Immunization of Chickens Against Adenovirus Infection R. W. WINTERFIELD, A. M. FADLY and F. J. HOERR School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907 (Received for publication January 7, 1977)
Poultry Science 56:1481-1486,1977 INTRODUCTION
The physical, chemical and biological characteristics of avian adenoviruses have been described in numerous publications and the latter have been reviewed extensively by Aghakhan (1974). Yates et al. (1954) first reported an infection of chickens by a virus now recognized as a member of the adenovirus genus. Subsequently, other investigators have studied the disease-inciting potential of certain adenovirus isolants. Berry (1969), Cook (1972), Gallina^ al. (1973) and Winterfield et al. (1973) described .the potential effects of infection on commercial layers. The cause of inclusion body hepatitis and aplastic anemia was found to be an adenovirus by Fadly and Winterfield (1973), Winterfield and Fadly (1973), Rosenberger et al. (1974) and Stein and Wills (1974). Some isolants were found to be pathogenic under experimental conditions whereas others proved relatively innocuous. Since little information was available on the immunity stimulated in chickens by adenovirus infection, or vaccination, this study was undertaken. The data are reported herein. MATERIALS AND METHODS
Viruses. Three different serotypes of adenoviruses, types 1, 2 and 3, were used as vaccines. Serological classification of these isolants was based on Calnek and Cowen's work (1975). Indiana C and Phelps-strain viruses represented
type 1 adenovirus (Winterfield et al., 1973; Yates et al, 1954). The Gal and Stein isolants were type 2 and Tipton represented type 3 (Sharpless and Jungherr, 1961; Stein, 1974; Fadly and Winterfield, 1973). Each was attenuated in virulence for chickens by chicken embryo passages. Where used in immunity challenge procedures, the unattenuated viruses were employed as inocula. Chickens and Embryos. Broiler-type chickens were vaccinated in each trial. High levels of VN antibodies were present to all serotypes at 1 day of age but were at insignificant levels 3 to 4 weeks later. All chickens were free of Mycoplasma gallisepticum and Mycoplasma synoviae as assessed serologically. Specific-pathogen-free (SPAFAS®) chicken embryos, devoid of detectable VN antibodies to adenovirus types 1, 2 and 3, were inoculated with the respective viruses at 6 t o 10 days of age. All virus propagation, VN tests and virus isolation attempts were done in embryos from the same source. Propagation of Viruses, Virus Neutralization and Titration. The Tipton and Stein isolants were grown in the yolk-sac of 6- or 7-day-old embryos as described previously (Fadly, 1973). Indiana C and Phelps CELO were propagated in the chorioallantoic sac of 9- to 11-day-old embryos (Winterfield et al., 1973). Harvested infective yolk and chorioallantoic fluids were stored in one-ml portions in screw-cap vials at —65 C. The respective virus harvests and pools
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ABSTRACT Chickens were vaccinated with monovalent and polyvalent adenovirus vaccines using three different serotypes, types 1, 2 and 3. No cross-protection was elicited'by the heterologous serotypes but satisfactory protection from lesions and the shedding of challenge virus was induced by the homologous serotypes in the monovalent and polyvalent vaccines. The virus neutralizing (VN) titers were occasionally lower where the polyvalent vaccines were used when compared to those from chickens given the monovalent vaccines. This suggested possible interference though protection did exist against the appearance of lesions. Where chickens were vaccinated at an early age and in the presence of congenital homologous VN antibodies, protection against the effects of infection was satisfactory whereas the VN antibody titers were low or insignificant by the assay methods employed. The vaccine virus was not shed beyond 28 days after vaccination. No physical effects of vaccination were noted in any of the chickens during the postvaccination period.
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R. W. WINTERFIELD, A. M. FADLY AND F. J. HOERR
nated chickens. Six or 7 days after challenge, all birds were necropsied and examined for the presence of gross evidence of liver lesions, or hepatitis. One or more focal areas of liver necrosis served as indicators of the effects of challenge. In addition, in one trial, swabs were taken from punctures of the seared liver of individual chickens and placed in 3 ml of nutrient broth containing antibiotics. Virus isolation (VI) attempts were made from these suspensions of the liver tissue to determine the presence or •absence of infection in individual chickens. Virus Persistence and Shed from Vaccinated Chickens. Groups of 4-week-old White Leghorns (SPAFAS©) were inoculated by eyedrop with the Indiana C, Tipton and Stein viruses and maintained in separate isolation rooms. Swabs were taken from the palatine clefts, tracheas, and cloacas at 7, 20, and 28 days postinoculation. At the 45th day the remaining birds were necropsied, examined for lesions and the livers were punctured with swabs. VI attempts were made from the collected material.
