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Antibody Response of Genetically Susceptible and Resistant Chickens to Cell-Free and Attenuated JM-V Leukosis Strain and Its Influence on Early Type II (Marek’s) Leukosis Infection
69
AMINO ACIDS IN VITELLINE MEMBRANES
McNalley, E. H., 1943. The origin and structure of the vitelline membrane of the domestic fowl's egg. Poultry Sci. 22: 40-43. Polin, D., 1957. Biochemical and weight changes of mottled yolks in eggs from hens fed nicarbazin. Poultry Sci. 36: 831-835. Powers, J. J., and E. S. Keith, 1967. Stepwise discriminant analysis of gas chromatographic data as an aid in classifying the flavor quality of foods. J. Food Sci. 33: 207-213. van Tienhoven, A., F. W. Hill, A. Prock and R. C. Baker, 1958. The effect of nicarbazin on yolk quality. Poultry Sci. 37: 129-132.
Antibody Response of Genetically Susceptible and Resistant Chickens to Cell-Free and Attenuated JM-V Leukosis Strain and Its Influence on Early Type II (Marek's) Leukosis Infection HAPPY K . SHIEH AND MARTIN SEVOIAN
Department of Veterinary and Animal Sciences, University of Massachusetts, 01002
Amherst,
Massachusetts
(Received for publication March 29, 1974)
ABSTRACT Experiments were carried out to: 1) determine the antibody response of chickens to cell-free and attenuated preparations of JMV leukosis strain, 2) determine the differences in antibody response to these antigens between susceptible (P-line) and resistant (N-line) chickens by means of serum neutralization and indirect fluorescent antibody tests, 3) investigate the influence of maternal (passive) antibody on early (day-old) JMV vaccination and 4) investigate the influence of maternal antibody in chicks naturally exposed continuously to JM virus from day-old to 8 weeks of age on the pathogenesis of Type II leukosis (Marek's) infections and oncogenesis. POULTRY SCIENCE 54: 69-77, 1975
he undiluted cell-free inoculum prepared Tfrom blood of moribund JM-V-infected birds was highly antigenic to all chicks but also lethal to a small percentage of day-old chicks. The pathogenicity of the agent was markedly decreased for day-old chicks, however, by serial passage of the JM-V agent in chicken embryos without decreasing the antigenicity. Cell-free JMV and JMV-A (embryo passaged) leukosis strains stimulated chickens Supported by grant-in-aid from Salsbury, Laboratories, Charles City, Iowa.
(both P- and N-line) to produce significantly (P < 0.001) high levels of neutralizing antibody and fluorescent antibody against JM leukosis virus. Vaccinated chicks were completely protected to challenge by virulent cellular JM-V. The vaccinated resistant Nline birds consistently produced higher levels of neutralizing antibody than the susceptible P-line birds. No virus was recovered in kidney cell cultures from JMV and JMV-A vaccinated and unvaccinated control birds after 3 weeks post vaccination. Progeny from cellfree JM-V vaccinated dams possessed high levels of maternal antibody which had a
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Britton, W. M., 1973. Vitelline membrane chemical composition in natural and induced yolk mottling. Poultry Sci. 52: 459-646. Dixon, W. J., 1970. BMD biomedical computer program. University of California Press, Los Angeles, California. Doran, B. M., and W. J. Muller, 1961. The development and structure of the vitelline membrane and their relationship to yolk mottling. Poultry Sci. 40: 474-478. Hale, K. K., Jr., W. J. Stadelman and V. D. Bramblett, 1973. Effect of dry-chilling on the flavor of fried chicken. Poultry Sci. 52: 253-262.
70
H. K. SHIEH AND M. SEVOIAN
INTRODUCTION By rapid passage of JM strain of Type II avian leukosis (Marek's disease) virus (Sevoian et al., 1962) in chicks, Sevoian (1967) established a virulent strain (JMV) which has been used experimentally for challenge and vaccine studies. Cellular JMV strain usually causes 100% mortality in approximately 4-7 days when given intraperitoneally to any genetic strain of day-old chicks. JMV strain has been demonstrated to be antigenically closely related, if not identical, to JM strain (Hamdy and Sevoian, 1973; Kenyon et al., 1969). It has also been shown to stimulate in dams high levels of neutralizing antibodies which were readily transmitted via embryo to offspring affording them protection during early life against natural exposure to JM virus (Sevoian, 1967). The present report describes 1) the immune response of genetically resistant and susceptible chickens to cell-free virulent and atten-
uated JM-V leukosis strain, 2) the influence of passive antibody on early vaccination and 3) the influence of passive antibody on the pathogenesis of Type II (Marek's) leukosis infections in young chicks. MATERIAL AND METHODS Chickens. The chickens used in these trials were JM-P and JM-N line, selected by Dr. R. K. Cole (1968), Cornell University, Ithaca, New York for susceptibility and resistance to JM virus challenge (Type II leukosis) respectively from the Cornell random bred population of chickens. Inocula. Cell-Free JM-V. JM-V whole blood with a titer of 105 5 day-old chick lethal doses fifty percent (CLD J0 ) per ml. was sonicated with sonifier cell disrupter (Bronwill Biosonik) with an intensity of 40 for 3 minutes, centrifuged at 1000 x g for 15 minutes. The supernatant was then passed through 1.2 u. Millipore filter which had been soaked in bovine serum for at least 3 hours. The titer of the filtrate was 104 chicken embryo infective doses fifty percent (CEID J0 ) per ml. Experimental birds were inoculated intraperitoneally with 0.2 ml. of the filtrate. JM-V Infected Chicken Embryo Harvests (JMV-A). Eighteenth and 21st embryo passages of JM-V strain, inoculated via the yolk sac at 4 days of incubation and harvested eight days later, was used for vaccination of day-old and 3 week-old chickens, respectively. Whole embryo harvests, including chorioallantoic membrane, yolk and embryo fibroblasts were diluted 1:1 in PBS (pH 7.2), homogenized with a Waring blender and then centrifuged at 2000 x gfor 10 minutes. Birds were intraperitoneally inoculated with 0.2 ml. of the supernatant containing 300 units of penicillin and 300 micrograms of streptomycin per ml. Harvests from embryos, when inoculated into 4-day old chicken embryos via yolk sac, caused death or typical JM-V
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restrictive effect on the antibody response of day-old chicks to cell-free JM-V and, to a lesser extent JMV-A vaccinations. Day-old progeny possessing JMV parental antibodies were afforded in excess of 3 log 10 of protection to ten-fold serial dilutions of JM virus challenge and sequential tumor development as compared to progeny from non-vaccinated dams and dams vaccinated with herpesvirus of turkeys (HVT). Challenged progeny with JMV maternal antibody had significantly less JM virus yield from their tissues as compared to much higher yields from tissues of progeny of HVT vaccinated and uninoculated dams. When dayold chicks were naturally exposed to heavy JM virus infection for 8 weeks, P-line progeny from JMV, HVT and non-vaccinated dams had gross oncogenic lesions (tumors) at levels of 5%, 67% and 77% respectively whereas N-line progeny from dams similarly vaccinated possessed lesions at levels of 7%, 15% and 14%.
