^?.>-.7* Circular No 94. United States Department of \gm EAU OK KNTOMOI i Bui \mi 1:11 w i oi I i:i:ooi). F. Win ik, Ph. I'.. \ii in Bactei iolo For Beveral reasons much confusion exists ae to the preBenl statue oi mir knowledge of bee diseases. It is hoped thai this circular will give information which will to Bomeextenl clear up the subject ol American foul brood from a bacteriological poinl of view. The Bymptoms of this re given in Circular No. 79 of the Bureau of Entomology, entitled "The Brood Diseases of Bees." American foul brood is the prevalent disease in America, and, judging from reports received from pe and from descriptions in European bee journals and hooks, it is the prevalent one there. Then' is another diseased condition, which Cheyne examined, and to which we now refer as European foul brood. When tlic author began his work on bee diseases in the Bummer of 1902 he observed, in combs containing American foul brood, in the dried remains of the dead larva', known as the Bcales, a very large num- ber of Bpores which failed to grow when inoculated into the media ordi- narily used in the laboratory. It was clear, then, that these Bpores are not Bacillus alvei and thai this disease is not the "foul brood " of Cheshire and Cheyne. The follow ing year the study was continued and a medium levised in which the Bpores found in this disease will germinate. This medium consist- of an agar made by following the directions ordi- narily used in the laboratory, with the exception that bee larva? are sub- stituted for meat. By the use of tins medium were obtained pure cul- tures of the microorganism which is found so abundantly, in the form of Bpores, in the dried BCaleS of American foul hrood. In reporting these findings the author referred to this organism as Bacillus \. " Further study was subsequently made, and the -pedes was given the permanent name Bacillus larva. The description of this species may he found in Technical Series No. 11 of this Bureau. In his publications the author has made no claim that Bacillus larva is the cause of American foul hrood. hut has made the statement that it is found to he present in all the samples of this disease which have 1 The author wishes to acknowledge the assistance "t I'r. K. F. Phillips, in Charge oi Apiculture, under whose direction tin.- «erk has been done. results have been reached since the author has been connected with the apicul- tural investigations of the Bureau oi Entomolo( been examined by him. No inoculation experiments were made, for the reason that sufficient cultures in suitable condition could not be obtained from any medium then known. Since the media used in former investigations are not suitable for obtaining cultures for purposes of inoculation, in taking up the further study it has been necessary to devise a medium which would be satis- factory in this respect. Such a medium has been discovered, and large amounts of the culture suitable for experimental inoculations have been obtained. This medium is prepared and used as follows : Healthy bee larva? or young pupa' are picked from the comb, crushed, strained through cheese cloth, diluted with 20 to 50 times their volume of water, filtered through ordinary filter paper, and then passed through an earthen- ware filter (the Berkefeld filter is satisfactory) to remove any bacteria which are present. The sterile filtrate thus obtained may be pipetted into tubes or flasks and stored until needed. When Bacillus larva is to be isolated, a tube of the ordinary agar of the laboratory is liquefied and cooled to 45° or 50° C. Then about 2 c.c. of the filtrate men- tioned above is added to it. A very small amount of the decaying larva' affected with American foul brood is then added. The procedure from this point is as usual in making agar plate cultures; these plates are afterward incubated. When a large amount of culture is desired for experimental purposes it is convenient to use the ordinary agar medium in large test tubes to which has been added, as above, about 2 c.c. of the sterile larva- filtrate. These agar tubes are then inclined and the surface of the congealed agar is inoculated. In no case should the larva 1 or filtrate reach a high temperature. The object, of course, is to obtain a medium which contains the food constituents which are afforded the bacteria in the living larva?. Inoculation experiments have been made by feeding to a healthy colony the scales from combs which had contained brood affected with American foul brood. The result of the feeding was that the colony became affected b} T disease, the symptoms of which were the same as those observed in the apiary where American foul brood is found. Like symptoms have been produced by feeding scales which had been put into ordinary meat bouillon, incubated for twenty-four hours, and then heated to 65° C. for twenty minutes. On microscopic examination of the decaying larva 3 dead from the disease thus produced experimentally, the same large number of spores and rods are seen as when samples are examined which are taken from an apiary affected with American foul brood. From these dead larva' pure cultures of Bacillus larva were obtained from plates, using the new