RESULTS
Table 1 demonstrates, in 3 trials, the protection of chickens against hepatic lesions of infection when they were vaccinated by the eyedrop route at 4 weeks of age. In trial 1, it is observed that GAL vaccine virus (serotype 2) prevented lesions from challenge with a homol-
TABLE 1 .—Protection of chickens against lesions of adenovirus infection when vaccinated by the eyedrop route with monovalent vaccines at 4 weeks of age. Homologous and heterologous challenge virus given 4 weeks postvaccination Gross lesions1 Trial and age of vaccination
Challenge virus Vaccine applied
Stein
Tipton
Indiana C
Trial 1 4 weeks
GAL virus (Type 2) Tipton virus (Type 3) Nonvaccinated
0/7 6/8 7/8
2/8 0/7 4/8
2
Trial 2 4 weeks
Indiana C virus (Type 1) Nonvaccinated
6/9 5/9
5/9 3/9
Trial 3 3 weeks
CELO-Phelps virus (Type 1) Nonvaccinated
5/12 3/9
1 2
Individuals with gross lesions/number necropsied. Blank space means not done.
0/11 2/4
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were titrated prior to vaccination and immunity challenge. Titrations were done with ten-fold dilutions of virus suspensions in nutrient broth. Neutralization tests were conducted with serums inactivated for 30 minutes in a water bath at 56 C. Each virus dilution was mixed 1:1 with serum and incubated for 30 minutes at room temperature. In titrations and VN tests, 5 embryos were inoculated per dilution, using 0.1 ml. per embryo. They were held 7 to 10 days postinoculation. Deaths that occurred within 24 hours postinoculation were regarded as nonspecific. Other deaths were recorded and the titer calculated according to the method of Reed and Muench (1938). Logjo VN indices were determined from the serum assays. Vaccination and Immunity Challenge. Each isolant used as a vaccine was diluted in nutrient broth to 10 4 embryo lethal dose 5 0 (ELD S 0 ) per ml. It was given by the eyedrop route, 0.02 ml. per chicken. Where a polyvalent vaccine was given, the respective isolants were mixed in equal proportions and then administered in a similar manner. Vaccinated and nonvaccjnated birds were kept in adjoining isolation rooms. Challenge virus was given 3 to 4 weeks after vaccination by the subcutaneous route in the dorsal part of the neck. A dosage of 0.2 ml. was injected with a tuberculin syringe and a 22 gage, 1 1/2 inch needle. This quantity contained between 10 4 and 10 5 E L D 5 0 of virus. Each challenge isolant was given separately to different groups of vaccinated and nonvacci-
ADENOVIRUS INFECTION
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TABLE 2.—Protection of chickens against adenovirus infection and lesions when vaccinated by the eyedrop route with monovalent and polyvalent vaccines at 4 weeks of age. Challenge virus given 4 weeks postvaccination
Vaccinated group
Indiana C
1 Indiana C virus (Type 1) 2 Stein virus (Type 2) 3 Tipton virus (Type 3) 4 Polyvalent vaccine 5 Nonvaccinated
0/12
Gross lesions1
Virus isolation2
Challenge virus
Challenge virus
Stein
Tipton
3
Indiana C
0/6 3/4
Tipton
0/5
0/11 0/5 1/4
Stein
0/5 0/11 0/6 5/5
0/5 3/4
0/5 3/4
0/5 0/5 3/5
Individuals with gross lesions/number necropsied. Individuals positive for virus/number examined. 3 Blank space means not done.
2
ogous serotype, the Stein isolant. However the same vaccine failed to protect against a serotype 3, heterologous Tipton virus. Similarly, the Tipton vaccinated birds were protected against homologous virus challenge but not against heterologous Stein virus. In trial 2, Indiana C vaccine virus, serotype 1, failed to protect against serotypes 2 and 3—the Stein and Tipton viruses. In trial 3, it was noted the CELO-Phelps vaccine virus (serotype 1) protected against the homologous serotype, Indiana C virus, but not against heterologous, the type 2 Stein. Thus no cross-protection was
demonstrated between heterologous vaccine and challenge adenovirus serotypes. The protection stimulated against infection and lesions when chickens were vaccinated by the eyedrop route with monovalent and polyvalent vaccines is given in Table 2. They were vaccinated at 4 weeks of age with challenge done 4 weeks later. It can be seen that Indiana C, Stein and Tipton viruses, types 1, 2 and 3 respectively, induced good protection from the development of lesions. In addition, protection from infection was in evidence since the challenge virus could not be recovered from the
TABLE 3.—Virus neutralizing serum titers of chickens vaccinated with monovalent and polyvalent adenovirus vaccines Log, 0 neutralization indices1 Vaccinated group and vaccine applied 1 Indiana C virus (Type 1)
Indicator viruses Indiana C
Stein
3=3.0, >3.0, >3.0 »=3.0, >3.0
2
Tipton
»3.6, »3.7, >3.7 3.9,3.9
2 Stein virus (Type 2) 3 Tipton virus (Type 3)
S*3.0, >3.0, >3.0 3.0, 3.0
4 Polyvalent vaccine
>3.0, 3*3.0, >3.0 S=3.0, >3.0
2.4, 2.7, 2.9 2.9, 3.8
1.5, >3.0, >3.0 >3.0, >3.0
5 Nonvaccinated
0,0,0 0.1,0.1
0.7,0.9, 1.02 1.5, 1.9
0,0,0 0.3,0.5
1 2
Neutralization index of each individual tested within the respective group. Blank space means not done.