71
ANTIBODY RESPONSE TO LEUKOSIS
lesions (enlarged liver and spleen), and titrated at approximately 10" CEID 5 0 per ml. JMV strain, passaged through avian embryos, is designated as JMV-A strain.
Serologic Response. Five chickens from eachgroup, randomly chosen, were bled from the wing vein or by cardiac puncture at day-old, 3-, 6- and 10-weeks of age in order to test the antibody titers. Serum samples were titrated by means of the plaque reduction neutralization test (Calnek, 1971; Shieh et al., 1973) and the indirect fluorescent antibody test* (Purchase et al., 1970). The neutralizing or FA antibody titers were expressed as the reciprocal of the highest serum dilution which caused at least 50% reduction in the virus titer, or which gave 2+ or more fluorescence (with scores ranging from 04+), respectively. The cell-free JM virus (Calnek, 1971) was used in the SN test whereas low passaged virus was grown in chick kidney (CK) cell cultures on 11- by 22-mm. coverslips and used in the indirect FA test. Challenge and Virus Recovery. Three chickens from each group were intraperitoneally challenged with 2000 CLD 5 0 of cellular *The conjugate, rabbit anti-chicken gamma globulin labelled with fluorescein isothiocyanate, was from Sigma Chemical Co., St. Louis, Mo.
Trial B. Influence of Passive Antibodies to JM Virus Infection and Tumorigenesis. Another trial was conducted to determine the influence of maternal antibodies in offspring of JM-V (2000 CEID 5 0 ), HVT (5000 plaque forming units/bird, FC-126) and non- vaccinated dams to JM virus infection and tumor formation. Both susceptible (JM-P) and resistant (JM-N) day-old chicks obtained from JM-V vaccinated, HVT-vaccinated, and control dams were inoculated intraperitoneally with ten-fold dilutions of cell-free JM virus, and then were kept in modified Horsfall units to prevent cross-infection. All chicks were necropsied at three weeks post-inoculation for examination of gross and/or microscopic lesions. Tumorous enlargements of dorsal root ganglia and gonads were considered characterisitic f or JM virus infection (Sevoian et al., 1962). The oncogenic dose fifty percent (OD 50 ) in both lines was calculated by the method of Reed and Muench (1938). In parallel, chicken kidneys of each dilution were removed aseptically and grown in vitro to quantitate levels of JM virus recovery. Day-old progeny of JMV and HVT-vaccinated and non-vaccinated dams were naturally exposed to JM virus for a longer period (8 weeks) at which time all chicks were necropsied and examined for gross lesions. RESULTS Trial A. Serum Neutralization (SN) Antibodies Against JM Virus. Antibody response to cell-free JMV: In trials using chicks from JM-V vaccinated dams, the maternal antibody titers against JM virus of N-line chicks at day-old was titered at 1:50 (geometric mean of titers of 5 serum samples), and
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Trial A. Vaccination. Forty-eight day-old chicks of each line from dams vaccinated with cell-free JM-V leukosis strain were divided into 3 equal groups. One group served as uninoculated controls, whereas the other two groups were intraperitoneally vaccinated at day-old and 3-weeks of age with approximately 2000 CEID J 0 of cell-free JMV strain or chicken-embryo passaged JMV (JMV-A). Parallel and comparable lots of thirty-nine chicks of each line from unvaccinated dams also were vaccinated. All birds were kept in Horsfall-Bauer isolators.
virulent JM-V strain at 6 weeks of age. Three chickens from each group, chosen at random, were sacrificed and their kidneys separately were trypsinized and cultured for virus recovery (Churchill and Biggs, 1967) at 3, 6, and 10-weeks of age respectively.
H. K. SHIEH AND M. SEVOIAN
72 la
a.
80
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FIG. 1. Serum neutralization titers of P-line (broken) and N-line (solid) chickens, with high (X) or low (O) passive (maternal) antibodies, vaccinated at day-old and 3-weeks of age with cell-free JM-V (la) or JM-V-infected embryo harvests (lb). Unvaccinated controls (D).
1:30 in P-line chicks. Three weeks after these chicks were vaccinated (at day-old) with cell-free JM-V, the SN titers of the N-line chicks had decreased to about 30, whereas P-line vaccinated chicks maintained their antibody titer at 1:30. At this time unvaccinated N-line and P-line chicks in isolation had lost almost all their SN maternal antibody. Revaccination at 3 weeks of age stimulated both lines of chickens to produce a higher level of SN antibody. Seven weeks after the second vaccination, or at ten-weeks of age, the SN titers of the cell-free JM-V vaccinated chicks of both lines declined somewhat (Fig. la) indicating that the host
was free of the vaccine viral antigen. Antibody of response to JMV-A: The Nline chicks from JM-V vaccinated dams had a mean maternal antibody titer of 1:50 at day-old at which time they were vaccinated with 2000 CEID J 0 of JMV-A. At 3 weeks of age, their SN titers remained at the same level whereas antibody titers in non-vaccinated siblings held in isolation decreased to insignificant levels. Re-vaccination with JMV-A at 3-weeks-old, increased the antibody titers to over 90 by 6 weeks of age. Seven weeks after the second vaccination, the SN titers had decreased (Fig. lb). When P-line chicks from JM-V-vaccinated
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//A
1
73
ANTIBODY RESPONSE TO LEUKOSIS
of both groups had increased to over 90 for N-line and 60 for P-line. Some decline in SN titers was also observed in these chickens at seven weeks after the second vaccination, presumably because of the disappearance of the vaccine viral antigen from within the host tissues (Fig. la, lb). Indirect Fluorescent Antibody (FA) Titers. The FA serum titer of the N-line chicks from JM-V vaccinated dams was 160 at one day of age, at which time they were vaccinated with cell-free JMV. At 3 weeks of age, the FA titer of vaccinated chicks was 95 whereas the titers in unvaccinated chicks had fallen
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FIG. 2. Indirect fluorescent antibody titers of P-line (broken) and N-line (solid) chickens, with high (X) or low (O) passive (maternal) antibodies, vaccinated at day-old and 3 weeks of age with cell-free JM-V (2a) or JM-V-infected embryo harvests (2b). Unvaccinated controls (•).
