EGG TRAN SMISSIGN OP AVIAN TUBERCULOSIS WITH OBSERVATIONS ON THE HEMAGGLUTINATION TEST By Dale James Richey AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHy Department of Bacteriology and Public Health Year Approved______l/J 1953 ProQuest Number: 10008414 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008414 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 -1346 ACKNOWLEDGMENTS The writer wishes to express his appreciation to Dr. W. N. Mack for his advice and encouragement offered during the course of this study. He is also greatly indebted to Dr. H. J. Stafseth for his kind guidance and valuable help in checking the disser­ tation. Grateful acknowledgment is also due Mr. Cleveland Allen, Department of Poultry Husbandry for his help in the insemination of the chickens. The writer deeply appreciates the financial support of the Graduate Assistantship from the Department of Bac­ teriology and Public Health and the scholarship provided by Michigan State College. Dale James Richey Two methods were selected to determine whether a tuber­ culous bird could be hatched from an artificially infected hen. They were 1) inoculation of embryonated eggs to determine the ability of a tuberculous embryo to hatch and develop active tuberculous; 2) inoculation of the ovary of normal hens, collecting and hatching their eggs, and finally reisolating and Identifying the tubercle bacilli from the hatched chick. The hemagglutination activity of several sera was studied according to the MIddlebrook-Dubos reaction. Inoculation of 21^2 normal embryonated eggs with tubercle bacilli resulted In the death of 115 or l\.7.5 percent. Only 85 of the 127 embryos which hatched were alive after eight months* Sixty or 70.5 percent of the birds gave a posi­ tive tuberculin reaction at eight months of age. Twenty-seven of these sixty tuberculin positive birds showed lesions charac­ teristic of the disease whereas 25 (2 9 .ll percent) of the 85 birds showed no reaction to the tuberculin test and were devoid of lesions. Inoculation of l6 normal hens with tubercle bacilli, directly into the ovary resulted in the development of progress fatal tuberculosis in six birds. Ten of the 16 hens were arti­ ficially inseminated once each week and eggs from each hen were collected. A total of Zl\X eggs were laid by these ten hens. Sixty-three were non-fertile, 18 fertile eggs failed to hatch Dale James Richey 2 because of death of the embryo, and l6o eggs hatched. All chickens hatched from these 160 eggs were examined micro­ scopically, culturally, and by embryo Inoculation of normal embryos for tubercle bacilli. Of the 81 eggs laid by these ten hens which failed to hatch, nine contained acid-fast bacilli. Three of these nine eggs contained virulent avian tubercle bacilli. One hundred and sixty eggs from the injected hens hatched. Twenty-three of the 160 chicks died within 11 days after hatching. One chick, which died on the fifth day after hatching contained acid-fast bacilli in its yolk sac. Three of 139 birds were tu­ berculin positive six months after hatching. These three birds were hatched from eggs of hens which also laid non-fertile eggs containing tubercle bacilli. A mixture of rooster semen and tubercle bacilli was intro­ duced into the oviduct of two birds in an attempt to hatch tuberculous birds. A hemagglutination reaction is described in which sera from tuberculous chickens and tuberculin sensitized sheep red blood cells are used. The sera of eight tuberculin negative chickens, previously inoculated in the embryonic stage, showed hemagglutinin in dilutions ranging from 1 sip through 1 :1 6 . There was no significant increase in hemagglutinins In the serum of six apparently healthy chickens "stimulated 11 with tuberculin. TABLE OP CONTENTS PAGE INTRODUCTION............................. REVIEW OP LITERATURE ..................................... k MATERIALS AND M E T H O D S .................................... lL Cultures . . ........................... Culture media . .........................‘........... Chicken embryos as cultures .......................... ^ Bacterial cell c o u n t s .......... . ................. l6 Inoculation of embryonated chicken e g g s ........... 18 Inoculation of adult hens .............. 19 Artificial insemination of h e n s .................... 22 Examination of eggs laid by inoculated h e n s .......23 Preparation of chicken sera .............. 25 Buffered saline solution .............................. Preparation of Alsever*s solution ................ Preparation of washed, packed sheep redblood cells . . 25 26 26 Adsorption of natural hemagglutinins from sera . . . . 26 Preparation of sensitized sheep red blood cells ... 27 . . . . . 28 Hemagglutination test procedure .......... R E S U L T S .................................................. 3k Inoculation of embryonated chicken eggs * .......... 34Inoculation of normal adult h e n s ................... 4^ Examination of 81 eggs, from injected hens, failing to h a t c h ................................................ 44 PAGE Examination of l6o eggs, from Injected hens, which h a t c h e d ............................... * ............ 49 Results of the hemagglutination tests with sera from chickensinjected with Mycobacterium avium . . . . 53 Tuberculin and hemagglutination reaction of 4^ chickens from eggs experimentally injected with tubercle b a c i l l i .............................................. 53 Hemagglutination results with sera from six chickens before and after the administration of avian tuberculin ..................................55 D I S C U S S I O N ................................................ 58 S U M M A R Y ......................................... 86 BIBLIOGRAPHY.............................................. 68 LIST OP TABLES TABLE I. II* PAGE Protocol of hemagglutination r e a c t i o n .............31 Mortality results of the inoculation of 2l}.2 embryonated e g g s ................................. 