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TABLE 4.—Protection of chickens against adenovirus lesions when vaccinated by the eyedrop route with monovalent and polyvalent vaccines at 4 weeks of age. Challenge virus given 4 weeks postvaccination Gross lesions1 Challenge virus
Vaccinated group and vaccine applied 1 2 3 4 5
Indiana C
Indiana C virus (Type 1) Stein virus (Type 2) Tipton virus (Type 3) Polyvalent vaccine Nonvaccinated
Stein
Tipton
0/11 0/6
Individuals with gross lesions/numbers necropsied. Blank space means not done.
livers of vaccinated chickens. Both t h e m o n o valent and polyvalent vaccines induced p r o t e c tion against a p p a r e n t infection as well as lesions of disease. Virus-serum neutralizing titers ( V N logi 0 ) from r a n d o m l y selected chickens in t h e vaccin a t e d a n d nonvaccinated groups listed in Table 2 are given in Table 3. Where t h e vaccines were given as m o n o v a l e n t preparations, in g r o u p s 1, 2 and 3, t h e neutralization indices were equal t o , or exceeded, 3.0. Likewise, t h e polyvalent vaccine induced significant VN t i t e r s b u t s o m e interference had possibly t a k e n place since t h e a n t i b o d y levels t o t h e Stein virus were lower t h a n where t h e m o n o v a l e n t Stein vaccine was given separately in g r o u p 2.
Table 4 indicates t h e p r o t e c t i o n of chickens in a trial where 4-week-old chickens were vaccinated with t h e m o n o v a l e n t and polyvalent vaccines. However, n o virus isolation a t t e m p t s were m a d e with challenge 4 weeks later. T h e criteria for p r o t e c t i o n was based solely o n t h e presence or absence of gross lesions. Again it is n o t e d t h a t g o o d i m m u n i t y was o b t a i n e d with b o t h monovalent and polyvalent vaccines. T h e virus neutralizing titers of individual birds in t h e previous trial are given in Table 5. While t h e neutralization indices were n o t as high as in a parallel e x p e r i m e n t (Table 3), t h e y were significant when c o m p a r e d t o t h e unvaccinated chickens. A t e n d e n c y t o w a r d s lower titers was again present w h e n t h e polyvalent
TABLE 5.—Virus neutralizing serum titers of chickens vaccinated with monovalent and polyvalent adenovirus vaccine Log, 0 neutralization indices1 Vaccinated group and vaccine applied 1
Indiana C virus (Type 1)
Indicator viruses Indiana C
Tipton virus (Type 3)
4
Polyvalent vaccine
5
Nonvaccinated
Tipton
>3.0, >3.2, »3.2 »3.0
2 Stein virus (Type 2) 3
Stein
2.3, 2.7,2.8,2.8 2.3,2.3,3.7 »4.3,»5.0 2.0, 2.0, 2.0 2.8, 3.2
2.2, 2.3, 2.5 2.7, 2.8
1.3,1.3,1.3 2.3,2.3
0.3,0.3,0.5
0.1,0.2,0.5
1.4, 1.4,1.4
1
Neutralization index of each individual tested within the respective group of chickens.
2
Blank space means not done.
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1 2
0/16 0/6 2/3
0/6 3/3
0/5 3/4
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ADENOVIRUS INFECTION
Vaccine virus shed in the postvaccination period at 7, 20, 28 and 45 days was studied with each of the serotype isolants, Indiana C, Tipton and Stein viruses. Seven days after vaccination with Indiana C, 5 of 5 chickens tested were positive on virus isolation attempts but were negative at 20, 28 and 45 days. With the Tipton virus, 2 of 5 were positive at 7 days but were negative at 20, 28 and 45 days. Following vaccination with Stein virus, 3 of 5 were positive at 7 days and 2 of 5 were positive at 20 days; all were negative at 28 and 45 days after vaccination. The isolation attempts were from cloacal, palatine cleft, tracheal and liver swabs, as previously mentioned.