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dams, were vaccinated with JMV-A at oneday, the SN titer increased to 40 by 3 weeks after vaccination. Revaccination at 3 weeks of age increased the titers further to about 60. As with the N-line chicks, a decrease in SN titers of vaccinated P-line chicks, maintained in isolators, was also noted at seven weeks after the second vaccination. The P- and N-line chicks, from unvaccinated dams, vaccinated with either cell-free JM-V or JMV-A at day-old produced relatively high levels of SN antibodies against JM virus at 3 weeks post-vaccination. Three weeks after re-vaccinating with the respective preparations at 3 weeks of age, the SN titers
74
H. K. SHIEH AND M. SEVOIAN
antibody levels increased by 3 weeks whereas the unvaccinated controls had no FA. Revaccination at this time, further increased the titers which peaked at 6 weeks of age. When the same vaccination procedure was followed for P- and N-line chicks (from unvaccinated dams) having a low maternal FA serum titer against JM virus at one day of age, a high level antibody was observed in 3 weeks. A second vaccination contributed to a further increase in FA titers (Fig. 2a, 2b). Clinical Manifestations of Chickens to Vaccination and Challenge. Most chickens vaccinated with virulent cell-free JMV at day-old and revaccinated at 3-weeks-old survived (Table 1). All of the chicks vaccinated with JMV-A inoculum survived with no clinical manifestations. Chickens vaccinated with either cell-free JMV or JMV-A completely withstood a challenge dose of 2000 CLD 5 0 of cellular JMV, whereas 58% of the unvaccinated controls died from the same challenge. Pathological Findings and Virus Recovery. During the 10 week experimental
TABLE 1.—Safety of cell-free JM-V or JMV-A strains in chickens vaccinated at one-day and 3 weeks of age By day-old vaccination Sources of chickens N-line progeny from JM-V vaccinated dams progeny from unvaccinated dams P-line progeny from JM-V vaccinated dams progeny from unvaccinated dams
*No. survival/No. vaccinated.
Revaccination at 3 weeks of age
JMV-A
Cell-free JM-V
JMV-A
10/10
13/14
7/7
7/9
10/10
5/5
7/7
13/15
15/15
10/11
12/12
13/18
15/15
10/11
12/12
Cell-free JM-V 16/18*
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to 40. Similar to the SN titer, a peak FA titer in N-line chicks was observed at 6 weeks of age after they were revaccinated at 3 weeks of age. A decline of the FA titer at 10-weeks old was also observed, again indicating the disappearance of the antigen. Day-old P-line chicks from JM-V vaccinated dams, with an initial FA serum titer of 80, were vaccinated with cell-free JMV. They maintained this antibody level till 3 weeks of age when they were revaccinated. At 6 weeks of age, the serum FA titer rose to 150 and then decreased to about 100 at 10 weeks of age (Fig. 2a, 2b). When day-old N-line chicks (from JM-V vaccinated dams) with a high initial FA serum titer (160) were vaccinated with JMV-A, they were able to maintain effective titers when tested at 3 weeks whereas the unvaccinated controls showed a sharp decrease in FA serum titer in the same time period. Revaccination with the same inoculum at 3-weeks of age stimulated the vaccinated birds to produce an even higher level of fluorescent antibody against JM (Fig. 2a, 2b). When day-old P-line chicks from JM-V vaccinated dams, with an original FA serum titer of 80, were vaccinated with JMV-A, the
75
ANTIBODY RESPONSE TO LEUKOSIS
Trial B. Experimental Challenge. The relationship of JM virus infection, neutralizing ability of JM virus by maternal antibody and tumor formation in the offspring of JM-V vaccinated, HVT-vaccinated, and control dams was studied. The day-old offspring of these groups inoculated intraperitoneally with serial dilutions of cell-free JM virus, and then were kept in modified Horsfall units to prevent cross-infection. All chicks were necrop-
sied at three weeks of age and examined for gross and/or microscopic Type II leukosis lesions. The resistance to JM virus challenge of JM-N and JM-P progenies from JM-V vaccinated dams was more than 3 x log 10 higher than of progeny from corresponding HVT-vaccinated and non-vaccinated dams (Table 2). In parallel, kidney samples from 3 birds of each challenge dilution from each group were removed aseptically for JM virus isolation at three weeks of age. In progeny from JM-V vaccinated dams, JM virus could only be recovered from those chicks that had been inoculated with the least diluted virus suspension. By comparison, JM virus was isolated from all the progeny of HVT-vaccinated and control dams that had been inoculated through 103 dilution in JM-N and JM-P lines. In other words, the progeny possessing JM-V parental antibodies inhibited JM virus challenge in excess of 3 x log 10 times greater
TABLE 2.—The comparative effects of passive (maternal) antibodies on cell-free JM virus challenge of day-old progeny chicks from JM-V and HVT vaccinated dams by virus recovery and tumor formation Dam vaccination
Challenge dilution JM virus 10" 1 10 " 2
****
io- 3
JM-V
Non-inoculated control
io-' 10 " 2
io -3 HVT
Non-inoculated control 10"'
io- 2
Non-vaccinated
10 " 3 Non-inoculated control
Infected total :*=*
Affected total * P
Plaques/*** 2 x 10" cells P 2 0 0
N 1 0 0
P 0/5 0/5 0/5
N 0/5 0/5 0/5
1/3 0/3 0/3
N 1/3 0/3 0/3
0/5 4/5 3/5 2/5
0/5
0/3
0/3
0
0
2/5 1/5 0/5
3/3 3/3 3/3
3/3 3/3 3/3
27 21 13
18 12 8
0/5
0/5
0/3
0/3
0
0
5/5 5/5 3/5
1/5 0/5 0/5
3/3 3/3 3/3
3/3 3/3 2/3
25 12 18
16 14 21
0/5
0/5
0/3
0/3
0
0
* Birds were necropsied and examined for gross and/or microscopic pathological changes at 3 weeks postinoculation. **JM virus recovery from chicken kidney cell cultures at 3 weeks postinoculation. *** Average number of plaques of total positive samples. ****Dams were inoculated with 0.25 ml. of cell-free JM-V strain (2000 CEID^/ml.) three times at monthly intervals.
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period, attempts were conducted in cell cultures for virus recovery from kidney of both JMV and JMV-A vaccinated and non-vaccinated chickens of each group at 3-, 6- and 10-weeks of age. No cytopathic effects were observed in these cultures maintained over a period of two weeks. When these same tissues were placed in chicken embryos, no lesions developed. The chickens sacrificed for virus recovery were also negative for gross lesions.
