TRANSMISSSON STUDIES ON AVIAN LY’MPHOMATOSIS Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE George Edward Coffral 1950 This is to certify that the thesis entitled Transmission Studies on Avian Lymphomatosis presented bg George E. Cottral has been accepted towards fulfillment of the requirements for M,S. degree in Animal Pathology raw-aura! m. ~93 .T—sfit' TRANSMISSION STUDIES ON AVIAN LYMPHOMAII‘OSIS TRANSMISSION STUDIES ON AVIAN LYMPHQ-IATOSIS 13? George Edward Qgttrel A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Pathology 1950 AC KNC‘WLED GLEN! The writer wishes to express his sincere appreciation to Dr. B. R. Burmster and Dr. Nelson F. Waters for their help and cooperation in carrying out these studies. Gratitude is also expressed to Mr. Barley Hinton, Dr. Frank Thorp, Jr., Dr. R. A. Runnells, Dr. A. M. Lucas, and Dr. E. F. Oakberg for their helpful and encouraging suggestions during the course of this study. Special thanks are due to Mr. B. M. Larkins for his technical assistance in the experimental work, and to Mrs. Hazel I. Garrison, Mrs. Florence F. Hickok, and Miss Patricia A. Soavarda for consultation on the preparation of the manuscript. "3.. v: £1.11", ~7‘Q TABLE OF CONTENTS Page INTR®UCTI _ 7.986 .. *t - significant at the 1.0 percent level. The results of the chi-square test for each experimenters given in table 8. The analysis was made at the 250 day point and at the termina- tion point (310 days). In experiment No. 1 there was no significant difference between the two control groups or between the control lot 3 and the inoculated lot 2. However, there was a significant difference between inoculated lot 1 and control lot 3. Both lots 1 and 2 received inoculum.C, the pooled intes- tines and trachea oi‘the donor chicks. However, lot 1 received the liver inoculum and lot 2 received the yolk sac incculume Thus, the higher incidence of lymphomatosis in lot 1 was attributed to the liver incculum. For this reason the liver of the donor'chicks and embryos was chosen.as the tissue to use as a source of inoculum for the experiments that followed. In experiment No. 2 there was no significant difference between the three control groups. All of the inoculated groups had a high incidence of lymphomatosis, varying from 76.5 to 100.0 percent. Thus, lymphomatosis - 41 - was transmitted by using liver tissue from.embryos 15 and 18 days old, chicks one day old, and by using a cell-free filtrate of the chick liver tissue. The controls in experiment No. 3 had a rather high incidence of lymphomatosis, 22.2 percent. This was the highest incidence for all of the control groups at the 250 day point. Only two inoculated groups, lots 12 and 13, had an incidence ol?lymphomatosis that was significantly high. The controls in experiment No. 4 had a low incidence of lympho- matosis and the incidence in the inoculated groups was also correspond- ingly lower than in the third experiment. Three inoculated groups (lots 23, 24, and 25) gave significant differences when compared to the controls. Experiment No. 5 was designed to detemmine at what ages the embryos and chicks would have the agent of lymphomatosis in their liver tissue. However, when the experiment was terminated, none of the inoculated lots differed significantly from.the controls. In experiment No. 6, some of'the previously used donor hens were retested. A filtrate prepared fronlthe livers of embryos of J709W pro- duced a significant increase in lymphomatosis in the inoculated birds, but the unfiltered preparation failed to give significant results. In the previous test of this hen's embryos the results were also positive (experimnt No. 4). The embryos of hen.No. J716R gave positive results this time for both the liver preparation and the amnionic fluid, but the filtrate group did not show a significant increase in the incidence of lymphomatosis. In the previous test (experiment No. 3) the results wdth the offspring of this bird were not significant. Embryo J404A liver tissue gave significant -42- results. This hen was not previously tested. J430H, which gave posi- tive results before, was negative in this test. J310R embryos were negative in both tests (experiments Nos. 3 and 6). In experiment No. 7 none of the inoculated lots had as high an incidence as the control lot. The embryos of these hens had been previously tested and they were negative in both tests. Unfortunately, the