DISCUSSION The results of these trials demonstrate the lack of cross protection among different adenovirus isolants of heterologous serotypes. This is in contrast to the observations made by this author, and others, and reported in numerous publications concerning the cross immunity existing among certain differing serotypes of infectious bronchitis virus. Therefore, the use of a polyvalent adenovirus vaccine may be indicated and dependent on the virus challenge present under field conditions. While the VN titers may be lower, in some instances, with use of a polyvalent vaccine, the protection stimulated appears satisfactory. Similarly, in the presence of congenital, or passive, antibodies at an early age, the serological response can be low. However, the protection
TABLE 6.—Protection of chickens against lesions of adenovirus infection when vaccinated by the eyedrop route with a polyvalent vaccine at 1 day of age in the presence of parentallyconferred immunity. Challenge virus given 4 weeks postvaccination Gross lesions' Challenge virus applied
Indiana C
Stein
Tipton
Polyvalent vaccine Nonvaccinated
0/15 3/15
0/15 5/15
0/15 3/15
1
Individuals with gross lesions/number necropsied.
against lesions and clinical disease may be satisfactory. This should not be regarded as unusual (Hilleman and Flatley, 1958). It is possible that if the experimental chickens had been on litter, or floor-reared, instead of wire, re-exposure to the excreted vaccine virus over an extended period of time would have resulted in higher VN titers. Avian adenoviruses are usually shed in the feces and are very stable under adverse conditions. Adenovirus vaccination of replacement breeder pullets may, at times, be needed since the progeny from immune dams are highly resistant to the effects of infection from pathogenic adenovirus (Fadly and Winterfield, 1973; Winterfield and Fadly, 1973). An age resistance factor apparently also exists (Fadly and Winterfield, 1973; Winterfield et at., 1973) to the effects of certain adenovirus infections and was confirmed in these experiments. Older, susceptible chickens had a considerably lower incidence of heptatic lesions than younger ones. Therefore, challenge procedures with adenoviruses may well include parallel virus isolation attempts from the birds to afford a more critical and sensitive means of evaluating the immune status. The Indiana C, Tipton and Stein viruses, employed as vaccines in these experiments, have not proven oncogenic (Fadly et al., 1976) and the viruses are shed for only a short period of time. Furthermore, no detrimental physical effects of the vaccine viruses were observed in the various experimental groups in this investigation. Field observations by the authors have also confirmed the safety of these attenuated vaccines when used in susceptible commercial flocks.
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vaccine was administered, suggesting that interference had taken place. The polyvalent vaccine was also given to 1-day-old chickens by the eyedrop route in the presence of parentally-conferred neutralizing antibodies (>2.00) to each serotype. Data from this trial are given in Table 6. Challenge with the respective viruses revealed that adequate protection against liver lesions was produced. At this time, VN titers were determined from the serums of 5 individuals in the vaccinated and nonvaccinated groups. None of the chickens had titers >2.0, whether the chickens were vaccinated or not, thus demonstrating the poor serological evidence of immunity from vaccination at an early age and in the presence of passive VN antibodies but in contrast to protection against the incitement of lesions from challenge.
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R. W. WINTERFIELD, A. M. FADLY AND F. J. HOERR REFERENCES
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Aghakhan, S. M., 1974. Avian adenoviruses. The Veterinary Bulletin, 9:531-552. Berry, D. M., 1959. Egg production and disease: adenovirus. Vet. Record. 84:397-398. Calnek, B. W., and B. S. Cowen, 1975. Adenoviruses of chickens: serologic groups. Avian Dis. 19:91-103. Cook, J. K. A., 1972. Avian adenovirus alone or followed by infectious bronchitis virus in laying hens. J. Comp. Pathol. 32:119-128. Fadly, A. M., and R. W. Winterfield, 1973. Isolation and some characteristics of an agent associated with inclusion body hepatitis, hemorrhages, and a p l a s t i c a n e m i a in chickens. Avian Dis. 17:182-193. Fadly, A. M., R. W. Winterfield and H. J. Olander, 1976. The oncogenic potential of some avian adenoviruses causing diseases in chickens. Avian Dis. 20:139-145. Gallina, A. M., R. W. Winterfield and A. M. Fadly, 1973. Adenovirus infection and disease. Avian Dis. 17:343-353. Hilleman, M. R., F. J. Flatley, S. A. Anderson, M. L. Luecking and D. J. Levinson, 1958. Antibody
response in volunteers to adenovirus vaccine and correlation of antibody and immunity, 1958. J. Immunol. 80:299-307. Reed, L. J., and H. Muench, 1938. A simple method of estimating 50 percent endpoints. Am. J. Hyg. 27:493^-97. Sharpless, G. R., and E. L. Jungherr, 1961. Characterization of 2 viruses obtained from lymphomatous liver. Amer. J. Vet. Res. 90:937-943. Stein, G., and C. R. Wills, 1974. Isolation and identification of infectious anemia virus. Abstract. 111th Annual Meeting of the Am. Vet. Med. Assoc, Denver, Colorado. Winterfield, R. W„ A. M. Fadly and A. M. Gallina, 1973. Adenovirus infection and disease. I. Some characteristics of an isolant from chickens in Indiana. Avian Dis. 17:334-342. Winterfield, R. W., and A. M. Fadly, 1973. Anemiainclusion body hepatitis syndrome: recognition and etiology. Proceedings 5th Internat. Cong. World's Vet. Poultry Assoc, Munich, West Germany: 111. Yates, V. J., D. E. Fry and B. Wasserman, 1954. A preliminary report on an apparendy new virus disease of chickens. In: Proc. of 26th Ann. Meeting Northeastern Conf. Lab. Workers in Pullorum Control.