76
H. K. SHIEH AND M. SEVOIAN
TABLE 3.— The comparative influence of maternal (passive) antibodies in progeny naturally exposed to JM leukosis virus infections from dams vaccinated with JMV leukosis strain and herpesvirus of turkeys (HVT)
than progeny from HVT-vaccinated and control dams. Although the JM-N line chicks were more resistant to Type II leukosis tumor development than the JM-P ones, both lines were equally sensitive to JM-virus infection and demonstrated by virus recovery technique (Table 2). Natural Exposure. Day-old progeny of JM-V vaccinated, HVT-vaccinated and control dams were naturally exposed to JM virus for an 8-week period. Table 3 shows results of the effectiveness of maternal antibody to early exposure to JM virus progeny of JM-V and HVT-vaccinated dams. The progeny possessing JM-V maternal antibody had significantly fewer oncogenic lesions (tumors) than progeny of HVT-vaccinated dams as well as of non-vaccinated dams in the first eight weeks of exposure. DISCUSSION Sevoian et al. (1962), reported that JM-V strain could immunologically stimulate dams to produce neutralizing antibodies which were readily transmitted via embryo to offspring, affording them protection during early life against natural exposure to JM virus. The data herein presented confirms the existence of high levels of neutralizing antibodies in day-old progeny from dams which had been
(7%) (15%) (14%)
vaccinated with JM-V, while little or no neutralizing antibodies were found in the day-old offspring from unvaccinated dams. Upon vaccination with cell-free JMV or JMV-A strains, day-old chicks with low (or no) maternal antibodies showed a marked increase in their serum antibody (both neutralizing and fluorescent antibodies) against JM virus within 3 weeks whereas day-old birds with high maternal antibodies either maintained their titer or slightly declined in the same time period. The decline in titers was more evident in birds vaccinated with cell-free preparation than with JMV-A. These results indicate that high levels of maternal antibodies had an inhibitory influence on the vaccine antigen which in turn restricted antibody response of the host. Day-old chicks (with low or no maternal antibodies) vaccinated with either cell-free JMV or JMV-A strains produced significantly high levels of serum neutralizing antibody in both P- and N-line chicks within 3 weeks postvaccination. This suggests that the JM-V agent is effective in inducing immunity against Type II (Marek's) leukosis. JM-V agent in its virulent form caused some losses in antibody free chicks vaccinated with cell-free inocula prepared from sonicated whole blood of
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Oncogenic lesions-progeny Treatment of progeny P-line N-line exposed8 1/20" (5%) 2731) non-exposed 0/10 0/15 HVT exposed 10/15 (67%) T/2Q non-exposed 0/10 0/20 Non-vaccinated exposed 23/30 (77%) 5730 non-exposed 0/15 0/10 "Naturally exposed to JM virus at day-old to 8 weeks of age. b No. positive/total—8 weeks of age. Vaccination of dams JMV
77
ANTIBODY RESPONSE TO LEUKOSIS
It was observed that most of the chickens vaccinated with JM-V strain showed a decline in antibody titers (both SN and FA) seven weeks after the second vaccination. This suggested that viral antigens from the vaccine disappeared within 3 weeks post-inoculation. All the serum samples that showed positive results in the SN test were also positive in the FA test. Serum titers as determined in the FA test were from 2 to 11 times higher than the titers from the SN test. There appears to be a direct correlation between serum neutralizing antibody and fluorescent antibody titers. Observations made during analysis of the FA results indicate that the best sources of viral antigens for indirect staining are from low passage cell cultures or new virus isolates. In the indirect FA test, using the same serum sample, the low passage antigen showed brilliant fluorescence whereas the high passage antigen (over 20 passages) gave dull or little fluorescence. Churchill and Chubb (1969) reported that the virulent strain of Type II leukosis (Marek's) virus was attenuated upon serial passage in cell culture with the loss of an antigen (A antigen) de-
monstrable in the virulent isolate. This may imply that the fluorescent antibody is related to the A antigen. REFERENCES Calnek, B. W., 1971. Antibody development in chickens exposed to Marek's disease. Symposium on Oncogenesis and Herpes-type Viruses. Christ's College, Cambridge, England. Churchill, A. E., and P. M. Biggs, 1967. Agents of Marek's disease in tissue culture. Nature, 215: 528-530. Churchill, A. E., R. C. Chubb and W. Baxendale, 1969. The attenuation, with loss of oncogenicity of the herpes-type virus of Marek's disease (strain HPRS-16) on passage in cell culture. J. Gen. Virol. 4: 557-564. Cole, R. D., 1968. Studies on genetic resistance to Marek's disease. Avian Dis. 12: 9-28. Hamdy, F. M., and M. Sevoian, 1973. Immunologic relationship between JM and JM-V leukosis strains and herpesvirus of turkeys. Avian Dis. 17: 476-485. Kenyon, A. J., M. Sevoian, M. Horvitz, N. D. Jones and C. F. Helmboldt, 1969. Lymphoproliferative disease of fowl-immunologic factors associated with passage of a lymphoblastic leukemia (JM-V). Avian Dis. 13: 585-595. Mikami, T., and R. A. Bankowski, 1971. The effect of DMSO and cultured behavior of two herpesviruses associated with Marek's disease. Avian Dis. 15: 242-253. Purchase, H. G., and G. H. Burgoyne. 1970. Immunofluorescence in the study of Marek's disease. Detection of antibody. Am. J. Vet. Res. 31: 117-124. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27: 493-495. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. I. Experimental reproduction of the neural and visceral forms. Vet. Med. 57: 500-501. Sevoian, M., 1967. Immunity studies in chickens inoculated with a virulent Type II avian leukosis strain (JM-V). Proc. 39th N.E. Conf. on Avian Disease. State Univ. of New York, Stony Brook, L.I.June 19-21. Shieh, H. K., and M. Sevoian, 1973. Antibody response of susceptible (P-line) and resistant (N-line) chickens to JM and JM-V leukosis strains and herpesvirus of turkeys (HVT). Avian Dis. (In press)
SEPTEMBER 5-11, 1976. FIFTH EUROPEAN POULTRY CONFERENCE, MALTA
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moribund JM-V infected birds. The pathogenicity of JM-V was markedly decreased by serial passage of the JM-V agent in chicken embryos as evidenced by the complete lack of morbidity and mortality of P- and N-line chicks when they were vaccinated with inocula prepared from 18th and 21st chicken embryo passages of JM-V strain. The serial passage of the agent in embryonated chicks modified only the pathogenicity, but not antigenicity, since the chickens vaccinated with this preparation produced neutralizing antibody titers (against JM virus) at least as high or higher than those vaccinated with the virulent cell-free JM-V inocula prepared from blood of moribund birds.