35 III* Development of tuberculosis in 127 chicks hatched from embryonated eggs injected with 50 tubercle bacilli IV. * • • • • • • • Tuberculin reaction of 85 chickens hatched from infected embryonated e g g s .................. V. 39 Results of the examination of six chickens developing rapidly fatal tuberculosis VI. 37 ........ lA Egg production and hatchability of eggs from 10 hens inoculated into the ovary with 100 avian tubercle b a c i l l i .....................* . * * • • VII* k-5 Summary of the examination of 81 eggs, from injected hens, failing to h a t c h ........................... Ij-6 VIII* Results of the examination of l6o eggs, from injected hens, which hatched IX. 50 * .................... Results of hemagglutination tests with sera from five hens injected with Mycobacterium a v i u m ........... ^ X* Tuberculin and hemagglutination reactions of 1+1 chickens hatched from eggs experimentally injected with tubercle bacilli ......................... £6 LIST OP FIGURES Figure 1* Page Kodachrome print of a positive and negative hemagglutination r e a c t i o n ............ . * * • 2* Diagrams illustrating the principles of the hemagglutination r e a c t i o n .................. * 3# . 33 A six-day-old chick hatched from an inoculated embryo* A six-day-old chick hatched from a cul­ ture medium inoculated embryo ........ lj_* 30 * ••* 36 Terminally ill bird inoculated experimentally with avian tubercle bacilli * ............ . . . i|-2 INTRODUCTION Tuberculosis in poultry, as observed by the poultry pathologist, comprises only a small percent of the specimens brought to the diagnostic laboratory. However, according to Smith (1914.7 ) tuberculin tests indicated that fifty percent of the flocks in 12 North Central States were affected; one state showed 65 percent infection with one or more birds diseased out of 51+2 flocks. The extent of infection may vary in different flocks; Smith (19^-7) reported seven percent infected in 88,000 older birds whereas he found only 0.1+ percent infection in 25,000 pullets. Ballantyne and Bigland (1951) reported that in Canada 11.8 percent of 170 chickens were infected with tuberculosis. Hinshaw, Niemann and Busic (1932) showed that there was a higher incidence of tuberculosis in the reporductive tract of turkeys than in the reproductive tract of chickens. Ducks may also exhibit tuberculous lesions caused by the avian tubercle bacillus. Rewell (191+8) reported a tuberculoma of the brain in a Mandarin duck in captivity from which the tubercle bacilli had spread to other organs of the body in­ cluding the ovary. Wild birds, pigeons, gulls and sparrows have been shown to be naturally infected with avian tubercle bacilli whereas some migratory birds revealed no evidence of such an infection (Plum, 191+2 ) . 2 Tuberculosis of swine is most often contracted either directly or indirectly from tuberculous chickens (Dickey* I 9I4-?) • The incidence of swine infection varies from less than one percent to 12 percent in the United States (Smith, 19^3) with about 21 percent infection of swine in Canada (McIntosh, 19^2)♦ mark. The highest incidence of swine tuberculosis is in Den­ Forty to 97 percent of the hogs are infected in that country and the great majority by Mycobacterium avium (Bankier, 19^6), and (H0eden, 19^4-1) * Although Mycobacterium avium can stimulate antibody formation in cattle with consequent sensitization to avian and 1 possibly mammalian tuberculin, widespread infection is not often encountered. Sporadic cases of tuberculosis in cattle caused by avian tubercle bacilli have been reported by Timothy (1939), Stuart and Marshall (1952), and Ottosen (19 ^ ) • Several recent cases of tuberculosis in humans, proved by laboratory and pathogenicity experiments to be caused by avian tubercle bacilli are described by Finlayson (I9lf8), Dragsted (19lj-9)* anc* Brandbury (19^6). Rich (1950) sums up the question of avian tubercle bacilli in humans with the following paragraph: ,fIn view of the various considerations presented in this discussion, one can only conclude that if progressive tuberculosis is ever produced in the human being by the avian tubercle bacillus it must be only very rarely, and it is highly desirable that more well-documented studies be brought to bear upon the question,f. 3 ■^t is generally assumed that the avian tubercle bacilli are transmitted directly from bird to bird by ingestion of infected droppings. The incidence of intestinal lesions and the presence of tubercle bacilli in the intestinal contents of infected fowl indicate that many new cases occur by such a method (Lichtenstein, 1933)* (Schalk et al. 1935)* and- (Feldman, 1938). Just what role ovarian transmission plays in the dis­ semination of avian tuberculosis is not completely known. A review of the literature indicates that there is need for a study of this method of dissemination. Such an opportunity existed principally because of housing facilities in a new building. In addition, test animals were available for the study of antibody reaction in avian tuberculosis. REVIEW OF LITERATURE Ovarian transmission of tuberculosis in the fowl has not been sufficiently established to say that this method is of major importance in the spread of avian tuberculosis. The possibility that the avian tubercle bacillus might be trans­ mitted through the egg from tuberculous hens has been suggested for over sixty years. Several factors are inherent in the study of natural or artificial transmission of avian tubercle bacilli through eggs. They include: 1) adequate isolation of infected birds, 2) Isolation and identification of tubercle bacilli and 3) establishing the pathogenicity of the bacilli for animals and other fowl. A review of the literature was made for information concerning previous study of this problem but little informa­ tion was found of an exacting nature that could not be challenged in relation to the factors listed above. Definite conclusions were, therefore, difficult to formulate. In part, this con­ fusion of results was due to various techniques utilized, dif­ ficulty in obtaining homogenous suspensions of the organism, strict isolation of test birds, and especially lack of Informa­ tion concerning the virulence of the isolated organisms. 