Poultry Science 56:1481-1486,1977 INTRODUCTION
The physical, chemical and biological characteristics of avian adenoviruses have been described in numerous publications and the latter have been reviewed extensively by Aghakhan (1974). Yates et al. (1954) first reported an infection of chickens by a virus now recognized as a member of the adenovirus genus. Subsequently, other investigators have studied the disease-inciting potential of certain adenovirus isolants. Berry (1969), Cook (1972), Gallina^ al. (1973) and Winterfield et al. (1973) described .the potential effects of infection on commercial layers. The cause of inclusion body hepatitis and aplastic anemia was found to be an adenovirus by Fadly and Winterfield (1973), Winterfield and Fadly (1973), Rosenberger et al. (1974) and Stein and Wills (1974). Some isolants were found to be pathogenic under experimental conditions whereas others proved relatively innocuous. Since little information was available on the immunity stimulated in chickens by adenovirus infection, or vaccination, this study was undertaken. The data are reported herein. MATERIALS AND METHODS
Viruses. Three different serotypes of adenoviruses, types 1, 2 and 3, were used as vaccines. Serological classification of these isolants was based on Calnek and Cowen's work (1975). Indiana C and Phelps-strain viruses represented
type 1 adenovirus (Winterfield et al., 1973; Yates et al, 1954). The Gal and Stein isolants were type 2 and Tipton represented type 3 (Sharpless and Jungherr, 1961; Stein, 1974; Fadly and Winterfield, 1973). Each was attenuated in virulence for chickens by chicken embryo passages. Where used in immunity challenge procedures, the unattenuated viruses were employed as inocula. Chickens and Embryos. Broiler-type chickens were vaccinated in each trial. High levels of VN antibodies were present to all serotypes at 1 day of age but were at insignificant levels 3 to 4 weeks later. All chickens were free of Mycoplasma gallisepticum and Mycoplasma synoviae as assessed serologically. Specific-pathogen-free (SPAFAS®) chicken embryos, devoid of detectable VN antibodies to adenovirus types 1, 2 and 3, were inoculated with the respective viruses at 6 t o 10 days of age. All virus propagation, VN tests and virus isolation attempts were done in embryos from the same source. Propagation of Viruses, Virus Neutralization and Titration. The Tipton and Stein isolants were grown in the yolk-sac of 6- or 7-day-old embryos as described previously (Fadly, 1973). Indiana C and Phelps CELO were propagated in the chorioallantoic sac of 9- to 11-day-old embryos (Winterfield et al., 1973). Harvested infective yolk and chorioallantoic fluids were stored in one-ml portions in screw-cap vials at —65 C. The respective virus harvests and pools
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ABSTRACT Chickens were vaccinated with monovalent and polyvalent adenovirus vaccines using three different serotypes, types 1, 2 and 3. No cross-protection was elicited'by the heterologous serotypes but satisfactory protection from lesions and the shedding of challenge virus was induced by the homologous serotypes in the monovalent and polyvalent vaccines. The virus neutralizing (VN) titers were occasionally lower where the polyvalent vaccines were used when compared to those from chickens given the monovalent vaccines. This suggested possible interference though protection did exist against the appearance of lesions. Where chickens were vaccinated at an early age and in the presence of congenital homologous VN antibodies, protection against the effects of infection was satisfactory whereas the VN antibody titers were low or insignificant by the assay methods employed. The vaccine virus was not shed beyond 28 days after vaccination. No physical effects of vaccination were noted in any of the chickens during the postvaccination period.