hens, whose embryos had given significant results in experiment No. 4, had not produced any fertile eggs by the time this experiment was scheduled, so they were not represented in experiment No. 7. The purpose of experiment No. 8 was to test the blood and respiratory washings of some of the donor hens that had been previously used in the other experiments. Hen No. J716R, which had given questionable results in experiment 3, and significant results in experiment 6, gave significant results this time. Hen J404N which also gave questionable results in experiment 3, was positive in this test. Hen No. J7010, whose embryos had shown significant results in experiment No. 3, gave only'questionable results this time. The respiratory washings of this bird and those of hen No. J7097.’ likewise produced questionable results. The results obtained with the other donor hens tested in this experiment were not significantly different from those of the control group. A significantly high incidence of lymphomatosis was transmitted to experimental birds by inoculating them with the liver tissue of newly-hatched chicks and embryos 15 and 18 days old. The logical conclu- sion was that the donor chicks and embryos were infected with the agent of lymphomatosis. Thus, if the donor embryos and chicks were infected, they must have acquired the infecting agent from their parents. Lympho- matosis was transmitted by using the blood of some of these donor hens. The respiratory washings of two of these hens gave only :qlestionable results. -43.. At the time they were used, the donor hens and sires were all clinically normal and most of them remained clinically normal or free of gross evidence of lymphomatosis. (Some of them are still living.) There did not appear to be any definite correlation between the infectivity of the embryo or chick tissue or the dam's blood and the subsequent development of gross lymphomatosis by the dam or sire. Thus, clinically normal dams and sires may be carriers of the agent of lymphomatosis. Some dcnor hens were checked several times, either by two tests with their embryos or by using the dam's blood for the re-test. The tests did not agree in all cases, however, one could not expect biological material to give constant results. Furthermore, the infected hens may be transmitting the disease to their offspring only at certain intervals or cycles. Thus, every egg laid by a carrier hen would not be infected. The work with pullcrum disease brought out the fact that Salmonella pullorum could not be isolated at every attempt from carrier birds or their offspring (Rettger and Stoneburn, 1909). In this study the work was greatly handicapped by the presence of naturally occurring lymphoratcsis in the birds that were used. Thus, lymphomatosis was transmitted to some of the birds, but in others we apparently were only adding to the infection that they already had; therefore, the interpretation of the results depended upon the inoculated lots developing an incidence of lymphomatosis that was significantly higher than that observed in the control groups. The concept of egg transmission of disease producing agents was established many years ago, in fact, the work of Pasteur in 1870 proved that pébrine, a protozoan disease of silkworms, caused by m bombycis, was egg-borne. Since then it has been shown that nearly all types of - 44 - disease producing agents--protozoa, fungi, bacteria, rickettsia, and viruses--have been proved to be transmitted through eggs or seeds. This type of transmission takes place in certain helmdnths, arthropods, birds, and plants (Cottral, 1949). There are a great many difficulties in unequivocally proving that a disease producing agent is transmitted through the egg. The ideal situation would be one in which the etiological agent could be demone strated in the dam, isolated from the interior of'the dam's eggs at different stages of?incubation, isolated from the hatched offspring, and, finally, in the ideal situation.the offspring would remain.carriers of the agent or subsequently develop the disease. Not all of these conditions have been satisfied in the study of lymphomatosis. However, three of the most important steps have been accomplished, namely, demon- stration that the agent is present in certain 15 and 18 day old embryos, and in newly-hatched chicks, and in the blood of‘certain dams. -45.. SUMMARY’AND'CONCLUSIONS