5 Avian tuberculosis was first described by Crisp (1868) who reported the occurrence of tubercles on and in the spleen, liver and peritoneal surface of diseased fowl. He was success­ ful, after two years experimentation, in transmitting the disease from infected to healthy birds. This work was done several years before Koch discovered and cultured the mammalian tubercle bacillus in 1882. Ribbert (1883), Simulatneously and independently Sutton (1881).), in two widely separated laboratories, established the bacterial nature of avian tuber­ culosis. One of the earliest publications concerning injection of embryonating chicken eggs with avian tubercle bacilli is that of Maffucci (I8 8 9 ), who inoculated 18 eggs. hatched. Eight chicks were Tubercle bacilli were demonstrated microscopically on necropsy in six of the eight hatched chicks. Baumgarten (I8 9 I) reported that only two eggs out of 12 inoculated with a culture of avian tubercle bacilli hatched. One of the chicks died after four and one-half months and was found to have generalized tuberculosis. The other chick died a short time later but was not infected. Maffucci (I8 9 2 ), and Milchner (190 !).) came to the same gen­ eral conclusion that chicks hatched from eggs injected with avian tubercle bacilli usually became tuberculous and died from the disease. Proof of the pathogenicity of the tubercle bacilli, the presence of which was determined by microscopic examination, was not discussed. 6 Inoculation of embryonating chicken eggs with avian, human and bovine tubercle bacilli was studied by Koch and Rabinowitsch (I9 0 7 ). Of llj., 11, and seven eggs respectively, which were inoculated with the above types of bacilli only eight chicks hatched* One chick died at two months of age with generalized tuberculosis; three chicks which were in­ jected with human bacilli developed no disease during four and one-half months; while of the four chicks injected with bovine bacilli, one died in one month with tuberculosis of the ceca and another in four and one-half months with tuber­ culous lesions in the liver. The two remaining chicks were sacrificed and no lesions noted. These authors did not discuss the infectivity of the different types of tubercle bacilli for the fowl. It Is interesting to note that two of the four embryos inoculated with bovine bacilli developed tuberculosis one and four and one-half months, respectively, after hatching. Information today concerning the infectivity of bovine bacilli for the fowl leads one to believe that the bovine tubercle bacillus, in small numbers, is non-pathogenic for the chicken. An experiment similar to that of Koch and Rabinowitsch (1907) 9 was carried out by Rautmann (1931). Three groups of 25 eggs each were inoculated with avian, bovine and human tubercle bacilli. Hatchability was forty, twenty, and fifty percent, respectively. Most of the chicks hatched from avian bacilli inoculated eggs died of extensive tuberculosis. Chicks hatched from the bovine and human bacilli inoculated eggs did 7 not die of tuberculosis and generally no tissue changes were noted. It was concluded that fowls are resistant to the human and bovine types of bacilli. Silva (1932) noted that inoculation of ten day-old embryonating chicken eggs resulted in a lower mortality as compared to that among those Inoculated at an earlier date; that is, the sixth to eighth day of incubation. This procedure enabled the bacilli to invade tissues of the embryo resulting in a tuberculous bird. There Is no doubt that inocu­ lation of embryonating chicken eggs with avian tubercle bacilli results in a lower hatchability and higher mortality of the embryo when compared to the mortality rate of controls (Thorp and Graham, 1930) and (Fitch and Lubb eh.ausen, 1 9 2 8 ). One of the earliest reports of apparent ovarian trans­ mission of tubercle bacilli In chickens is that of Sibley (1893)9 who observed tuberculosis In chickens hatched from eggs laid by naturally infected hens. He attempted to explain the origin of the disease by stating that ’’the disease appears to be a clear case of heredity’1. Gartner (1893) was one of the first to scientifically carry out experiments to determine if tubercle bacilli could be transferred through the egg. In these experiments he inocu­ lated one male and 12 female canaries intraperitoneally with virulent avian tubercle bacilli. He observed, microscopically, the presence of acid-fast bacilli in the peritoneal cavity of two guinea pigs inoculated with portions of each egg. In a 8 second experiment, two of 2)± eggs contained acid-fast bacilli. Artault (l895>), and Mohler and Washburn (1906)'also observed by microscopic examination, acid-fast bacilli in rabbits and guinea pigs following inoculation of portions of one egg laid by a known tuberculous hen. However, these and the two pre- ceeding experiments failed to mention ovary or oviduct infec­ tion. Higgenfs (1912) report is similar to those of Gartner (1893), and Artault (1895) hut he mentioned seeing tubercles microscopically in two of 11 guinea pigs inoculated with mater­ ial from three apparently infected eggs. The apparent transmission of avian tubercle bacilli through the egg was summarized by Van Es and Schalk (I9ll|-)l seems thus that transmission by means of the eggs must be given con­ sideration, although the data are not sufficiently numerous to enable us to correctly estimate the extent of the danger". Little work was done in this field during the next decade until the publication of Pitch et al,. (I92l|_). This group published results of a systematic experiment to determine whether avian tuberculosis is transmitted through the eggs of tuberculous fowls. They examined 876 eggs, 367 by cul­ tural methods and 509 by inoculation of poultry. They found that the composite of nine eggs obtained from two of i_|_3 tuber­ culous hens contained tubercle bacilli. It is interesting to note in this report one of the hens on being necropsied gave no evidence of