AMINO ACIDS IN VITELLINE MEMBRANES
McNalley, E. H., 1943. The origin and structure of the vitelline membrane of the domestic fowl's egg. Poultry Sci. 22: 40-43. Polin, D., 1957. Biochemical and weight changes of mottled yolks in eggs from hens fed nicarbazin. Poultry Sci. 36: 831-835. Powers, J. J., and E. S. Keith, 1967. Stepwise discriminant analysis of gas chromatographic data as an aid in classifying the flavor quality of foods. J. Food Sci. 33: 207-213. van Tienhoven, A., F. W. Hill, A. Prock and R. C. Baker, 1958. The effect of nicarbazin on yolk quality. Poultry Sci. 37: 129-132.
Antibody Response of Genetically Susceptible and Resistant Chickens to Cell-Free and Attenuated JM-V Leukosis Strain and Its Influence on Early Type II (Marek's) Leukosis Infection HAPPY K . SHIEH AND MARTIN SEVOIAN
Department of Veterinary and Animal Sciences, University of Massachusetts, 01002
Amherst,
Massachusetts
(Received for publication March 29, 1974)
ABSTRACT Experiments were carried out to: 1) determine the antibody response of chickens to cell-free and attenuated preparations of JMV leukosis strain, 2) determine the differences in antibody response to these antigens between susceptible (P-line) and resistant (N-line) chickens by means of serum neutralization and indirect fluorescent antibody tests, 3) investigate the influence of maternal (passive) antibody on early (day-old) JMV vaccination and 4) investigate the influence of maternal antibody in chicks naturally exposed continuously to JM virus from day-old to 8 weeks of age on the pathogenesis of Type II leukosis (Marek's) infections and oncogenesis. POULTRY SCIENCE 54: 69-77, 1975
he undiluted cell-free inoculum prepared Tfrom blood of moribund JM-V-infected birds was highly antigenic to all chicks but also lethal to a small percentage of day-old chicks. The pathogenicity of the agent was markedly decreased for day-old chicks, however, by serial passage of the JM-V agent in chicken embryos without decreasing the antigenicity. Cell-free JMV and JMV-A (embryo passaged) leukosis strains stimulated chickens Supported by grant-in-aid from Salsbury, Laboratories, Charles City, Iowa.
(both P- and N-line) to produce significantly (P < 0.001) high levels of neutralizing antibody and fluorescent antibody against JM leukosis virus. Vaccinated chicks were completely protected to challenge by virulent cellular JM-V. The vaccinated resistant Nline birds consistently produced higher levels of neutralizing antibody than the susceptible P-line birds. No virus was recovered in kidney cell cultures from JMV and JMV-A vaccinated and unvaccinated control birds after 3 weeks post vaccination. Progeny from cellfree JM-V vaccinated dams possessed high levels of maternal antibody which had a
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Britton, W. M., 1973. Vitelline membrane chemical composition in natural and induced yolk mottling. Poultry Sci. 52: 459-646. Dixon, W. J., 1970. BMD biomedical computer program. University of California Press, Los Angeles, California. Doran, B. M., and W. J. Muller, 1961. The development and structure of the vitelline membrane and their relationship to yolk mottling. Poultry Sci. 40: 474-478. Hale, K. K., Jr., W. J. Stadelman and V. D. Bramblett, 1973. Effect of dry-chilling on the flavor of fried chicken. Poultry Sci. 52: 253-262.
70
H. K. SHIEH AND M. SEVOIAN
INTRODUCTION By rapid passage of JM strain of Type II avian leukosis (Marek's disease) virus (Sevoian et al., 1962) in chicks, Sevoian (1967) established a virulent strain (JMV) which has been used experimentally for challenge and vaccine studies. Cellular JMV strain usually causes 100% mortality in approximately 4-7 days when given intraperitoneally to any genetic strain of day-old chicks. JMV strain has been demonstrated to be antigenically closely related, if not identical, to JM strain (Hamdy and Sevoian, 1973; Kenyon et al., 1969). It has also been shown to stimulate in dams high levels of neutralizing antibodies which were readily transmitted via embryo to offspring affording them protection during early life against natural exposure to JM virus (Sevoian, 1967). The present report describes 1) the immune response of genetically resistant and susceptible chickens to cell-free virulent and atten-
uated JM-V leukosis strain, 2) the influence of passive antibody on early vaccination and 3) the influence of passive antibody on the pathogenesis of Type II (Marek's) leukosis infections in young chicks. MATERIAL AND METHODS Chickens. The chickens used in these trials were JM-P and JM-N line, selected by Dr. R. K. Cole (1968), Cornell University, Ithaca, New York for susceptibility and resistance to JM virus challenge (Type II leukosis) respectively from the Cornell random bred population of chickens. Inocula. Cell-Free JM-V. JM-V whole blood with a titer of 105 5 day-old chick lethal doses fifty percent (CLD J0 ) per ml. was sonicated with sonifier cell disrupter (Bronwill Biosonik) with an intensity of 40 for 3 minutes, centrifuged at 1000 x g for 15 minutes. The supernatant was then passed through 1.2 u. Millipore filter which had been soaked in bovine serum for at least 3 hours. The titer of the filtrate was 104 chicken embryo infective doses fifty percent (CEID J0 ) per ml. Experimental birds were inoculated intraperitoneally with 0.2 ml. of the filtrate. JM-V Infected Chicken Embryo Harvests (JMV-A). Eighteenth and 21st embryo passages of JM-V strain, inoculated via the yolk sac at 4 days of incubation and harvested eight days later, was used for vaccination of day-old and 3 week-old chickens, respectively. Whole embryo harvests, including chorioallantoic membrane, yolk and embryo fibroblasts were diluted 1:1 in PBS (pH 7.2), homogenized with a Waring blender and then centrifuged at 2000 x gfor 10 minutes. Birds were intraperitoneally inoculated with 0.2 ml. of the supernatant containing 300 units of penicillin and 300 micrograms of streptomycin per ml. Harvests from embryos, when inoculated into 4-day old chicken embryos via yolk sac, caused death or typical JM-V
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restrictive effect on the antibody response of day-old chicks to cell-free JM-V and, to a lesser extent JMV-A vaccinations. Day-old progeny possessing JMV parental antibodies were afforded in excess of 3 log 10 of protection to ten-fold serial dilutions of JM virus challenge and sequential tumor development as compared to progeny from non-vaccinated dams and dams vaccinated with herpesvirus of turkeys (HVT). Challenged progeny with JMV maternal antibody had significantly less JM virus yield from their tissues as compared to much higher yields from tissues of progeny of HVT vaccinated and uninoculated dams. When dayold chicks were naturally exposed to heavy JM virus infection for 8 weeks, P-line progeny from JMV, HVT and non-vaccinated dams had gross oncogenic lesions (tumors) at levels of 5%, 67% and 77% respectively whereas N-line progeny from dams similarly vaccinated possessed lesions at levels of 7%, 15% and 14%.
