments where pure cultures of either or both organisms were used. These facts were sufficient to eliminate tentatively Bacillus alvei and Streptococcus apis from the list of possible causes of European foul brood, and to justify a strong suspicion that the microorganism which was referred to as “ Bacillus Y ” bore a causal relation to the disease. It was necessary, however, to reckon with other factors before a more definite statement could be made. A continuation of the work on the cause of European foul brood has yielded some interesting results. These will be briefly con- sidered in this preliminary paper. .It has been observed in the examination of diseased brood that Bacillus alwei is frequently either absent, or present only in small numbers, in many of the larvee which seem from gross appearance to be dead of European foul brood. Such samples have been received as a rule from localities in which apparently the disease had only recently appeared. Frequently, also, Streptococcus apis seemed to be absent, or present only in small numbers, in many of the larve which froin gross appearance gave strong evidence of European foul brood. These bacteriological findings further strengthened the theory that neither Bacillus alvet nor Streptococcus apis is the pri- mary exciting cause of European foul brood. .Other inoculation experiments were performed, using pure cultures of these two species. THE CAUSE OF EUROPEAN FOUL BROOD. 3 The results were always negative, confirming further similar results that had been previously obtained. RECENT WORK BY THE WRITER. While these facts were in a measure satisfactory, as tending strongly to indicate certain conclusions, there was still wanting that degree of conclusiveness which is always desired. By experi- mental inoculation and by the study of the brood sick or dead of the disease which was artificially produced, however, considerable’ in- formation of the character hoped for has been obtained. The details of the technique used in making the inoculation will not be given in this brief report. EXPERIMENTAL INOCULATIONS. Diseased material from various localities was used for these inocu- lations. Some colonies were fed diseased brood that contained, as revealed by cultural examinations, large quantities of Bacillus alvei; other colonies were fed diseased material containing large numbers of Streptococcus apis as shown by cultures, and still others were fed diseased material which was demonstrated to contain neither Bacillus alvei nor Streptococcus apis. It was found that at the first appearance of the disease in each class of experiments the symptoms manifested by the sick larvae were the same. Larvee showing these early symptoms were studied bacteriologically. The examinations showed that whether or not the diseased material fed to the bees contained Bacillus alvei or Streptococcus apis these species were in the early stages of the disease either absent, or present in small numbers only. It is quite evident that the disease was not pro- duced by species of bacteria which were absent at this early stage of the disease. Continuing the bacteriological study of the larve in the early stages of the disease, some new species were found to be present. One bacterium especially is frequently encountered. This species is a small, slender rod, apparently nonmotile and nonspore bearing. It is to be known by the name Bacterium eurydice. Its description will appear in a later publication. Experimental colonies have been fed pure cultures of this species, but no disease has been produced. Tentatively, therefore. this species is not to be regarded as the cause of European foul brood. Two other species of bacteria might be mentioned here as being of interest in connection with the study of the brood diseases. The first to be mentioned is a motile, spore-bearing, easily cultivatable rod. It is to receive the name Bacillus orpheus. It also will be de- scribed later. This species is occasionally found in very large num- 4 THE CAUSE OF EUROPEAN FOUL BROOD. bers in samples of European foul brood. Feeding it in pure cultures has so far given negative results. This organism, then, can also be eliminated tentatively from the list of possible causes of this disease. The other species, mentioned by Lambotte in 1902 as Bacillus mesentericus vulgaris, may be said to belong to a group of bacteria found quite widely distributed in the apiary. Its infre- quency in diseased brood and its occurrence in small numbers readily eliminates this species from the list of possible causes. Most of the bacteria that are met with in the study of European foul brood were therefore excluded tentatively from the list of possible causes of the disease. The possibility of an ultramicroscopic virus was also considered. Brood sick or dead of European foul brood were removed from