tuberculosis but was tuberculin positive. Pitch and Lubbehausen (1928) completed experiments on the 9 same group of chickens and. hatched 697 birds from two thou­ sand eggs laid by 88 tuberculin positive hens. two-thirds of the chicks died. Approximately A bacteriological examination was performed on each bird and no evidence of tubercle bacilli was noted microscopically or culturally. The other one-third of the chicks matured normally and were later tuberculin tested. above. On necropsy, each bird was carefully examined as Tubercle bacilli were not found in any birds of this last group. The investigators concluded that transmission of tuberculosis through the egg, rarely, if ever occurs. Raebiger (193^) reported the largest number of eggs re­ vealing tubercle bacilli microscopically or by cultural methods. The hens that laid these eggs were experimentally infected. Approximately 36 percent of the eggs contained tubercle bacilli. The virulence of the acid-fast organisms isolated was not mentioned. Organisms resembling tubercle bacilli have been reported present in eggs laid by hens. The number of organisms reported by various investigators varied from zero to 13*9 percent (Harshfield, 1937; Eber, 1932; Klimmer, 1930; Liverani, 193^; Bigger, 1932; and Raebiger, 1 9 2 9 )# The bacilli isolated were not proved to be virulent avian tubercle bacilli. Bunyea (1 9 2 9 ) hatched 276 chicks from eggs obtained from a flock of 1,075 hens. These hens were either tuberculin positive or revealed lesions of the disease when necropsied. 10 The experiment also included three generations of newly hatched chicks from the tuberculous parent stock* Of 361 chicks reared, noh£ gave a positive tuberculin reaction and no tuberculous lesions were found. Beller and Henninger (1930) conducted an ingenious ex­ periment whereby they fed large numbers of virulent tubercle bacilli to 18 hens over a period of two years. Six hens were given avian, six bovine, and six human tubercle bacilli. Twelve contact controls were added to the group and all thirty birds placed in the same area. At necropsy, lesions of tuber­ culosis were found in only three birds and these had received avian tubercle bacilli. Only two of the 620 eggs laid by the thirty hens, contained acid-fast bacilli, and these organisms were identified as avian species by microscopic and cultural exaimination. The possibility of rearing tuberculous chickens from eggs of artificially infected hens was studied by Stafseth ejb a l . (193^1-) • They reported no tuberculous birds after the hatched chicks were seven months of age. Complete identification of tubercle bacilli is lacking in much of the literature reviewed. Infectivity studies of the isolated bacilli are essential for positive identification. Electron microscopic studies of pathogenic mycobacteria in­ dicate that human tubercle bacilli are generally longer and more slender than bovine bacilli, and usually, that avian bacilli are more pleomorphic than mammalian bacilli, (Soltys, 11 Hill and Ansell, 1952) • These investigators mentioned the variable microscopic characters of the three types of bacilli and stated that it is impossible to decide on morphological grounds alone whether the bacilli are of human, bovine, or avian origin* It is apparent that morphological characteristics of tubercle bacilli depend on the medium on which they are growing, the age of the culture, and on the variation of strains. Middlebrook and Dubos (19^8) described a hemagglutination reaction with the sera from experimental animals or from tuber­ culous patients. The reaction involved tuberculin treated sheep red blood cells in the presence of tuberculous antiserum. The authors did not elaborate on this reaction as a diagnostic procedure but presented results which showed that the sera from six patients with active pulmonary tuberculosis had titers of 1:8 or higher; whereas sera from eleven non-tuberculous patients showed no titer higher than 1 slf.• Rothbard, Doonief and Hite (1950) tested sera from 216 normal adults and patients with syphilis or other non-tuberculous chest diseases in an attempt to demonstrate the presence of tuberculosis antibodies by the hemagglutination reaction. Their results showed that 203 of 2l6 sera gave no hemagglutination reaction, 12 showed reactions in serum dilutions of 1:2, and one showed a reaction in a I:!}, dilution. Ninety-two percent of the sera from l68 patients with active tuberculosis gave hemagglutination reactions. These reactions occurred in 12 dilutions ranging from 1:8 to 1:512. Thirty-one sera from 33 supposedly cured tuberculous individuals revealed no reaction. The other two sera showed slight reactions in a 1:2 serum dilution. They concluded that the hemagglutination reaction is apparently a relatively specific test for active tuber­ culosis. Smith and Scott (1950) reported that the hemagglutinatinn test was usually negative in the far advanced or arrested types of tuberculosis. They theorized that the antibodies of the first type were neutralized by adsorption of products produced by the growing tubercle bacilli while agglutinins were absent in the second type because there was not sufficient antigenic stimulation from healed tuberculous lesions. also found that of They students with positive tuberculin tests, 12 (22 percent) had hemagglutinins before "stimulation" with tuberculin. Of the remaining ij.6, 15 (32 percent), had no de­ tectable hemagglutinins before stimulation with tuberculin, whereas, after stimulation the hemagglutinins were detectable. They also reported eighty percent positive hemagglutination reactions with 10l|_ sera from active tuberculous patients. Gernez-Rieux and Tacquet (1950), Sohier et a]L. (1950), Scott and Smith (1950), and Chang _et al_. (1952) presented similar results with sera from actively tuberculous patients. Kirby, Bunnell, and 0 1Leary (1951) found that the hemagglu­ tination reaction was positive in 123 of 251 sera from patients 13 exhibiting acute non-tuberculous infections. In a second group of 5^ patients, all with active tuberculosis, 