1482
R. W. WINTERFIELD, A. M. FADLY AND F. J. HOERR
nated chickens. Six or 7 days after challenge, all birds were necropsied and examined for the presence of gross evidence of liver lesions, or hepatitis. One or more focal areas of liver necrosis served as indicators of the effects of challenge. In addition, in one trial, swabs were taken from punctures of the seared liver of individual chickens and placed in 3 ml of nutrient broth containing antibiotics. Virus isolation (VI) attempts were made from these suspensions of the liver tissue to determine the presence or •absence of infection in individual chickens. Virus Persistence and Shed from Vaccinated Chickens. Groups of 4-week-old White Leghorns (SPAFAS©) were inoculated by eyedrop with the Indiana C, Tipton and Stein viruses and maintained in separate isolation rooms. Swabs were taken from the palatine clefts, tracheas, and cloacas at 7, 20, and 28 days postinoculation. At the 45th day the remaining birds were necropsied, examined for lesions and the livers were punctured with swabs. VI attempts were made from the collected material.
RESULTS
Table 1 demonstrates, in 3 trials, the protection of chickens against hepatic lesions of infection when they were vaccinated by the eyedrop route at 4 weeks of age. In trial 1, it is observed that GAL vaccine virus (serotype 2) prevented lesions from challenge with a homol-
TABLE 1 .—Protection of chickens against lesions of adenovirus infection when vaccinated by the eyedrop route with monovalent vaccines at 4 weeks of age. Homologous and heterologous challenge virus given 4 weeks postvaccination Gross lesions1 Trial and age of vaccination
Challenge virus Vaccine applied
Stein
Tipton
Indiana C
Trial 1 4 weeks
GAL virus (Type 2) Tipton virus (Type 3) Nonvaccinated
0/7 6/8 7/8
2/8 0/7 4/8
2
Trial 2 4 weeks
Indiana C virus (Type 1) Nonvaccinated
6/9 5/9
5/9 3/9
Trial 3 3 weeks
CELO-Phelps virus (Type 1) Nonvaccinated
5/12 3/9
1 2
Individuals with gross lesions/number necropsied. Blank space means not done.
0/11 2/4
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were titrated prior to vaccination and immunity challenge. Titrations were done with ten-fold dilutions of virus suspensions in nutrient broth. Neutralization tests were conducted with serums inactivated for 30 minutes in a water bath at 56 C. Each virus dilution was mixed 1:1 with serum and incubated for 30 minutes at room temperature. In titrations and VN tests, 5 embryos were inoculated per dilution, using 0.1 ml. per embryo. They were held 7 to 10 days postinoculation. Deaths that occurred within 24 hours postinoculation were regarded as nonspecific. Other deaths were recorded and the titer calculated according to the method of Reed and Muench (1938). Logjo VN indices were determined from the serum assays. Vaccination and Immunity Challenge. Each isolant used as a vaccine was diluted in nutrient broth to 10 4 embryo lethal dose 5 0 (ELD S 0 ) per ml. It was given by the eyedrop route, 0.02 ml. per chicken. Where a polyvalent vaccine was given, the respective isolants were mixed in equal proportions and then administered in a similar manner. Vaccinated and nonvaccjnated birds were kept in adjoining isolation rooms. Challenge virus was given 3 to 4 weeks after vaccination by the subcutaneous route in the dorsal part of the neck. A dosage of 0.2 ml. was injected with a tuberculin syringe and a 22 gage, 1 1/2 inch needle. This quantity contained between 10 4 and 10 5 E L D 5 0 of virus. Each challenge isolant was given separately to different groups of vaccinated and nonvacci-
ADENOVIRUS INFECTION
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TABLE 2.—Protection of chickens against adenovirus infection and lesions when vaccinated by the eyedrop route with monovalent and polyvalent vaccines at 4 weeks of age. Challenge virus given 4 weeks postvaccination
Vaccinated group
Indiana C
1 Indiana C virus (Type 1) 2 Stein virus (Type 2) 3 Tipton virus (Type 3) 4 Polyvalent vaccine 5 Nonvaccinated
0/12
Gross lesions1
Virus isolation2
Challenge virus
Challenge virus
Stein
Tipton
3
Indiana C
0/6 3/4
Tipton
0/5
0/11 0/5 1/4
Stein
0/5 0/11 0/6 5/5
0/5 3/4
0/5 3/4
0/5 0/5 3/5
Individuals with gross lesions/number necropsied. Individuals positive for virus/number examined. 3 Blank space means not done.
