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Sys 3 eee & —* eee : a aan as =F ~s CES a 3. 2 FB yA. 2 eas ~" SRE OE rs aed ea ee Ss Saye ote PhS Re oe a ~ YY a Ie tees ae Sete segs. = ee oe ~ mt Se aH + Mii: i a iat +e nik re j if I 1H) ¢ i he bai a ¢ K 3a) este es = aoa 5 EN passes at eee aS ms Saree pees f nn at aie = a gas ° * lt — set a Sf ObaaA Ar eae Fia. 1.—COMB CONTAINING LARV4 DEAD OF SACBROOD. N Natural size. (White.) se #0 “gf CONG s* Mee y é ,o 4, aK . Win S258 af 4 LR Fe ONG a 4 3? 4 Fea at 8 44 59) Pe, ~~ ¢. -s ®% a. ca 4 ~ % “-? at tne og att ‘ EF et Pe bear 2G 00 OU Se i? Ol Ve 4 ait one = re ea oe IG! Fic. 2.—STAINED SMEAR PREPARATION SHOWING SPORES OF NOSEMA APIS. Highly magnified. (Original.) SACBROOD AND NOSEMA SPORES. DIAGNOSIS OF BEE DISEASES. 9 that on thrusting the forceps into the brown larval remains and withdrawing them a portion of the decaying mass adheres and is drawn out, often to a distance of 3 or 4 inches. The viscidity is often referred to by the term “ropiness” in beekeeping literature. In the rare instances in which young uncapped affected larve are en- countered the ropiness is less pronounced. (¢) Odor—The odor is characteristic and may be described as unpleasant. Often it is feeble or absent altogether, probably having disappeared after the removal of the diseased brood from the hive. Affected brood-comb will absorb other odors if given the oppor- tunity, thus masking the original characteristic odor. Such dis- appearance and masking of the odor has been observed where speci- mens of diseased brood in combs from various sources had been thrown together and allowed to le about preparatory to being de- stroyed (McCray, 1916). (h) Kind of brood.—lIt is affected worker-brood that is most often encountered in American foulbrood samples, although drone-brood is sometimes affected. (z) Scales—tThe scales of American foulbrood are distinctive and are characteristic of the disease. If they are present in sufficient numbers the disease can be diagnosed from the gross appearance alone. The lower cell walls can be easily illumined by tilting the upper portion of the comb toward the observer, as previously de- scribed. The scales appear extended along the lower cell wall, are quite dark in color, and adhere closely to the floor and base of the cell. Sometimes they adhere so closely as to break when an attempt is made to remove them from the cell. Often a semblance of the form of the pup is evident in the dried-down mass forming the scale. Some of the mouth rarts of the pup sometimes protrude sufficiently to adhere to the root of the cell. MICROSCOPIC FINDINGS. In considering the microscopic appearance of stained smears from infected brood in this disease, there usually is only Bacillus larvae in the spore form to engage the attention of the observer. In some imstances, however, samples containing the disease in its earlier stages are received and then Bacillus larvae in the rod or vegetative form may be encountered. Such a sample usually is more difficult to diagnose. To receive a sample in which some older larve con- taining spores of Bacillus larvae can not be found, however, is a very rare occurrence. In a stained smear made from an infected larva and mounted in water, the microscopic picture is rather characteristic. The most striking feature of the mount is the large number of spores adher- ing to the cover glass, floating with the current, or dancing free in 10 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE. the water medium. Many of the spores stain slightly about the periphery, which aids somewhat in the observation. Good results may be obtained from an unstained water mount. The vegetative forms of Bacillus larvae, when they are present, are observed to be slender rods, which tend to occur in chains. CULTURES. In culturing affected larvee it is the absence of growth on the agar plates that is important in the diagnosis of American foulbrood. This is because the spores of Bacillus larvae will not germinate and grow on the ordinary media of the laboratory, and other growth is absent because there are seldom secondary invaders present. Occa- sionally there will be a spreading growth of B. vulgatus, or B. mesen- tericus, and very rarely of B. alvei. On quite rare occasions a con- siderable number of colonies of B. vulgatus or B. mesentericus have been found. As both of these species form spores, as a rule, within 24 hours, their differentiation from B. alvez usually can be made in this way. A report on a sample of American foulbrood, therefore, nearly always can be made within a day. SACBROOD. Sacbrood is an infectious brood disease of bees caused by a filter- able virus (White, 1913 and 1917). GROSS CHARACTERS. (a) Character of caps and regularity of the brood.