the combs and crushed. An aqueous suspension of this diseased material was then made in boiled water and filtered with the Berkefeld filter. The filtrate remained clear when incubated at different temperatures and cultures made from it produced no growth. Separate filtrations have been made of diseased brood received from various localities, but in no instance where healthy colonies were fed filtrate obtained in this way was European foul brood produced. The results of the ex- periments therefore justify the tentative conclusion that there is no filterable virus in European foul brood capable of producing the disease. To this extent, then, has the possibility of an ultramicro- scopic virus been eliminated. Having thus tentatively eliminated all he microscopically visible organisms except Bacillus Y from the list of possible causes and like- wise eleminated the probability of an ultramicroscopic virus, the tentative conclusion was naturally reached that this remaining micro- organism probably plays an important role in the etiology of Euro- pean foul brood. Such a conclusion was all the more imperative since this organism had been encountered so frequently in the brood of this disease and since, moreover, there had been no other. factor observed to which the exciting cause could be attributed. SYMPTOMS MANIFESTED BY LARV SICK OF EUROPEAN FOUL BROOD. This conclusion led to a more extended study of this microorganism in the disease produced experimentally. The presence of disease can usually be detected in the experimental colony during the week that the feeding is begun. The first indication of it may be that only a portion of a larva is seen in a cell (fig. 1), the remaining por- tion having been removed by the bees. Aside from an observation of this kind the earliest indication one gets from the macroscopic (gross) examination is that sick larvee are found among the uncapped brood. One should acquaint himself, therefore, with certain symp- toms or signs manifested by sick larve during the course of the dis- THE CAUSE. OF EUROPEAN FOUL BROOD. 5 ease by which its presence can be diagnosed while the larve are still alive. Some of these will now be considered. le [a | yy Fic. 1.—Larva sick of European foul Fic. 2.—Healthy larva of the age repre- brood, partly removed by the _ bees. sented in figure 4. (Original.) (Original. ) The length of time that a developing bee is sick of European foul brood is variable. It can be stated in a general way that the three. RY URS = SNVASSANYNY SS NSS a1 we : Fie. 3.—Sick larva of the age represented in figures 2 and 4. (Original.) days just preceding the time when a larva would ordinarily be capped is the most favorable period for making a diagnosis from the gross Wy examination alone. When healthy larve of the age nepnesencul a nics ab es: 2 and Fic. 4.—Sick larva with roof of cell 5 are shghtly magnified a peristal- removed. (Onisindl) 6 THE CAUSE OF EUROPEAN FOUL BROOD. sislike motion of their bodies is easily seen, but larve of this same age when sick frequently exhibit a marked peristalsislike motion of their bodies which can be easily seen with the unaided eye. Some- Fic. 5.—Sick larva which is more trans- Fic. 6.—Healthy larva with dorsal wall parent than a healthy larva of the turned toward the observer showing Same age. ~ (Original.) the narrow transparent area along the median dorsal wall. (Original.) times the color of the larve assists in the selection of those that are diseased. If, instead of the glistening white or bluish-white appear- ance of healthy larvee, one observes some that are more transparent (fig. 5), or that possess a very sheht yellowish tint, frequently such larvee are diseased. In the absence of the exaggerated peri- stalsislike movement, however,: other tests should be applied, as the color symptom is at times de- ceptive. Figures 6 and 7 represent older larve than the preceding. These have turned themselves in the cell so as to present a dorsal portion to the observer. The narrow and quite transparent area frequently seen along the dorsal median line of a larva serves often a useful Hig. Re Dick Iitva Of theese Temiesented «ir pose ai time Guan ates b h atciags in figure 6. (Original.) pean foul brood. In a healthy larva (fig. 6) a pollen-colored intestinal mass is frequently plainly visible through this transparent dorsal area. Microscopically this mass is easily demonstrated to be largely pollen. If, however, upon THE CAUSE OF EUROPEAN FOUL BROOD. Ff inspection this intestinal mass appears white or yellowish white, the presence of European foul brood is almost certain. A modification of this simple inspection method may often be profitably used. This consists simply in turning the larva in the cell with a pair of forceps until the median dorsal line is exposed to the observer. More frequently