31 ' 0 ml. of five percent aqueous phenol was prepared. These re­ agents were mixed and filtered through No. 1 Whatman filter paper until free from small particles. Dilutions of the standard 17 suspension prepared above were made in one percent formalized saline solution by adding one ml. of the bacterial suspension to nine ml. of formalized saline solution. bacterial suspension. This gave a 1:10 One ml. of this dilution was added to ten ml* of saline solution. To this tube was added one ml. of the filtered gentian violet solution, giving a final 1:120 dilution of the standard ninety percent light transmittancy suspension. This final suspension containing the mixture of organisms and stain, was heated until the liquid boiled. The suspension was allowed to stand for three minutes before pro­ ceeding with the bacterial count. The Petroff-Hausser counting apparatus was cleaned and a cover glass placed over the chamber. A capillary pipette was filled with the well-mixed and stained suspension, and the chamber filled by capillary attraction. The small 1/20 mm* squares in the chamber were observed with the high, dry objective. By raising and lowering the objective slightly the bacterial cells were counted in each small square until 20 squares had been counted. The results were calculated by dividing the total number of bacterial cells counted by 20 to obtain the average number for each square. The average number of cells per square was multiplied by 20,000 (which is the cubic dimension of each square in millimeters), by 120 (the dilution of tubercle suspension), by 1,000 (the number of cubic millimeters in a cubic centimeter). This result was 18 round to b© the bacterial count per ml* of the ninety percent light transmittancy tubercle culture* Inoculation of embrvonated chicken eggs: Fertile white leghorn eggs, from disease free chickens were placed in the hatching incubator. The eggs were candled every day to observe development of the embryo. All eggs with dead or abnormal embryos were discarded. When the embryonated chicken eggs were seven days old they were candled with the long axis in the horizontal plane and the yolk sac located. A mark was made on the shell over the yolk sac about half-way from the small end of the egg to the apex of the curvature of the shell. A small opening was drilled through the shell leaving the membrane intact. Tinc­ ture of metaphen (1 to 200) was applied to the opening and with the long axis of the egg in the horizontal plane, the inoculum was introduced slowly into the yolk sac. Injection was made with a 27 gauge, 1/2 inch needle on a ml. syringe, the needle being inserted the full length. l/l± Approximately 50 tubercle bacilli were introduced into the yolk sac of each embryo. The opening was sealed with melted paraffin and the inoculated eggs returned to the incubator. All inoculated eggs were candled each day -until hatching occurred. Dead em­ bryos were subjected to bacterial, cultural, and microscopic examination. Chicks hatched were isolated in a heated brooder which had been thoroughly disinfected. Birds dying before eight months of age were examined for tuberculous lesions and other gross pathological symptoms. 19 Unabsorbed yolk material, yolk stalk tissue and its contents were subjected to microscopic and cultural examina­ tion for tubercle bacilli. Digestion of a portion of the above material with four percent sodium hydroxide for thirty minutes at 37° C. was routine practice. The sediment was neutralized with dilute hydrochloric acid. A portion of the sediment was stained by the ZIehl-Neelsen method and the rest distributed over the surface of four tubes of Petragnani*s medium. The tubes were incubated for ten weeks at 37° C. until acid-fast bacilli were observed. Tubes exhibiting growth of organisms other than acid-fast bacilli were discarded. Yolk stalk, portions of the digestive tract, liver, and spleen were examined in the older birds. All birds alive after eight months of age were bled for collection of serum and then necropsied. Any suspicious lesions were subjected to cultural examina ti on • Inoculation of adult hens; Eighteen normal New Hampshire red hens, nine months of age, were purchased locally, wingbanded and isolated in individual cages. The cages were placed in a room that had never been used for animal quarters. Each bird was given an intradermal injection of approximately 0 .2 5 avian tuberculin. Injection was made with a small syringe and 27 gauge needle into the skin of one wattle. was produced at the site of injection. A small bleb The injected wattle of each bird was examined at 24 and 4 ® hours using the other wattle for comparison. None of the 18 birds was sensitive to 20 the tuberculin. A sensitivity reaction produces a soft, edematous swelling, marked by discoloration, ranging from dirty pale yellow to dirty greenish yellow. All 18 birds were quarantined under excellent housing conditions and did not experience any disease other than the one to which they were exposed. The ovary of 16 of the 18 hens!was inoculated with approximately 100 tubercle bacilli. The hens were deprived of food and water 12 to 18 hours prior to the ovarian inocul- . ation. This permitted the intestinal contents to be re­ duced to a minimum resulting in a clear field of operation in the abdominal cavity and less danger of puncturing the intestine when the intercostal incision was made. The hen was placed on the operating table on its right side. The wings were securely held together above the body with the legs fully extended. Assistance in restraining the bird during the operation eliminates the need of an anes­ thetic * Some of the feathers were plucked from the region of the hip and ribs. of metaphen. The area was washed with 1:200 tincture The narrow region between the sixth and seventh ribs (last two) was located with the fingers of the left hand. The skin was drawn back over the hip and with it the thigh muscle. An incision was made about an inch long through the skin and intercostal structures which reach­ ed almost to the dorsal rib attachments or vertebral column. 