—Uarve usually die after capping in this disease, some of the dead brood being un- capped by the bees later. Occasionally the caps are punctured. An area of comb affected with sacbrood therefore presents an irregularity. So far as the age of the affected larve themselves is concerned, there is considerable uniformity owing to the fact that death in this disease occurs after sealing during the two-day period of rest just preceding pupation. The affected brood, however, is interspersed among healthy brood of varying age, which adds to the irregular appearance of the affected comb (PI. II, fig. 1). (0) Proportion of affected brood. —As a rule there is not a large proportion of affected brood in a given comb area. Often there will be an affected larva only here and there. (c) Position within the cell_—The position of the affected larve is that of extension lengthwise along the floor of the cell, against which the dorsal portion of the larva lies. The head is turned upward, to- ward the roof of the cell. (d) Age—The brood dies after it has been sealed. (e) Color—uUsually by the time brood is received for diagnosis the color of the affected larve is brown or quite dark—often almost black. If the brood is in the earlier stages of decay, however, the DIAGNOSIS OF BEE DISEASES. ah color may be light yellow, light gray,.or ight brown. The lighter shades soon deepen to the darker ones. (7) Conststency.—The consistency is characteristic. The cuticular portion of the body wall of an affected larva is decidedly resistant so that the larva may be grasped with forceps and removed from the cell intact. After removal from the cell the larva has the appear- ance of a small closed sac. When the sac is ruptured the contents will be seen to be watery. Suspended in the waterlike fluid will be noted numerous fine brown granules. (g) Odor.—There is no distinctive odor to sacbrood combs. (h) Kind of brood.—The greatest ravages occur in the worker- brood. Affected drone-brood may be encountered. (¢) Scales——The scales when dried down are quite black and the surface appears somewhat roughened. They separate readily from the cell wall and may be lifted out intact by means of forceps. MICROSCOPIC FINDINGS. The striking feature of the microscopic examination is the absence of microorganisms. Rarely a few rods may be observed. A large amount of detritus is always in evidence, consisting of the brown granular material seen on gross examination after rupturing the body wall of the larva. These granules are in a large part the re- sult of the disintegration of the fat body of the larva. CULTURES. As might be expected from the microscopic examination, agar plates inoculated with infected material are practically always nega- tive as to bacterial growth. Even the presence of organisms of the vulgatus group is rare. No other growth occurs unless from chance contamination. OTHER ABNORMAL CONDITIONS OF THE BROOD. There are noninfectious abnormal conditions of the brood of bees which have been confused with one or more of the infectious diseases. ’ Among the more important of these may be mentioned chilled brood, starved brood, overheated brood, drone-brood resulting from laying workers, and brood dying after removal from the hive. The names given to most of the foregoing conditions are sufficient to indicate in a general way the probable cause of death. These conditions are less likely to be confused with American foulbrood than with either one of the other two infectious brood diseases of bees. The specimens that resemble European foulbrood in the gross consist of soft, easily ruptured, gray, yellow, and light-brown larve. The irregularity of the brood, the age, the color, and sometimes the scales bear a strik- ing resemblance to many cases of the disease. On microscopical examination of the affected larve the smear often discloses microorganisms, vet they lack the definite, clear-cut, 12 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE. positive picture desired in the diagnosis of the disease. If, on cultur- ing, the agar plates are free from Bacillus alvei the specimen is usu- ally considered negative. Occasionally specimens are received resembling sacbrood that tend to cause confusion. The head in some of these has a tendency to turn upward, resembling sacbrood, but unless there is present the tough body wall and watery granular contents, a diagnosis of sacbrood should not be made. A resemblance to the disease is sometimes noted after the brood dries down to form a scale, dark in color and sepa- rating readily from the cell wall. The microscopic examination and the cultures are often negative as in sacbrood. In such cases, when there are only a few affected larve, it is impossible to make a diag- nosis. Samples of comb containing only pollen without brood or scales have been received for diagnosis. Such specimens are always unsatisfactory and insufficient for diagnosis. TABLE 1.—Differential features in the diagnosis of the brood diseases of bees by laboratory methods. European foulbrood. | | American foulbrood. General appearance of brood. Proportion of affected brood. Position within cell... -_- Age of the larve...._..