still, it will be found advantageous to remove the larva from the cell with the forceps. With a little care this can be done, leaving the larva intact. If the larva is diseased and the dis- ease is sufficiently advanced, a whitish intestinal content can very often be plainly observed. In response to the muscular action of the larva this mass is frequently seen to be moved to and fro. A POSITIVE TEST FOR THE DISEASE IN LIVING LARVA. There is a sign represented in figure 8 which, in the experience of the writer, has proven thus far to be a positive symptom of Euro- pean foul brood. When the age and condition of the diseased larva are favorable—and these frequently are—the sign can be quite easily and conveniently demonstrated in this way: Select a larva to be tested, approximately of the age represented by figures 2, 3, and 4; remove it from the cell and place it upon glass, preferably with a dark background; with a dissecting needle in each hand and with _ their points near together, pierce with both needles the wall of the larva near its head, avoiding the intestine; separate now the points of the needles so as to tear the body wall crosswise and continue to separate the two portions of the larva. If the larva is diseased and one is successful in applying the test, it will be found that the in- testinal content will be stripped from and pulled out of the posterior and blind end (0, fig. 10) of the canal, obtaining results as repre- sented in figure 8. In case of living, healthy larve the intestinal — content can not be removed in this way. | This mass thus removed from the intestine, if examined micro- ecopically, will be found, in general, to consist of a white or shghtly yellowish-white mass along the longitudinal axis. This central mass is surrounded by a substance which is more or less transparent and mucuslike in appearance. The appearance of this outer portion, however, will vary in detail, depending in a great measure upon the stage of the disease when the examination is made. The force which is applied in pulling the mass from the intestine frequently causes this enveloping substance to stretch and the in- closed whitish substance to break into segments as represented in a of figure 8. This is an earlier stage of the disease than that repre- sented in either 6 or ¢ of the same figure. 38171°—Cir. 157—12——2 THE CAUSE OF EUROPEAN FOUL BROOD. Fic. 8.—The intestinal content removed from larve sick of European foul brood but not yet dead of the disease. (Original.) THE CAUSE OF EUROPEAN FOUL BROOD. 9 If the disease is more advanced than either stage represented in figure 8 when this test is applied, a portion of the intestinal content may flow out in the form of a sac, the wall of which is very easily broken. When broken the content of this saclike structure will flow out as a rather thin whitish or yellowish-white fluid containing small whitish granules that vary in size. If the disease is far advanced and the larva probably dead, the enveloping substance of the intes- tinal content is so easily broken that often only whitish or yellowish- white fluid with its granular content flows from the ruptured wall of the larva. } Figures 2 and 6 represent healthy larvee, and at these ages the seg- ments of the body are strongly marked off. Living larve at these ages, if suffering from European foul brood, frequently show these markings less distinctly as represented in figures 3, 4, and 7. This sign, too, may asssist in the selec- tion of larve that are suspected of being diseased. THE VALUE OF EARLY SYMPTOMS IN THE DIAGNOSIS OF EUROPEAN FOUL BROOD. These symptoms of European foul brood are some of the more important ones that are observed in sick larve or in those only recently dead. They are espe- cially valuable in the study of the disease in the experimental Fic. 9.—Bacillus pluton in a stained smear colony. They have not been used Dean Piaaeiscatee Tonnainye pals by the apiarist for making a diagnosis. The symptoms of European foul brood that have been looked for by the bee keeper for the most part are the evidences of disease which obtain as a result of the death of the brood. The post-mortem symptoms as manifested by the dead larvee themselves have been the most positive evidences used by the bee keeper in diagnosing the disease. It is hoped, however, that when they are well learned, the symptoms of European foul brood observed in living larve and in those very recently dead may prove of value in the apiary as well as in the experimental colony. Practically all the later symptoms of European foul brood have also been observed during the course of the disease in the experi- mental colony. This fact is used as evidence that the disease which was produced in the experimental colony was the same as that encoun- tered in the apiary. Since the diseased material for making the inoculations has been received from various sources and the disease produced was apparently the same in every case, the conclusion that 10 THE CAUSE OF EUROPEAN FOUL BROOD. there is but one disease present in the condition which is being called European foul brood is, therefore, still further confirmed. 