71
ANTIBODY RESPONSE TO LEUKOSIS
lesions (enlarged liver and spleen), and titrated at approximately 10" CEID 5 0 per ml. JMV strain, passaged through avian embryos, is designated as JMV-A strain.
Serologic Response. Five chickens from eachgroup, randomly chosen, were bled from the wing vein or by cardiac puncture at day-old, 3-, 6- and 10-weeks of age in order to test the antibody titers. Serum samples were titrated by means of the plaque reduction neutralization test (Calnek, 1971; Shieh et al., 1973) and the indirect fluorescent antibody test* (Purchase et al., 1970). The neutralizing or FA antibody titers were expressed as the reciprocal of the highest serum dilution which caused at least 50% reduction in the virus titer, or which gave 2+ or more fluorescence (with scores ranging from 04+), respectively. The cell-free JM virus (Calnek, 1971) was used in the SN test whereas low passaged virus was grown in chick kidney (CK) cell cultures on 11- by 22-mm. coverslips and used in the indirect FA test. Challenge and Virus Recovery. Three chickens from each group were intraperitoneally challenged with 2000 CLD 5 0 of cellular *The conjugate, rabbit anti-chicken gamma globulin labelled with fluorescein isothiocyanate, was from Sigma Chemical Co., St. Louis, Mo.
Trial B. Influence of Passive Antibodies to JM Virus Infection and Tumorigenesis. Another trial was conducted to determine the influence of maternal antibodies in offspring of JM-V (2000 CEID 5 0 ), HVT (5000 plaque forming units/bird, FC-126) and non- vaccinated dams to JM virus infection and tumor formation. Both susceptible (JM-P) and resistant (JM-N) day-old chicks obtained from JM-V vaccinated, HVT-vaccinated, and control dams were inoculated intraperitoneally with ten-fold dilutions of cell-free JM virus, and then were kept in modified Horsfall units to prevent cross-infection. All chicks were necropsied at three weeks post-inoculation for examination of gross and/or microscopic lesions. Tumorous enlargements of dorsal root ganglia and gonads were considered characterisitic f or JM virus infection (Sevoian et al., 1962). The oncogenic dose fifty percent (OD 50 ) in both lines was calculated by the method of Reed and Muench (1938). In parallel, chicken kidneys of each dilution were removed aseptically and grown in vitro to quantitate levels of JM virus recovery. Day-old progeny of JMV and HVT-vaccinated and non-vaccinated dams were naturally exposed to JM virus for a longer period (8 weeks) at which time all chicks were necropsied and examined for gross lesions. RESULTS Trial A. Serum Neutralization (SN) Antibodies Against JM Virus. Antibody response to cell-free JMV: In trials using chicks from JM-V vaccinated dams, the maternal antibody titers against JM virus of N-line chicks at day-old was titered at 1:50 (geometric mean of titers of 5 serum samples), and
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Trial A. Vaccination. Forty-eight day-old chicks of each line from dams vaccinated with cell-free JM-V leukosis strain were divided into 3 equal groups. One group served as uninoculated controls, whereas the other two groups were intraperitoneally vaccinated at day-old and 3-weeks of age with approximately 2000 CEID J 0 of cell-free JMV strain or chicken-embryo passaged JMV (JMV-A). Parallel and comparable lots of thirty-nine chicks of each line from unvaccinated dams also were vaccinated. All birds were kept in Horsfall-Bauer isolators.
virulent JM-V strain at 6 weeks of age. Three chickens from each group, chosen at random, were sacrificed and their kidneys separately were trypsinized and cultured for virus recovery (Churchill and Biggs, 1967) at 3, 6, and 10-weeks of age respectively.
H. K. SHIEH AND M. SEVOIAN
72 la
a.
80
60
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E
V/
40
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FIG. 1. Serum neutralization titers of P-line (broken) and N-line (solid) chickens, with high (X) or low (O) passive (maternal) antibodies, vaccinated at day-old and 3-weeks of age with cell-free JM-V (la) or JM-V-infected embryo harvests (lb). Unvaccinated controls (D).
1:30 in P-line chicks. Three weeks after these chicks were vaccinated (at day-old) with cell-free JM-V, the SN titers of the N-line chicks had decreased to about 30, whereas P-line vaccinated chicks maintained their antibody titer at 1:30. At this time unvaccinated N-line and P-line chicks in isolation had lost almost all their SN maternal antibody. Revaccination at 3 weeks of age stimulated both lines of chickens to produce a higher level of SN antibody. Seven weeks after the second vaccination, or at ten-weeks of age, the SN titers of the cell-free JM-V vaccinated chicks of both lines declined somewhat (Fig. la) indicating that the host
was free of the vaccine viral antigen. Antibody of response to JMV-A: The Nline chicks from JM-V vaccinated dams had a mean maternal antibody titer of 1:50 at day-old at which time they were vaccinated with 2000 CEID J 0 of JMV-A. At 3 weeks of age, their SN titers remained at the same level whereas antibody titers in non-vaccinated siblings held in isolation decreased to insignificant levels. Re-vaccination with JMV-A at 3-weeks-old, increased the antibody titers to over 90 by 6 weeks of age. Seven weeks after the second vaccination, the SN titers had decreased (Fig. lb). When P-line chicks from JM-V-vaccinated
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//A
1
73
ANTIBODY RESPONSE TO LEUKOSIS
of both groups had increased to over 90 for N-line and 60 for P-line. Some decline in SN titers was also observed in these chickens at seven weeks after the second vaccination, presumably because of the disappearance of the vaccine viral antigen from within the host tissues (Fig. la, lb). Indirect Fluorescent Antibody (FA) Titers. The FA serum titer of the N-line chicks from JM-V vaccinated dams was 160 at one day of age, at which time they were vaccinated with cell-free JMV. At 3 weeks of age, the FA titer of vaccinated chicks was 95 whereas the titers in unvaccinated chicks had fallen
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FIG. 2. Indirect fluorescent antibody titers of P-line (broken) and N-line (solid) chickens, with high (X) or low (O) passive (maternal) antibodies, vaccinated at day-old and 3 weeks of age with cell-free JM-V (2a) or JM-V-infected embryo harvests (2b). Unvaccinated controls (•).