21 Severing of the cutaneous vein which runs diagonally across the body from the thigh to the wing was avoided* A spreader was placed in the incision and the peritoneal membrane which forms the abdominal air sac punctured* The abdominal organs were visible after the membrane was punc­ tured* The single ovary that develops to maturity is situ­ ated in the sub lumbar region of the abdominal cavity. It lies at the anterior end of the left kidney and slightly to the left of the midline of the body. The heavy stalk, or base, of the ovary is attached to the dorsal wall of the abdominal cavity by a fold of peritoneum, called the mesovarium. This fold keeps the ovary in position, suspended in the cavity. The inactive ovary is composed of many ova, gray-white in color, which becomes more intensely colored, soft to bright yellow, as they increase in size and age. This change in coloration indicates that yolk material is forming. One-tenth ml. of a suspension of the organisms (100) was injected into the yolk stalk of each hen. After inoculation, the spreaders were removed from the incision and the bird released from the table. Suture of the incision was not found necessary unless the tissues were badly torn. The skin and thigh muscle slipped back in place over the in­ cision as the limbs regained their normal positions. Hard crusts or scabs were formed of exudate from the incised 22 tissue and healing took place rapidly. After inoculation the birds were transferred to isolated cages. Some of the birds developed wind puffs which were released by puncturing the skin with a 22 gauge, two inch needle. Two of the 18 hens, not included in the group, were artificially inseminated with 0 .2 ml. of semen from the roosters mixed with one hundred tubercle bacilli. This pro­ cedure was performed on the two hens each time the entire group was inseminated. Artificial insemination of the hens: Four normal, well-developed male chickens were kept segregated in quar­ antine and used solely for the collection of semen. The semen was artificially introduced into the oviduct of the 18 hens. This method of insemination was desirable because the cages were too small for a rooster and a hen. Also there was less chance of cross infection. The technique of collection of the semen and insemina­ tion of the hens was as follows: the male chicken was held under the right arm while resting on the right knee. left hand held the legs securely. The Stimulation of the bird was accomplished by placing the right hand on the bird’s back and sliding the thumb and fingers backward toward the base of the tail. Stroking the male in this manner caused the protrusion of the copulatory organ. Extending the thumb and forefinger around the base of the tail brought about ejection 23 of the semen* The average volume of semen collected from each male was 0 * 5 nil* to 1 *0 ml* Insemination of the hen was performed by extruding the oviduct through the vent and injecting 0 .2 ml* semen directly into the vagina with a one ml* syringe without needle. In order to extrude the oviduct, the hen was held under the left shoulder with the arm extending tinder the keel* Its head was placed in a downward position to the rear of the operator. The side of the hen was placed against the operator's body, the left thumb and fingers were used to exert pressure around the vent. The sudden and simultaneous pressure exerted by both hands caused the ovi­ duct to protrude* Each hen was inseminated once a week as long as she was in production. Examination of eggs laid by inoculated hens; The hens band number and date of laying were recorded on each egg collected* The eggs were then placed in the incubator and candled each day until non-fertility was established. For the bacteriological examination, the shells of the non-fertile eggs were washed with 70 percent alcohol by immersion for 15 minutes* The egg shell was broken and the contents released into a sterile Petri dish. A five ml. portion of each yolk was digested with an equal volume of four percent sodium hydroxide. After digestion of the specimen, the tube and its contents were centrifuged at 2500 revolutions for thirty minutes. The supernatant fluid (not the fatty material at 2k the surface) was discarded, and the sediment and fat mater­ ial neutralized with 2 . 5 percent hydrochloric acid with brom thymol blue as the indicator. A portion of the sedi­ ment was streaked upon three tubes of Petragnani’s medium and upon a clean glass slide. The inoculated culture tubes were incubated for eleven weeks at 37° C. A microscopic examination was made of the slide after staining by the Ziehl-Neelsen acid-fast method. Two normal embryonated eggs were inoculated with 0 .2 ml. of the sediment as des­ cribed under Chicken embryos as cultures. In order to de­ termine the efficiency of these methods of isolating acidfast bacilli from yolk material, control eggs were inoculated with approximately fifty tubercle bacilli. The yolks of two inoculated control eggs were processed, as described above, for every ten test eggs. All fertile eggs laid by the inoculated hens were kept in the incubator until the embryos died or the chicks hatched. Each dead embryo was immediately removed, from the incubator and necropsied. The unabsorbed yolk and any tissue suspected of containing tubercle bacilli were examined microscopically and cultured as described above. Hatched chicks were isolated In a brooder and quarantined from all sources of avian tuber­ cle bacilli. As soon as any bird showed symptoms of the dis­ ease it was bled and the serum stored in the frozen state. A thorough bacteriological examination was made of suspicious lesions observed in each bird. Gross pathology was recorded. (Hemagglutination Studies) Preparation of chicken sera: Approximately five ml* of blood was drawn by cardiac puncture from each chicken and allowed to clot at room temperature. The serum was re­ moved after centrifugation and stored in the frozen state* Stored in this manner the titer remains constant for several months. Each serum was diluted with an equal volume of buffered saline solution and inactivated by heating to 6 5 ° three minutes. for This temperature was slightly higher than that often employed in inactivating sera. The chicken serum was not harmed by this inactivation procedure and time was saved by employing this method. The inactivated serum-saline solution was stored in the frozen state. Adsorption of natural hemagglutinins with normal sheep red blood cells was performed before proceeding with the test proper. Buffered saline solution: The buffered saline solution was made as follows: Nacl kh 2 po^ Na 2 HP 0^ 170 grams 2*7 11 11.3 " Distilled water to 1000 ml* Before use, It was diluted 1:20 with distilled water. 