7 MICROSCOPIC STUDIES OF DISEASED LARV. Returning now to the discussion of European foul brood in the earlier stages, it should be emphasized that by a macroscopic exami- nation alone it is not always possible to make a positive diagnosis of the presence or absence of disease in a larva. During the very. earliest period of infection it 1s impossible from the gross examina- tion alone to make a positive diagnosis of the presence of disease. Such is to be expected. About the time the larva dies there is a period at which one can not always be sure that the disease is present from a macroscopic examination alone. Between these stages there is a period in the course of the disease in the larvee during which it is usually possible to make a diagnosis positive from the gross exami- nation. Since a macroscopic examination alone is not always sufli- cient for making a positive diagnosis, one looks naturally to a micro- scopic examination for assistance. During the course of the disease in the experimental colony the microscopic picture presented in the examination of diseased larvee changes markedly. To begin the microscopic study, it is well to obtain the intestinal content as represented in a, figure 8. If a thin smear is made of the white growth-mass of this content and stained, it is found to consist almost entirely of forms represented in fig- ure 9. This organism is the one that the writer referred to in an earlier paper as “ Bacillus Y.” All attempts to cultivate this new species on artificial media have thus far been unsuccessful. Since consider- able information has now been obtained concerning this organism the specific name “ pluton” is now substituted for the “ ¥””’ in the term “ Bacillus Y ” and the species will now be known as Bacillus pluton. This organism is an unusual one and the classification has not yet been definitely determined. The generic term “ Bacillus,” therefore, may, and probably will, be changed later. At the stage of the disease represented in a, figure 8, the majority of the individuals of this new species in general appear in stained preparations to be pointed at the ends (fig. 9). Some show both ends rather sharply pointed, others show only one end so pointed, the other end being rounded, while still others show both ends rounded. The individuals having.this general form vary much in size. They are as a rule 1 p or less in length, the breadth being about one-half the length. Forms in pairs frequently occur in a smear preparation made at this stage of the disease. These paired forms vary markedly in size and shape. (See fig. 9.) Accompany- THE CAUSE OF EUROPEAN FOUL BROOD. 18s ing Bacillus pluton, Bacterium eurydice is frequently found at this stage of the disease, but in comparatively small numbers. If the intestinal content in a later stage of the disease—for exam- ple, that represented in 0, figure 8—is examined microscopically, Bacillus pluton is still found in very large numbers, and Bacterium eurydice when present will be relatively very much increased in num- bers. A similar examination of the intestinal content represented in ¢, figure 8, will usually show Bacillus pluton in large numbers, Bac- terium eurydice in increased numbers, and in addition one may find Bacillus alvei in comparatively small numbers. | Fic. 10.—Schematie drawing representing a longitudinal section of a larva at an early stage of infection. The position of the invading organism, Bacillus pluton, is along and near the peritrophic membrane. (Original. ) By examining the fluid mass which flows from the body of a larva when the disease is far advanced and the body wall is broken, one usually finds, together with Bacillus pluton, bacteria of different species in considerable numbers. From this point on in the decay of the larve the relative propor- tion of the different microorganisms present varies markedly. When Bacillus alvet is present it increases very rapidly in proportion to the others. This rapid increase of Bacillus alvei in the larve after the death of the larve accounts in a large measure for the frequency with which this species is mentioned in reports on this disease. 