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dams, were vaccinated with JMV-A at oneday, the SN titer increased to 40 by 3 weeks after vaccination. Revaccination at 3 weeks of age increased the titers further to about 60. As with the N-line chicks, a decrease in SN titers of vaccinated P-line chicks, maintained in isolators, was also noted at seven weeks after the second vaccination. The P- and N-line chicks, from unvaccinated dams, vaccinated with either cell-free JM-V or JMV-A at day-old produced relatively high levels of SN antibodies against JM virus at 3 weeks post-vaccination. Three weeks after re-vaccinating with the respective preparations at 3 weeks of age, the SN titers
74
H. K. SHIEH AND M. SEVOIAN
antibody levels increased by 3 weeks whereas the unvaccinated controls had no FA. Revaccination at this time, further increased the titers which peaked at 6 weeks of age. When the same vaccination procedure was followed for P- and N-line chicks (from unvaccinated dams) having a low maternal FA serum titer against JM virus at one day of age, a high level antibody was observed in 3 weeks. A second vaccination contributed to a further increase in FA titers (Fig. 2a, 2b). Clinical Manifestations of Chickens to Vaccination and Challenge. Most chickens vaccinated with virulent cell-free JMV at day-old and revaccinated at 3-weeks-old survived (Table 1). All of the chicks vaccinated with JMV-A inoculum survived with no clinical manifestations. Chickens vaccinated with either cell-free JMV or JMV-A completely withstood a challenge dose of 2000 CLD 5 0 of cellular JMV, whereas 58% of the unvaccinated controls died from the same challenge. Pathological Findings and Virus Recovery. During the 10 week experimental
TABLE 1.—Safety of cell-free JM-V or JMV-A strains in chickens vaccinated at one-day and 3 weeks of age By day-old vaccination Sources of chickens N-line progeny from JM-V vaccinated dams progeny from unvaccinated dams P-line progeny from JM-V vaccinated dams progeny from unvaccinated dams
*No. survival/No. vaccinated.
Revaccination at 3 weeks of age
JMV-A
Cell-free JM-V
JMV-A
10/10
13/14
7/7
7/9
10/10
5/5
7/7
13/15
15/15
10/11
12/12
13/18
15/15
10/11
12/12
Cell-free JM-V 16/18*
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to 40. Similar to the SN titer, a peak FA titer in N-line chicks was observed at 6 weeks of age after they were revaccinated at 3 weeks of age. A decline of the FA titer at 10-weeks old was also observed, again indicating the disappearance of the antigen. Day-old P-line chicks from JM-V vaccinated dams, with an initial FA serum titer of 80, were vaccinated with cell-free JMV. They maintained this antibody level till 3 weeks of age when they were revaccinated. At 6 weeks of age, the serum FA titer rose to 150 and then decreased to about 100 at 10 weeks of age (Fig. 2a, 2b). When day-old N-line chicks (from JM-V vaccinated dams) with a high initial FA serum titer (160) were vaccinated with JMV-A, they were able to maintain effective titers when tested at 3 weeks whereas the unvaccinated controls showed a sharp decrease in FA serum titer in the same time period. Revaccination with the same inoculum at 3-weeks of age stimulated the vaccinated birds to produce an even higher level of fluorescent antibody against JM (Fig. 2a, 2b). When day-old P-line chicks from JM-V vaccinated dams, with an original FA serum titer of 80, were vaccinated with JMV-A, the
75
ANTIBODY RESPONSE TO LEUKOSIS
Trial B. Experimental Challenge. The relationship of JM virus infection, neutralizing ability of JM virus by maternal antibody and tumor formation in the offspring of JM-V vaccinated, HVT-vaccinated, and control dams was studied. The day-old offspring of these groups inoculated intraperitoneally with serial dilutions of cell-free JM virus, and then were kept in modified Horsfall units to prevent cross-infection. All chicks were necrop-
sied at three weeks of age and examined for gross and/or microscopic Type II leukosis lesions. The resistance to JM virus challenge of JM-N and JM-P progenies from JM-V vaccinated dams was more than 3 x log 10 higher than of progeny from corresponding HVT-vaccinated and non-vaccinated dams (Table 2). In parallel, kidney samples from 3 birds of each challenge dilution from each group were removed aseptically for JM virus isolation at three weeks of age. In progeny from JM-V vaccinated dams, JM virus could only be recovered from those chicks that had been inoculated with the least diluted virus suspension. By comparison, JM virus was isolated from all the progeny of HVT-vaccinated and control dams that had been inoculated through 103 dilution in JM-N and JM-P lines. In other words, the progeny possessing JM-V parental antibodies inhibited JM virus challenge in excess of 3 x log 10 times greater
TABLE 2.—The comparative effects of passive (maternal) antibodies on cell-free JM virus challenge of day-old progeny chicks from JM-V and HVT vaccinated dams by virus recovery and tumor formation Dam vaccination
Challenge dilution JM virus 10" 1 10 " 2
****
io- 3
JM-V
Non-inoculated control
io-' 10 " 2
io -3 HVT
Non-inoculated control 10"'
io- 2
Non-vaccinated
10 " 3 Non-inoculated control
Infected total :*=*
Affected total * P
Plaques/*** 2 x 10" cells P 2 0 0
N 1 0 0
P 0/5 0/5 0/5
N 0/5 0/5 0/5
1/3 0/3 0/3
N 1/3 0/3 0/3
0/5 4/5 3/5 2/5
0/5
0/3
0/3
0
0
2/5 1/5 0/5
3/3 3/3 3/3
3/3 3/3 3/3
27 21 13
18 12 8
0/5
0/5
0/3
0/3
0
0
5/5 5/5 3/5
1/5 0/5 0/5
3/3 3/3 3/3
3/3 3/3 2/3
25 12 18
16 14 21
0/5
0/5
0/3
0/3
0
0
* Birds were necropsied and examined for gross and/or microscopic pathological changes at 3 weeks postinoculation. **JM virus recovery from chicken kidney cell cultures at 3 weeks postinoculation. *** Average number of plaques of total positive samples. ****Dams were inoculated with 0.25 ml. of cell-free JM-V strain (2000 CEID^/ml.) three times at monthly intervals.
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period, attempts were conducted in cell cultures for virus recovery from kidney of both JMV and JMV-A vaccinated and non-vaccinated chickens of each group at 3-, 6- and 10-weeks of age. No cytopathic effects were observed in these cultures maintained over a period of two weeks. When these same tissues were placed in chicken embryos, no lesions developed. The chickens sacrificed for virus recovery were also negative for gross lesions.