2
ogous serotype, the Stein isolant. However the same vaccine failed to protect against a serotype 3, heterologous Tipton virus. Similarly, the Tipton vaccinated birds were protected against homologous virus challenge but not against heterologous Stein virus. In trial 2, Indiana C vaccine virus, serotype 1, failed to protect against serotypes 2 and 3—the Stein and Tipton viruses. In trial 3, it was noted the CELO-Phelps vaccine virus (serotype 1) protected against the homologous serotype, Indiana C virus, but not against heterologous, the type 2 Stein. Thus no cross-protection was
demonstrated between heterologous vaccine and challenge adenovirus serotypes. The protection stimulated against infection and lesions when chickens were vaccinated by the eyedrop route with monovalent and polyvalent vaccines is given in Table 2. They were vaccinated at 4 weeks of age with challenge done 4 weeks later. It can be seen that Indiana C, Stein and Tipton viruses, types 1, 2 and 3 respectively, induced good protection from the development of lesions. In addition, protection from infection was in evidence since the challenge virus could not be recovered from the
TABLE 3.—Virus neutralizing serum titers of chickens vaccinated with monovalent and polyvalent adenovirus vaccines Log, 0 neutralization indices1 Vaccinated group and vaccine applied 1 Indiana C virus (Type 1)
Indicator viruses Indiana C
Stein
3=3.0, >3.0, >3.0 »=3.0, >3.0
2
Tipton
»3.6, »3.7, >3.7 3.9,3.9
2 Stein virus (Type 2) 3 Tipton virus (Type 3)
S*3.0, >3.0, >3.0 3.0, 3.0
4 Polyvalent vaccine
>3.0, 3*3.0, >3.0 S=3.0, >3.0
2.4, 2.7, 2.9 2.9, 3.8
1.5, >3.0, >3.0 >3.0, >3.0
5 Nonvaccinated
0,0,0 0.1,0.1
0.7,0.9, 1.02 1.5, 1.9
0,0,0 0.3,0.5
1 2
Neutralization index of each individual tested within the respective group. Blank space means not done.
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R. W. WINTERFIELD, A. M. FADLY AND F. J. HOERR
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TABLE 4.—Protection of chickens against adenovirus lesions when vaccinated by the eyedrop route with monovalent and polyvalent vaccines at 4 weeks of age. Challenge virus given 4 weeks postvaccination Gross lesions1 Challenge virus
Vaccinated group and vaccine applied 1 2 3 4 5
Indiana C
Indiana C virus (Type 1) Stein virus (Type 2) Tipton virus (Type 3) Polyvalent vaccine Nonvaccinated
Stein
Tipton
0/11 0/6
Individuals with gross lesions/numbers necropsied. Blank space means not done.
livers of vaccinated chickens. Both t h e m o n o valent and polyvalent vaccines induced p r o t e c tion against a p p a r e n t infection as well as lesions of disease. Virus-serum neutralizing titers ( V N logi 0 ) from r a n d o m l y selected chickens in t h e vaccin a t e d a n d nonvaccinated groups listed in Table 2 are given in Table 3. Where t h e vaccines were given as m o n o v a l e n t preparations, in g r o u p s 1, 2 and 3, t h e neutralization indices were equal t o , or exceeded, 3.0. Likewise, t h e polyvalent vaccine induced significant VN t i t e r s b u t s o m e interference had possibly t a k e n place since t h e a n t i b o d y levels t o t h e Stein virus were lower t h a n where t h e m o n o v a l e n t Stein vaccine was given separately in g r o u p 2.
Table 4 indicates t h e p r o t e c t i o n of chickens in a trial where 4-week-old chickens were vaccinated with t h e m o n o v a l e n t and polyvalent vaccines. However, n o virus isolation a t t e m p t s were m a d e with challenge 4 weeks later. T h e criteria for p r o t e c t i o n was based solely o n t h e presence or absence of gross lesions. Again it is n o t e d t h a t g o o d i m m u n i t y was o b t a i n e d with b o t h monovalent and polyvalent vaccines. T h e virus neutralizing titers of individual birds in t h e previous trial are given in Table 5. While t h e neutralization indices were n o t as high as in a parallel e x p e r i m e n t (Table 3), t h e y were significant when c o m p a r e d t o t h e unvaccinated chickens. A t e n d e n c y t o w a r d s lower titers was again present w h e n t h e polyvalent
TABLE 5.—Virus neutralizing serum titers of chickens vaccinated with monovalent and polyvalent adenovirus vaccine Log, 0 neutralization indices1 Vaccinated group and vaccine applied 1
Indiana C virus (Type 1)
Indicator viruses Indiana C
Tipton virus (Type 3)
4
Polyvalent vaccine
5
Nonvaccinated
Tipton
>3.0, >3.2, »3.2 »3.0
2 Stein virus (Type 2) 3
Stein
2.3, 2.7,2.8,2.8 2.3,2.3,3.7 »4.3,»5.0 2.0, 2.0, 2.0 2.8, 3.2
2.2, 2.3, 2.5 2.7, 2.8
1.3,1.3,1.3 2.3,2.3
0.3,0.3,0.5
0.1,0.2,0.5
1.4, 1.4,1.4
1
Neutralization index of each individual tested within the respective group of chickens.