The history of the etiological concepts of the avian leukosis complex is presented, and the literature dealing with the transmission of avian lymphomatosis is reviewed. The problem of egg transmission of avian lymphomatosis is discussed and new evidence is presented. The problem.of egg transmission of avian lymphomatosis was studied by attempting to transmit the disease to experimental birds by making use of inocula prepared from.the tissues of newly-hatched chicks and embryos l5 and 18 days old, and from the blood and respiratory washings of certain selected donor hens. Eight experiments were conducted and the results indicate that certain clinically normal dams and sires may produce offspring infected with the agent of'lymphomatosis. Furthermore, in two experimental groups lymphomatosis was produced with a filtrate prepared from embryo and chick liver tissue. In another test, the amnionic fluid from.eggs of a certain dam was infective. The blood from.two donor-hens produced lymphomatosis when inocu- lated into susceptible birds. The results obtained with respiratory washings from donor'hens were not statistically significant. The interpretation of the results is rendered difficult due to the presence of the agent of naturally occurring lymphomatosis in the experi- mental birds that were used. Thus, the disease produced by inoculation was superimposed on the infection.already present in the birds. If stock free of the agent of lymphomatosis could be found, studies of this kind would be much easier to carry out and interpret. Many experiments have been conducted which suggest that lymphoma- tosis may be egg-borne, such as: epiornithic Observations, flock isolation -45- studies, incubator exposure studies, histological studies and trans- mission studies. The results of these studies in conjunction with the result of the present study justify the conclusion that avian lympho- matosis is transmitted from carrier parents to the offspring through the egg. - 47 - BIBLIOGRAPHY Andersen, C. W. and 0. Bang. La leucémie cu leucose transmissible des poules. Festkrif til B. Bang. 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N .02 pnofiflomfim aom corpse was 3333 aoeoo mo mpqoaom .wo haopmfim .m Emma TABLE h.--History of parents of donor (Experiments 3, 6, and 8) embryos Days lymphomatosis in sibs Age at lived Age of dams Dams and first after killed Number Percent Sires use use or died Diagnosis of sibs lymphomatosis Dam: J70lO 301 259 560 Cannibalism 16 56.25 Sires: K 315 177 h92 Normal T 301 177 h78 " V 301 53 35h Visceral lymph. Dam: JLl3OH 309 365+ alive Normal 17 £11.18 Sire: Q 350 152 502 Neural lymph. Dam: Jllolm 350 168 518 No gross lesions 15 116.67 Sires: P 350 177 527 Normal V 336 129 M65 No gross lesions Dam: J716R 282 365+ alive Normal 11 72.73 Sire: E 329 163 h92 " Dam: J313N 309 365+ alive " 10 60.00 Sire: G 321 178 A99 " ' Dam: J310R 301 365+ alive " 1h 28.57 Sires: D 3L6 " " " N 320 n H u 0 320 N n n Dam: J2560 350 365+ alive " 12 25.00 Sire: D 350 177 527 Peritonitis Dam: J726Q 29h 17 311 Reproductive 11 36.36 disorder Sire: E 329 163 L92 Normal Dam: J709W 316 365+ alive " 1h 50.00 Sire: E 329 163 1.92 " Dam: JhOhA h96 365+ alive " 15 h6.67 SirethO3P h55 72 527 No gross lesions V be 25 h65 Normal A2 N40 73 513 " Dam: J306P L20 365+ alive " 26 15.38 SileszJ3l6C M40 73 513 " JBZOG h26 73 h99 " J321L 14110 75 513 " Dam; J709D2 392 365+ alive " 1h 50.00 smmm:J707H bl} 72 h85 " J7151 h13 72 h85 " TABLE 5.--History of parents of donor embryos (Experiments h, 7, and 8) Age at Days lived Age killed Dams and Sires first use after use or died Diagnosis Dam: 111111.112 711; 365+ alive Normal Sire: R2 696 31 727 Bacterial infection Dam: I1h06U2 71b 38 752 Visceral lymphomatosis Sire: * Dam: Ilhl382 7111 365+ alive Normal Sire: * Dam: J1318K2 23o 111b, 3711 Visceral and neural lymphomatosis Sires : T 230 1L1; 37h Norma 1 T2 230 21 251 Neural lymphomatosis L 230 1hh 37h Visceral and neural lymphomatosis V 230 9 239 Neural lymphomatosis Dam: J131882 203 136 339 Visceral lymphomatosis Sires: ** Dam: J131882 259 365+ alive Normal Sires: ** Dam: J1301Wé 231 365+ alive Normal Sires: ** Dam: J1301M2 259 365+ alive Normal Sires: ** Dam: J131hW 231 365+ alive Normal Sires: Q2 2hh. 28h. 528 " L2 2hb- 389 633 " D2 216 175 391 No gross lesions v2 230 215 L115 Neural lymphomatosis Y2 2hb. 320 56h. 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