76
H. K. SHIEH AND M. SEVOIAN
TABLE 3.— The comparative influence of maternal (passive) antibodies in progeny naturally exposed to JM leukosis virus infections from dams vaccinated with JMV leukosis strain and herpesvirus of turkeys (HVT)
than progeny from HVT-vaccinated and control dams. Although the JM-N line chicks were more resistant to Type II leukosis tumor development than the JM-P ones, both lines were equally sensitive to JM-virus infection and demonstrated by virus recovery technique (Table 2). Natural Exposure. Day-old progeny of JM-V vaccinated, HVT-vaccinated and control dams were naturally exposed to JM virus for an 8-week period. Table 3 shows results of the effectiveness of maternal antibody to early exposure to JM virus progeny of JM-V and HVT-vaccinated dams. The progeny possessing JM-V maternal antibody had significantly fewer oncogenic lesions (tumors) than progeny of HVT-vaccinated dams as well as of non-vaccinated dams in the first eight weeks of exposure. DISCUSSION Sevoian et al. (1962), reported that JM-V strain could immunologically stimulate dams to produce neutralizing antibodies which were readily transmitted via embryo to offspring, affording them protection during early life against natural exposure to JM virus. The data herein presented confirms the existence of high levels of neutralizing antibodies in day-old progeny from dams which had been
(7%) (15%) (14%)
vaccinated with JM-V, while little or no neutralizing antibodies were found in the day-old offspring from unvaccinated dams. Upon vaccination with cell-free JMV or JMV-A strains, day-old chicks with low (or no) maternal antibodies showed a marked increase in their serum antibody (both neutralizing and fluorescent antibodies) against JM virus within 3 weeks whereas day-old birds with high maternal antibodies either maintained their titer or slightly declined in the same time period. The decline in titers was more evident in birds vaccinated with cell-free preparation than with JMV-A. These results indicate that high levels of maternal antibodies had an inhibitory influence on the vaccine antigen which in turn restricted antibody response of the host. Day-old chicks (with low or no maternal antibodies) vaccinated with either cell-free JMV or JMV-A strains produced significantly high levels of serum neutralizing antibody in both P- and N-line chicks within 3 weeks postvaccination. This suggests that the JM-V agent is effective in inducing immunity against Type II (Marek's) leukosis. JM-V agent in its virulent form caused some losses in antibody free chicks vaccinated with cell-free inocula prepared from sonicated whole blood of
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Oncogenic lesions-progeny Treatment of progeny P-line N-line exposed8 1/20" (5%) 2731) non-exposed 0/10 0/15 HVT exposed 10/15 (67%) T/2Q non-exposed 0/10 0/20 Non-vaccinated exposed 23/30 (77%) 5730 non-exposed 0/15 0/10 "Naturally exposed to JM virus at day-old to 8 weeks of age. b No. positive/total—8 weeks of age. Vaccination of dams JMV
77
ANTIBODY RESPONSE TO LEUKOSIS
It was observed that most of the chickens vaccinated with JM-V strain showed a decline in antibody titers (both SN and FA) seven weeks after the second vaccination. This suggested that viral antigens from the vaccine disappeared within 3 weeks post-inoculation. All the serum samples that showed positive results in the SN test were also positive in the FA test. Serum titers as determined in the FA test were from 2 to 11 times higher than the titers from the SN test. There appears to be a direct correlation between serum neutralizing antibody and fluorescent antibody titers. Observations made during analysis of the FA results indicate that the best sources of viral antigens for indirect staining are from low passage cell cultures or new virus isolates. In the indirect FA test, using the same serum sample, the low passage antigen showed brilliant fluorescence whereas the high passage antigen (over 20 passages) gave dull or little fluorescence. Churchill and Chubb (1969) reported that the virulent strain of Type II leukosis (Marek's) virus was attenuated upon serial passage in cell culture with the loss of an antigen (A antigen) de-
monstrable in the virulent isolate. This may imply that the fluorescent antibody is related to the A antigen. REFERENCES Calnek, B. W., 1971. Antibody development in chickens exposed to Marek's disease. Symposium on Oncogenesis and Herpes-type Viruses. Christ's College, Cambridge, England. Churchill, A. E., and P. M. Biggs, 1967. Agents of Marek's disease in tissue culture. Nature, 215: 528-530. Churchill, A. E., R. C. Chubb and W. Baxendale, 1969. The attenuation, with loss of oncogenicity of the herpes-type virus of Marek's disease (strain HPRS-16) on passage in cell culture. J. Gen. Virol. 4: 557-564. Cole, R. D., 1968. Studies on genetic resistance to Marek's disease. Avian Dis. 12: 9-28. Hamdy, F. M., and M. Sevoian, 1973. Immunologic relationship between JM and JM-V leukosis strains and herpesvirus of turkeys. Avian Dis. 17: 476-485. Kenyon, A. J., M. Sevoian, M. Horvitz, N. D. Jones and C. F. Helmboldt, 1969. Lymphoproliferative disease of fowl-immunologic factors associated with passage of a lymphoblastic leukemia (JM-V). Avian Dis. 13: 585-595. Mikami, T., and R. A. Bankowski, 1971. The effect of DMSO and cultured behavior of two herpesviruses associated with Marek's disease. Avian Dis. 15: 242-253. Purchase, H. G., and G. H. Burgoyne. 1970. Immunofluorescence in the study of Marek's disease. Detection of antibody. Am. J. Vet. Res. 31: 117-124. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27: 493-495. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. I. Experimental reproduction of the neural and visceral forms. Vet. Med. 57: 500-501. Sevoian, M., 1967. Immunity studies in chickens inoculated with a virulent Type II avian leukosis strain (JM-V). Proc. 39th N.E. Conf. on Avian Disease. State Univ. of New York, Stony Brook, L.I.June 19-21. Shieh, H. K., and M. Sevoian, 1973. Antibody response of susceptible (P-line) and resistant (N-line) chickens to JM and JM-V leukosis strains and herpesvirus of turkeys (HVT). Avian Dis. (In press)
SEPTEMBER 5-11, 1976. FIFTH EUROPEAN POULTRY CONFERENCE, MALTA
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moribund JM-V infected birds. The pathogenicity of JM-V was markedly decreased by serial passage of the JM-V agent in chicken embryos as evidenced by the complete lack of morbidity and mortality of P- and N-line chicks when they were vaccinated with inocula prepared from 18th and 21st chicken embryo passages of JM-V strain. The serial passage of the agent in embryonated chicks modified only the pathogenicity, but not antigenicity, since the chickens vaccinated with this preparation produced neutralizing antibody titers (against JM virus) at least as high or higher than those vaccinated with the virulent cell-free JM-V inocula prepared from blood of moribund birds.