medium described above. These experiments show that by the feeding method the disease may he produced and that the contagion is found in the scales. The second experiment tends to indicate that the cause of American fool brood as found in thi ie not killed by heal .1! ■ pplied for twenty minuti Up to the presenl time there 1- no authentic record of thie disi having been produced by experimental inoculations of pure cultui Knowing thai by the feeding method the disease may be produced, pure cultures of Bacillus larva have been mixed with sterile sugar sirup and fed to healthy colonies with the result thai the die appeared in the colonies within three weeks with symptome identical with those produced by feeding the Bcales of the disease. In the ropj brown mass of the decaying larvae in the disease which is produced experimentally by feeding pure cultures of Bacillus larva there are found the same large number of spores and rods as when the disease ie produced by feeding th>- or when the disease is found in an apiary. Pure cultures ol Bacillus larva have been obtained from the larva' dead from the disease produced experimentally by feeding pure cultures of Bacillus larva . Some European investigators of brood diseases omil the symptoms, so thai it is impossible to tell which disease they are investigating. Their descriptions of microorganisms also are entirely too brief. These facts have led to much confusion, and they necessitate much additional work on the part of other investigators. They have also added to the presenl confusion. From what can be gained from their papers, the author is inclined to believe thai Burri has been working with Bacillus larva and lias Keen referring to it as the "bacillus difficult of cultiva- tion;" thai Maassen has been working with Bacillus larva and has been referring to it as Bacillus brandenburgiensis, and thai von Butte) Reepen lias referred to Bacillus larva as "/»'. fwrri." It is hoped thai this confusion may soon cease to exist. In the study of Bacillus larva on this new medium Borne interesting additional facts have been observed in the morphology and cultural characters of this organism which will be given in a bulletin from this Bureau in the near future. One fact i- mentioned now because it seems to have caused one German investigator, Dr. Allien Maassen, to fall into error in the interpretation of certain findings. This fact is that this specie.-. Bacillus larva, produce- a large number of gianl whips. (Gianl whips are at present believed to he in Borne way a modification of Qagella, the motile organs of bacteria.) These gianl whip- appear in pure culture- of Bacillus larva and persist there for a long time. structures which Maassen evidently saw and reported in two diiTer- ent publications, naming them 8pirocha>ta apis, are nothing other than gianl whips which normally belong to Bacillus larva and which an' formed by the growth of Bacillus larva in the larva' of the bi Maassen Beems t<> have no further evidence that the structures which w are spirochetes than what could he gained by a microscopic examination of the remains of the dead larva' which had suffered from UNIVERSITY OF FLORIDA 3 1262 09216 4614 this disease. The appearance which he interprets as a spirochete in the process of division can be seen in the giant whips obtained from pure cultures of Bacillus larva. These giant whips are found in the decaying larvae which are dead from American foul brood experimentally produced by feeding pure cultures of Bacillus larva . The author has observed these structures in a large number of exam- inations of American foul brood, especially in the hanging-drop prepa- rations made directly from the dead larva'. There is nothing else contained in the dead larva' which can be seen that resembles a spiro- chete, and since Maassen made no mention of the giant whips found there so abundantly, it is quite certain that he has made this mistake. This preliminary note will be followed by a bulletin which will con- tain in full the results of recent investigations by others on the brood diseases of bees and a detailed account of the work done here. The results may lie summarized as follows: (1) In previous publications the author has made no claim that Bacillus larva is the cause of American foul brood. (2) A medium has been devised by which cultures of Bacillus larva may be obtained in large quantities suitable for experimental inocula- tion. This medium consists of the sterile filtrate obtained by diluting and filtering the crushed bodies of bee larva- through a Berkefeld or other fine filter. (3) American foul brood has been produced by feeding pure cultures of Bacillus larva , and the symptoms of the disease are the same as those produced by feeding the scales of this disease and as those ol .served in the apiary where colonies are affected with this disease. (4) The structures described by Doctor Maassen, of Dahlem, Ger- many, as spirochetes and named by him Spirocha la apis are not spi- rochetes, but normal structures, produced by the growth of Bacillus larva. These are known in bacteriology as giant whips. Approved : W. M. Hays. Acting Secretary of Agriculture. Washington, D. C, July 15, W<>7. O