- Colorationes=s—eae eee Kind of brood affected.. Sealesiey sso eee Nok ee Microscopic findings... Culfurest eee see eee Usually die before capping... .| Bacillus Brood irregular. Large amount of afiected brood unsealed. Varying number of young ae affected, usually Usually curled at bottom. Larve soft, with melting appearance. Larve yellow, and brown. gray, Slight, inoffensive Soft, rather friable..........- Often considerable amount of drone-brood as well as worker-brood. Usually small and lie at bot- tomofcell. Yellow, gray, or brownin color. Some- times a few larger, brown, rubberlike scales. All scales separate readily from cell wall. pluton always. Bacillus alvei usually. Streptococcus apis some- times. Bacillus orpheus, Bacterium eurydice, Bacil- lus vulgatus, and Bacillus mesentericus, occasionally. Any of the above organisms except Bacillus pluton. Very irregular; affected brood sealed, sunken and perforated caps present. Usually a large amount of brood affected. Extension along lower cellwall. Larvz soon become a _ shapeless mass. Usually die after capping Usually dark chocolate... Usually strong charac- teristic odor. More or less offensive. Viscid, can be ‘‘roped”’ out a distance of 3 or 4 inches. Any considerable amount of drone-brood less likely to be seen. Extension along lower cell wall dark brown in color. Sur- faces Somewhat smooth. Separate from cell with diffi- culty. Usually only Bacillus larvae. Occasionally Bacillus vulgatus and Bacillus mesentericus. Frequently negative. Sacbrood. Brooaless irregular, perforated caps pres- ent, dark sunken caps not so pro- nounced asin Ameri- ean foulbrood. Small amount of brood affected. Extension along ioavor cell wall. Head turned upward. Normal form main- tained. Almost invariably die after capping. Scon become dark brown to almost black. None. Contents watery and granular. Laryve can be removed from cell ‘without rupturing body wall. Greatest ravages among worker-brood. Extension along lower cell wall. Dark in color, often black. Somewhat rough- ened appearance. Separate readily from cell wall. Negative as a rule. | Nearly always wholly Never Bacillus larvae | on common media. | negative. DIAGNOSIS OF BEE DISEASES. en NOSEMA DISEASE. Nosema disease is an infectious disease of adult bees. It is the only adult disease which at the present time can be diagnosed by labora- tory methods (White, 1918). Sixty years ago Doénhoff (1857) ob- served an infectious condition among adult bees in which, upon examining the stomach of affected bees, small oval bodies were found. This work had been practically forgotten until Zander (1909) reported some interesting findings in a disorder of adult bees. ‘He found that the stomach wall of bees taken from colonies suffering from what he called “malignant dysentery” contained a. protozoan parasite. To this parasite he gave the name Nosema apis. In England (Graham-Smith, Fantham, Porter, Bullamore, and Malden, 1912) Nosema infection in bées has been associated with a disorder referred to as the Isle of Wight bee disease. Recent in- vestigations in Scotland (Anderson and Rennie, 1916) have lead to a somewhat different view. As Nosema apis occurs in the group Microsporidia the name “microsporidiosis” has been given to the disease (Fantham and Porter, 1912). Nosema disease is widely distributed. It occurs in Germany, Australia, Switzerland, and England at least. The junior author (White, 1914) has found the parasite Vosema apis in samples of bees from a large number of the States of the United States and from Canada. The disease weakens and even kills colonies and is there- fore one of interest to beekeepers. The exact losses from it are not known, but in America they are less than has been attributed to it in some other countries. OBTAINING THE BEES. Hither dead or living bees are suitable for examination. Dead bees may be dry and still be suitable material. Living bees for examination can be sent very satisfactorily in mailing cages such as are used by queen breeders; dead ones may be sent in any con- venient way. A complete history of the colony and apiary as to disease should accompany the bees. GROSS CHARACTERS. The presence of various symptoms has been mentioned as being of importance in the diagnosis of Nosema infection. Among these are noted the spotting of the hive with feces, abdominal distention, the presence of shiny bees devoid of hair, and the activity of the bees, either in the cages or when free. These are of questionable value. It is upon the presence or absence of Nosema spores that the diagnosis is based. Bees otherwise apparently healthy may, upon examination of the stomach, show the presence of spores of Nosema apis in large numbers. 