2
Blank space means not done.
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1 2
0/16 0/6 2/3
0/6 3/3
0/5 3/4
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ADENOVIRUS INFECTION
Vaccine virus shed in the postvaccination period at 7, 20, 28 and 45 days was studied with each of the serotype isolants, Indiana C, Tipton and Stein viruses. Seven days after vaccination with Indiana C, 5 of 5 chickens tested were positive on virus isolation attempts but were negative at 20, 28 and 45 days. With the Tipton virus, 2 of 5 were positive at 7 days but were negative at 20, 28 and 45 days. Following vaccination with Stein virus, 3 of 5 were positive at 7 days and 2 of 5 were positive at 20 days; all were negative at 28 and 45 days after vaccination. The isolation attempts were from cloacal, palatine cleft, tracheal and liver swabs, as previously mentioned.
DISCUSSION The results of these trials demonstrate the lack of cross protection among different adenovirus isolants of heterologous serotypes. This is in contrast to the observations made by this author, and others, and reported in numerous publications concerning the cross immunity existing among certain differing serotypes of infectious bronchitis virus. Therefore, the use of a polyvalent adenovirus vaccine may be indicated and dependent on the virus challenge present under field conditions. While the VN titers may be lower, in some instances, with use of a polyvalent vaccine, the protection stimulated appears satisfactory. Similarly, in the presence of congenital, or passive, antibodies at an early age, the serological response can be low. However, the protection
TABLE 6.—Protection of chickens against lesions of adenovirus infection when vaccinated by the eyedrop route with a polyvalent vaccine at 1 day of age in the presence of parentallyconferred immunity. Challenge virus given 4 weeks postvaccination Gross lesions' Challenge virus applied
Indiana C
Stein
Tipton
Polyvalent vaccine Nonvaccinated
0/15 3/15
0/15 5/15
0/15 3/15
1
Individuals with gross lesions/number necropsied.
against lesions and clinical disease may be satisfactory. This should not be regarded as unusual (Hilleman and Flatley, 1958). It is possible that if the experimental chickens had been on litter, or floor-reared, instead of wire, re-exposure to the excreted vaccine virus over an extended period of time would have resulted in higher VN titers. Avian adenoviruses are usually shed in the feces and are very stable under adverse conditions. Adenovirus vaccination of replacement breeder pullets may, at times, be needed since the progeny from immune dams are highly resistant to the effects of infection from pathogenic adenovirus (Fadly and Winterfield, 1973; Winterfield and Fadly, 1973). An age resistance factor apparently also exists (Fadly and Winterfield, 1973; Winterfield et at., 1973) to the effects of certain adenovirus infections and was confirmed in these experiments. Older, susceptible chickens had a considerably lower incidence of heptatic lesions than younger ones. Therefore, challenge procedures with adenoviruses may well include parallel virus isolation attempts from the birds to afford a more critical and sensitive means of evaluating the immune status. The Indiana C, Tipton and Stein viruses, employed as vaccines in these experiments, have not proven oncogenic (Fadly et al., 1976) and the viruses are shed for only a short period of time. Furthermore, no detrimental physical effects of the vaccine viruses were observed in the various experimental groups in this investigation. Field observations by the authors have also confirmed the safety of these attenuated vaccines when used in susceptible commercial flocks.
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vaccine was administered, suggesting that interference had taken place. The polyvalent vaccine was also given to 1-day-old chickens by the eyedrop route in the presence of parentally-conferred neutralizing antibodies (>2.00) to each serotype. Data from this trial are given in Table 6. Challenge with the respective viruses revealed that adequate protection against liver lesions was produced. At this time, VN titers were determined from the serums of 5 individuals in the vaccinated and nonvaccinated groups. None of the chickens had titers >2.0, whether the chickens were vaccinated or not, thus demonstrating the poor serological evidence of immunity from vaccination at an early age and in the presence of passive VN antibodies but in contrast to protection against the incitement of lesions from challenge.
1486
R. W. WINTERFIELD, A. M. FADLY AND F. J. HOERR REFERENCES
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