26 Preparation of Alseverts solution Dextrose 2 .0 5 grams Na citrate 0 *8 Na chloride 0 .1|-2 " Citric acid 0 .0 0 5 " Distilled water to 100 ml. The Alseverfs solution was sterilized by Seitz filtration. Preparation of washed, packed sheep red blood cells: Approximately 100 ml. of sheep*s blood was collected aseptically in a flask containing 100 - 200 ml* Alsever*s solution. The sheep red blood cells can be used satisfactorily for several months if kept in this solution in the refrigerator at i{-° C. When ready for use, the necessary amount was cen­ trifuged at 2000 revolutions per minute for 15 minutes. The supernatant fluid was removed and the sheep cells were washed three times with about five volumes of buffered sa­ line solution. After the fourth washing and centrifugation as much as possible of the supernatant fluid was removed, leaving the packed, washed sheep red blood cells. These sheep red blood cells were used within i|.8 hours. Adsorption of natural hemagglutinins from the sera: Natural hemagglutinins were removed from the chicken sera by adding normal, washed and packed sheep red blood cells in 0 . 2 ml. amounts to each tube containing the serum-saline solution. The tube was shaken and allowed to stand at room 27 temperature for twenty minutes. The mixture was centrifuged at 2500 revolutions per minute for six minutes and the super­ natant fluid transferred to another tube containing 0 .2 ml* amounts of previously washed, unsensitized sheep red blood cells. Two adsorptions do not always remove all the natural hemagglutinins from the serum and more adsorptions may be necessary until no agglutination occurs. The adsorbed serum * was used to test for anti-tuberculosis antibodies by the hemagglutination test. This serum can be stored indefinitely in the frozen state. Preparation of sensitized sheen red blood cells; ^ / Old Tuberculin (Lederle L a b o r a t o r i e s I p C strength, in 0 .8 ml. amounts, was added to 1 1 .2 ml. buffered saline solution. After shaking this mixture, 0^2 ml. of washed, packed sheep red blood cells was added to the mixture and incubated in a 3 7 ° C. water bath for two hours. Settling of the sheep red blood cells can be avoided by shaking the mixture every minutes during the incubation period. l£ Adequate sensitization was accomplished during the two hour period. The mixture was centrifuged at low speed (about 1000 revolutions per min­ ute) for six to eight minutes and supernatant fluid discarded. The sheep red blood cells were washed three times by resuspending in 12 ml. buffered saline solution and centrifuging as above. Low speed centrifuging prevented the formation of -^-Courtesy of Dr. H. D. Piersma, Lederle Laboratories, Pearl River, N. Y. 28 tightly packed sheep red blood cells which were difficult to resuspend without producing hemolysis* centrifuging the cells were suspended in After the last $0 ml* of buffered saline solution and used as an antigen in the hemagglutina­ tion test* This final concentration of sensitized red blood cell suspension was approximately 0 .1|. percent, and may be used up to three days if stored at four degrees centigrade. Hemagglutination test procedures The protocol for the test procedure is presented in Table I*' Wassermann tubes were placed in a rack* Nine standard Tubes one through seven contained serial serum dilution tubes, tube nine con­ tained unsensitized sheep red blood cells-serum control, and tube eight contained sensitized sheep red blood cells-saline control. Tubes one, two and eight received 0.5 nil* adsorbed test serum diluted 1:2. Serial dilutions were then made from tube two by transferring 0 . 5 ml* of that mixture to tube three, 0 . 5 ml. of the mixture in tube three to tube four and so through tube seven, the content of which was reduced to the standard volume by discarding 0.5 ml. All tubes received 0*5 ml. of the sensitized sheep red blood cells except tube eight which received 0 . 5 ml. of unsensitized sheep red blood cells. Pinal dilutions of the adsorbed serum were from l:Ij_ in tube one to 1 :2 5 6 in tube seven. *With permission of the authors and publisher, Isabelle Gil­ bert Schaub and M. Kathleen Foley, chap. 15. Diagnostic Bacteriology. Ij-th ed. St* Mo., The C. V. Mosby Co. p* 290. 29 All tubes were shaken and then Incubated for two hours in a 37° C. water bath* After partial settling of the sheep red blood cells the tubes were shaken vigorously and left undisturbed at room temperature overnight. Figure 1 shows the appearance of a positive and negative hemagglutination reaction. Figure 2 procedure* shows diagrams of the hemagglutination text They are similar to those of the usual research and laboratory diagnostic hemagglutination reactions. ^Modified from chart by Armstrong, A. R . and J. Orlicki, 1951. The technique of the sensitized sheep cell hemagglu­ tination test for tuberculosis. Can. Jour. Med. Tech. 2:67-75. 