12 THE CAUSE OF EUROPEAN FOUL BROOD. In living larvee, therefore, in which European foul brood can be diagnosed from gross examination, it is found that bacteria usually accompany Bacillus pluton. This fact made desirable the study of the diseased larve in still earlier stages of the infection, i. e., during the period of incubation. This was done culturally in part, but prin- cipally by fixing and sectioning the younger larve from strongly infected experimental colonies. From such sections it was observed that Bacillus pluton was the first invader of the healthy larve. Figure 10 represents schematically the condition in the larve at an early stage of infection. In this figure fg represents the foregut; mg the midgut, and hg the hindgut. At this age of the larve the posterior end of the midgut is closed, as represented at >. In the same figure, m represents that portion of the intestinal content lying in contact with the wall of the intestine; 7, the central portion of the drawing, represents the food taken at this age; and p represents what seems to be a peritrophic membrane between the enveloping substance, m, and the paplike food substance, 7, of the midgut. In the growth of Bacillus pluton this parasite very early takes a position along the peritrophic membrane p, and just central to it (fig. 10). At this early stage of its growth this microorganism pre- sents in general an appearance of being rod shaped with a strong tendency to grow in chains. As the disease advances and the growth- . mass of this organism increases, the central portion of the lumen of the intestine becomes filled by a solid growth which is made up very largely of Bacillus pluton. During this stage of the disease the con- tent can be removed from the posterior blind end of the midgut, as shown in figure 8. The relation of the central growth-mass to the surrounding mucuslike-appearing mass represented in a, b, and ¢ of figure 8 is well demonstrated microscopically by sectioning these intestinal masses. From the studies made thus far it would seem that Bacillus pluton is easily killed by heat. PROBABLE EXPLANATIONS OF ERRORS AS TO THE EXCITING CAUSE OF EUROPEAN FOUL BROOD. It is quite probable that others at different times have observed this new species, Bacillus pluton, but have failed to differentiate it from bacteria which were present and which appeared in the cultures made, leading them thus to erroneous statements concerning the dis- ease and its exciting cause. For example, Wiliam R. Howard may have seen this organism microscopically in his so-called * black brood.” but failed to differentiate it from some bacterium—Sacil/us milii or Bacillus alvei—which he cultivated on artificial media. Burri may have seen it in the so-called “sour brood ” and mistaken it THE CAUSE OF EUROPEAN FOUL BROOD. 13 for the “ guntheri-forms” which he observed in his cultures. Maassen mentions some difficulty experienced at times in obtaining Strepto- coccus apis from brood which on microscopic examination seemed to contain this bacterium. To explain this difficulty, he advanced the supposition that the Streptococcus was probably killed by acid pro- duced by itself. The difficulty probably could be as well explained by supposing that Maassen failed to differentiate this parasite from the bacterium which he cultivated and described as Streptococcus apis. IS THERE MORE THAN ONE DISEASE IN THE CONDITION KNOWN AS EUROPEAN FOUL BROOD 2 The question now arises whether or not there is more than one disease in the condition now known as European foul brood. In Switzerland and in Germany there has been a tendency to diagnose the diseased brood in which Bacillus alvei is found as the foul brood of Cheshire and Cheyne and the diseased brood in which Strepto- coccus apis is found as “sour brood.” From the facts at hand the writer is strongly inclined to believe that these two conditions are only the one disease, known in America as European foul brood. Enough evidence has not yet been obtained, however, to speak with complete positiveness on this point. As secondary invaders some of the species of bacteria mentioned ~in this paper may and probably do exert an influence on the course of the disease in the larva and in the colony. To what extent these bacteria modify the disease is yet to be determined. Should it be found that Bacillus alvei actually causes an infectious brood disease, then such a disease should be called European foul brood, and the , disease caused by Bacillus pluton would have to be differentiated from it. Further details will not be given in this preliminary announce- ment but will be included in more technical papers which are being prepared. SUMMARY AND CONCLUSIONS. The steps taken in the writer’s endeavor to find the cause of EKuro- pean foul brood may be briefly summarized as follows: (1) Bacillus alvet, which has been so generally spoken of as the cause of foul brood, was isolated from diseased brood, and pure cul- tures of the organism in both the vegetative and spore forms were repeatedly fed to colonies of healthy bees with the result that foul brood was not produced in any instance. This fact cast a suspicion that Bacillus alvei was probably not the cause of a disease. (2) By