14 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE. MICROSCOPIC FINDINGS. The bees if alive may be killed easily by crushing the thorax be- tween the jaws of a pair of dissecting forceps. Then the thorax is grasped by the thumb and finger of one hand, the tip of the abdomen is grasped with a pair of forceps held in the other, and by gentle traction the ventriculus (stomach) and hind gut usually come away entirely and may be teased apart for examination, or the whole gut may be crushed under a cover glass and examined. In making a diagnosis at least 10 bees should be examined. Spores of Nosema apis if present are easily recognized, being oval, highly refractile bodies (PI. II, fig. 2). Usually they occur in large numbers crowd- ing the field. They stain with difficulty, and for diagnostic pur- poses water mounts unstained are satisfactory. The young forms of the parasite when present are quite difficult of detection, and should not be depended upon in the diagnosis. Occasionally protozoa other than Nosema apis have been en- countered in the examination of adult bees. These have no relation to Nosema disease, however, and may be disregarded in its diagnosis. LITERATURE. Much has already been written on bee diseases. The journals on beekeeping contain numerous articles pertaining to them. Bulletin No. 98 of the Bureau of Entomology briefly reviews a number of papers, published prior to 1912, dealing with the causes of these diseases. The papers reviewed and the publications cited, together with the papers in the following list and the references which they contain, comprise a fairly comprehensive résumé of all the literature detailing work done on these diseases. With regard. to further papers to appear soon, it is announced that studies have been made on American foulbrood and European foulbrood, similar to those on sacbrood (White, 1917) and Nosema disease (White, 1918) and that the results are now being prepared for publication. ANDERSON, JOHN, and RENNIE, JOHN. 1916. Observations and experiments bearing on ‘“ Tai of Wight” disease in hive bees. Jn Proc. Roy. Phys. Soc. Edinb., Session 1915-1916, v. 20, pt. 1, p. 23-61, 1 pl. ARISTOTLE. (B. C. 384-822.) 1783. Histoire des Animaux d’Aristote avec la Traduction Francoise (Notes sur Histoire des Animaux d’Aristote) par M. Camus [Greek and French on opposite pages]. Paris. 2yv. 4°. Volume 1, Book IX, page 615: Bee diseases. 1910. The works of Aristotle Translated into English under the Editorship of J. A. Smith [and] W. D. Ross. Oxford. v. 4, Historia animalium by D’Arcy Wentworth Thompson. Book IX, p. 626b: Bee diseases. DIAGNOSIS OF BEE DISEASES. 15 CHESHIRE, F. R., and CHEYNE, W. W. 1885. The pathogenic history and history under cultivation of a new bacillus (B. alvei), the cause of a disease of the hive bee hitherto known as foul brood. Jn Jour. Roy. Micros. Soe. [London], ser. 2, v. 5, pt. 2, p. 581-601, pl 10, 11. DzIERZON, JOHANNES. 1882. Dzierzon’s Rational Bee Keeping; or the Theory and Practice of Dr. Dzierzon. Translated from the latest German edition by H. Dieck and S. Stutterd. Edited and revised by Chas. Nash Abbott. London. DONHOFF and LEUCKART. 1857. Ueber die Fadenpilz im Darm der Biene. Jn Bienen-zeitung [Hich- stiidt], v. 18, no. 6, p. 66-67. FANTHAM, H. B., and PorTEer, ANNIE. 1912. The morphology and life history of Nosema apis and the significance of its various stages in the so-called “Isle of Wight” disease in bees (Microsporidiosis). Jn Ann. Trop. Med. and Parasit., v. 6, no. 2, p. 163-195, col. pl. 14-16. Page 189: References. GRAHAM-SmiTH, G. S., FANTHAM, H. B., PorTER, ANNIE, BULLAMORE, G. W., and MaALpEen, W. 1912. Report on the Isle of Wight bee disease (Microsporidiosis). In Sup. 8 to the Jour. Bd. Agr. [London], v. 19, no. 2. 148 p., 5 pl. Pages 139-143: Bibliography. McCray, A. H. 1916. Some difficulties in gross diagnosis of the infectious brood diseases of bees. Jn Jour. Econ. Ent., v. 9, no. 1, p. 192-196. 1917. The spore-forming bacteria of the apiary. Jn U. S. Dept. Agr. Jour. Agr. Research, v. 8, no. 11, p. 399-420, 6 fig., pl. 938-94. Mo.ritTor-MUHLFELD. 1868. Die Faulbrut, ibre Entstehung, Fortpflanzung und Heilung. In Bienen-zeitung [EHichstédt], Jahrg. 24, Nro. 8, p. 938-97. ScHIRACH, A. G. 1771. Histoire Naturelle de la Reine des Abeilles, avec l’Art de Former des Essaims. Le Haye. WHITE, G. F. 1907. The cause of American foul brood. U.S. Dept. Agr. Bur. Ent. Cire. 94. 4p. 1912. The cause of European foul brood. U. S. Dept. Agr. Bur. Ent. Cire. 157. 15 p., 10 fig. 1913. Sacbrood, a disease of bees. U.S. Dept. Agr. Bur. Ent. Cire. 169. 5 p. 1914. Destruction of germs of infectious bee diseases by heating. U. S. Dept. Agr. Bul. 92. (Contribution from the Bureau of Entomology.) 8 p. 1917. Sacbrood. U. S. Dept. Agr. Bul. 431. (Contribution from the Bu- reau of Entomology. Professional paper.) 54 p., 33 fig., 4 pl. Literature cited, p. 538-54. 1918. Nosema disease. U.S. Dept. Agr. Bul. —. (Contribution from the Bureau of Entomology. Professional paper.) (In press.) Literature cited. ZANDER, ENOCH. 1909. Tierische Parasiten als Krankheitserreger bei der Biene. Jn Miinch- ener Bienen-zeitung, 1909, Heft 9. 11 p., 3 fig. ADDITIONAL COPIES OF THIS PUBLICATION MAY BE PROCUEED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 5 CENTS PER COPY NATIONAL AGRICULTURAL LIBRARY WM 1022829783