30 . ' • v , Figure 1 s psiiiH'iflllHiB Kodochrome print from transparency showing a positive and a negative hemagglutination reaction 31 o CO M rH tA• O 1 1 rH £ •L• A O 1 1 • <—1 £ lA • O I 1 rH £ -drH • • ia • rH £ \A • O * vO i —1 £ MIN. SERUM A T ROOM TEMP. AND CENTRIFUGED TREATED A G AIN REM OVED SERUM CELLS W IT H COMPLEMENT w NATURAL INACTIVATEL AN TIBOD IE S // M IX E D P R E P A R A T IO N OF 2 IN C U B A T E D + A N T IG E N O O - o 3x I'15 O ld C E N T R IF U G E D WASHED SHEEP CELLS D IL U T E D T u b e r c u lin C E L LS VASHED 3X RESUSPENDFD N BUFFE RE D I S OT ONI C SALINE S E N S IT IZ E D CELLS DIAGRAM 8 I - 16 l 64 I 128 PATIENT'S R EMOVED W ITH F R E S H DIAGRAM OF I 256 S a l in e Se r u m CONTROL C O N TR O L 3 AN D REVCVEE RESULTS Inoculation, of embryonated chicken eggs: A total of 2ij.2 seven-day-old embryonated chicken eggs were inoculated each with approximately fifty tubercle bacilli by the yolk sac method. The mortality of the infected embryos was high; 115 or lj.7.5 percent failed to hatch* In comparison, the mortality of the culture medium inoculated controls was two out of l 6 or 12.5 percent. II. These results are given in Table There is no doubt that inoculation of embryonated chicken eggs with avian tubercle bacilli affects their hatchability, and that the action of the virulent tubercle bacilli was instru­ mental in their destruction. Figure 3 illustrates a six-day- old chick hatched from an Inoculated embryo and a six-day-old chick hatched from a culture medium inoculated control. Because of the action of the tubercle bacilli many of the 127 hatched chicks died during the first few weeks of life. They exhibited general weakness, inappetence, pasty vent and were often found dead in the cage. A composit sum­ mary of the development of tuberculosis in these birds is given in Table III. It will be noticed that the mortality of the hatched chicks showing infection was highest during the first week. Approximately fifty percent of the deaths occurred within the first week. usually survived. Birds living to be six weeks of age 35 TABLE II MOHTALITI' RESULTS OF THE INdCXJLATIOH . ,r -• ' _ s - ' ‘v • • OP 2^2 EMBBYCHATED -EGGS ; • v ’* . Embryo age (days) Nhmber dead (2l|.2 Inoculated.) 1-5 33 6-10 2k 0 V V-■* 1 11-15 18 i 16-21 40 0 Mortality totals 115 Total no. hatching 1 2 7 ($2.5%) ‘j,;.vQe►ntrols dead (li> inocull.atad) . Cif.7.5#) 2 tl2.5%) ik 187.5^) I chick / II hatched from from embryo embryo inoculated inoculated bacilli a culture medium a tubercle 1 A six-day-old hatched 1 chick ’ BBSBLIS OP '®EB”^AMIHAa!XCaj OP S IX C H ld E p S DEV^CiOPIlIG' HAPXDLY FAKHii TUBERCULOSIS , . t '■* . -V■.' .V'.V’-Y-’ ■' 1 * • . '' ",' ' 1.V ■v< "- f. / ' '" V . v.\- • 1. , . . .■ *■■'• ,•/: ^7fjj' ; ..v Y->.. ■■■■ ‘ . % . ■ .*v . -V. ' . (IST© eggs collected from this group) v .* ■ ; ,-fc■ , • ^ .y. , -.-V'. ,V . ‘• --.V. .• . •\ ■ -;V- " :* * ' Tike @f . Results p t v-1> death i !' . . «...-------- • .■'tyt' ~/y '.•'1 Bird No. , 1917 If days •• . 1995. : 20 * 1906 21 * 1989 ■:* -Yy ':v: J ■■ vv ■: vvExtensive ovarian and some digestive IVsiohs. N© i^dules ©n liver © £ spleen,, Qvafiesnorv? functional at death* A’ e ld-fast organisms observed in stained smears* ’a weeks >:V; ■'v; ■ . 'v■ ", ■ : ' -1 •' ‘- ' yf'-:.■ : 1935 ; '‘ 8 % weeks 1933 12 :‘ " /Y " "r ... . As above plus small miliary foci in liver and apleen. Many nodules on peritonerfcand mesentery * ill bird inoculated experimentally -d- Terminally Figure with aviaa tubercle bacilli kz lj-3 The other ten hens of this group were inoculated with approximately 100 tubercle bacilli directly into the ovary and isolated in individual cages* Each bird was inseminated artificially with 0 . 2 ml* of pooled semen obtained from three healthy roosters. Insemination was performed each week while the birds were in production. A total of 2lfl eggs were collected during the eight months of observation. One egg was the smallest number collected from an individual hen. months. One bird produced eggs in eight Pour eggs were found broken giving a total of 237 eggs incubated for hatching. One hundred and sixty (6 7 per­ cent) of these eggs hatched (Table VI). Hens numbered 1909# 19^2* and 1970 did not lay eggs at constant intervals of time as did the other seven hens. The egg laid by hen 1909 was obtained four weeks after inoculation. Three of the six eggs obtained from hen 1970 were collected four weeks after Inoculation, two nine weeks, and one eleven weeks after Inoculation. Hen 1942 laid five of its six eggs 16 weeks after inocu­ lation and the last egg 21 weeks after inoculation. Although each hen was inoculated with approximately the same number of organisms at the same time, and in the same organ, there was much variation as to the progress of the disease in the indi­ vidual birds. It can be observed from Table VI that 160 chicks were hatched from 2lfl eggs laid by ten hens. Of the 81 eggs failing Mi- to hatch, 63 were found to be non-fertile. The remaining 18, which were fertile, failed to hatch either because of a dead embryo or death during hatching. A representative portion of the contents of each egg failing to hatch was examined microscopically and culturally for acid-fast bacilli re­ sembling Mycobacterium avium. The stage of development of the ovary at the time of inoculation of each hen seemed to have no correlation with its ability to lay eggs. Hens 19^2 and 1970 each laid six eggs; the ovary of the former hen was not mature while that of hen 1970 was mature. It can be noticed from Table VI also that the two hens laying the largest number of eggs did not possess ovaries in the same stage of development. Examination of 81 eggs, from injected hens, failing to hatch: Every egg which failed to hatch laid by these injected hens was examined microscopically, culturally, and by embryo inoculation for tubercle bacilli. It was approxi­ mately four weeks after injection of the ovary of the hens that acid-fast organisms were first observed in some of the eggs failing to hatch. Table VII presents the results of the examination of 81 eggs failing to hatch. Prom this table it can be seen that three hens, 19 2lj_, 1 9 7 7 , and 1 2 6 , laid eggs in which acid-fast bacilli were detected. Hen 192lj. laid one non^fertile egg and one fertile egg (not hatching) which contained acid-fast bacilli. 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Yegian, D., and J. Kurung, 19^7* Morphology of Mycobac­ terium tuberculosis. Am* Rev. Tub ere. 5>6:36-1^0. Avian tuberculosis.