a study of many larve in samples of European foul brood it was frequently found that there were larve apparently dead of the disease that contained Bacillus alvei only in small numbers or not at. 14 THE CAUSE OF EUROPEAN FOUL BROOD. all. This increased the suspicion that Bacillus alvei was not the exciting cause of the disorder. (3) In 1907 the writer proved that by feeding pure cultures of Bacillus larve to healthy bees American foul brood could be produced. This fact still further emphasized the doubt that was already enter- tained concerning the possibilities of Bacillus alvei in the etiology of European foul brood. (4) By feeding diseased larvee to healthy colonies it was found that European foul brood could be artificially produced, showing that this disease, too, could be produced by feeding, and that ee virus was contained in the diseased brood. (5) The sick larve of the disease thus artificially produced were » frequently found, when examined, to be free from Bacillus alvei. This evidence, too, was damaging to the theory that Bacillus alvei is the cause of a brood disease. (6) Bacillus alvei in this way was tentatively eliminated from the list of possible exciting causes of European foul brood. In a quite similar manner the other bacteria—Streptococcus apis, Bacillus me- sentericus vulgaris, Bacillus orpheus, and Bacterium eurydice—were likewise eliminated from the list. | (7) Considerable quantities of filtrate from aqueous suspensions of crushed diseased larvee were fed to healthy colonies and in no instance - was European foul brood produced. This eliminated tentatively the probability of there being an ultramicroscopic virus in European foul brood capable of producing the disease. (8) Bacillus pluton, therefore, was the only factor that was not so eliminated from the lst of possible exciting causes of the disease and became thus the probable exciting cause of European foul brood. (9) When this organism was studied in larvee in which the disease could be suspected by inspection alone, one or more species of bacteria were sometimes found to be present also. These, when present, how- ever, occurred in relatively small numbers. (10) The disease was then studied in a still earlier stage; i. e., before its presence could be detected by gross examination of the larve. This was done by cultures in part, but principally by fixing and sectioning larve during the incubation period of the disease. This study demonstrated that inthe production of the disease Bacillus pluton was the first invader of the healthy larve. Tt will be noticed, therefore, that in the determination of the pri- mary exciting cause of European foul brood two objects were accom- plished: (1) All the factors in the list of possible exciting causes of the disease were eliminated except the one organism Bacillus pluton, and (2) by the study of infected larvee soon after the infection took place, this parasite was found to be the first invader. THE CAUSE OF EUROPEAN FOUL BROOD. 15 As a conclusion, it is the belief of the writer that sufficient evidence has now been obtained to justify the statement that Bacillus pluton is the primary exciting cause of a brood disease. This brood disease is now generally known in America as European foul brood. This opinion is rendered in accordance with views now generally accepted relative to the etiology of animal diseases. There are, then, three principal brood diseases. Two of these— American foul brood, caused by Bacillus larvw, and European foul brood, caused by Bacillus pluton—are known to be infectious. From these two diseases there must be differentiated the third one, an apparently noninfectious disorder, the so-called “pickled brood.” Larve dead of this latter disease are practically free from micro- organisms. The exciting cause of this disorder is not yet known. Approved: JAMES WILSON, Secretary of Agriculture. WasurineTon, D. C., dlarch 28, 1912. DDITIONAL COPIES ofthis publication may be procured from the SUPERINTEND- ENT OF DOCUMENTS, Government Printing Office, Washington, D. C., at 5 cents per copy daaoa Sal : Fie Th Dy o ihe i ee mete ie ain eae eae CMM on) oe CeE a it te Pa is aes i Pe > {haa ee wet bi Rot iets hk Joie Ne al Kira Nhe Opie » eae een) Tae OF | ae a EG oa : ee ni HD wee ; ; "us We “ie te BaD ce re ‘ter cy ae re Pe oe ace 2 see 2 : Pa 5ar wy oa ane . vies ie 2 : BH een f a ; i TNS : Nepraverireciver | af +) Bf 3 OES ish ite " OY NSS Sones abe ~ ~~ me x) «th Seats SY jute J aad, os, as pea = — <1 neers Renan 5 a" wee ¢ OY erikesegt iS: ee, ty ) v ‘2, : ; Cane (s We nares ~ So Ars eas ~ ewe ne Taser = ’ ep tt ELT Tt Lae re mor Sha (oe) LOA . pen NY ; Srey f Arsh ; Beth alse a bees ae ms em, ~ Dye pore —) hin nn vit ‘ j f Nreiace $144 {i ist i wit : NY : Pei IESE t ii Hf aed a i Sy: nih i) i aN Mh Wie Bi i Hus Atala Me eo er} ‘