J. Comp. Path. 1992 Vol. 107, 285-294
Effects
of Fowl
Adenovirus Infection System of Chickens M. Saifuddin
on the Immune
and C. R. Wilks
Department of Veterinary Pathology and Public Health, Massey Universip, .New Zealand
Palmerston North,
Summary
A virulent strain of serotype 8 fowl adenovirus (FAV) was isolated from an outbreak of inclusion body hepatitis (IBH) in broiler flocks. Post-mortem changes included characteristic liver lesions with intranuclear inclusion bodies in the hepatocytes and severe lymphocytic depletion in the bursa, thymus and spleen. The packed cell volume was reduced by 50 per cent or more and varying amounts of cell depletion were observed in the bone marrow. Typical IBH was reproduced in specific pathogen-free chickens inoculated orally with the FAV isolated from the natural infection. There was severe depletion of lymphocytes in the bursa, thymus and spleen of the experimentally infected birds and FAV antigens were detected by ELISA and immunocytochemical staining in various lymphoid tissues. Humoral antibody responses against sheep red blood cells, detected by the haemagglutination test, were decreased in the chickens infected with FAV. These findings suggest that the damage caused by replication of this virulent strain of FAV in lymphoid tissues compromises the immunological capabilities of infected chickens.
Introduction Inclusion body hepatitis (IBH) is an acute infectious disease of young chickens normally of 5 to 7 weeks of age (Howell, MacDonald and Christian, 1970; MacPherson, McDougall and Laursen-Jones, 1974). It is caused by several serotypes of Group I avian adenovirus including serotype 8 (McFerran, 198 1) . Natural outbreaks are characterized by a sudden onset of sharply increased flock mortality (10 to 30 per cent), a short clinical course, anaemia and necrotic hepatitis. Although hepatitis with intranuclear inclusion bodies (INIBs) in the hepatocytes is characteristic of IBH (Howell et al., 1970; Pettit and Carlson, 1972)) the involvement of bone marrow and lymphoid organs, particularly the bursa, thymus and spleen, has also been reported by several workers (MacPherson et al., 1974; Fadly, Riegle, Nazerian and Stephens, 1980). Similar lesions in lymphoid organs following experimental infection have also been observed (Hoffmann, Wessling, Dorn and Dangschat, 1975; Grimes, King, Kleven and Fletcher, 1977; Fadly et al., 1980). It has been suggested that concurrent infection with infectious bursal disease (IBD) virus enhances the pathogenicity of adenovirus and that the bursal Address for correspondence: Dr Mohammed Saifuddin, Department of Immunology/Microbiology, Medical School, 1653 West Congress Parkway, Chicago, IL 60612, U.S.A. 0021p9975/92/070285 + 10 $08,00/O
Rush
@ 1992 Academic Press Limited
286
M. Saifuddin
and C. R. Wilks
lesions described in natural outbreaks of IBH may have been due to IBD virus infection (Rosenberger, Klopp, Eckroade and Krauss, 1975; Fadly, Winterfield and Olander, 1976). However, results from both natural outbreaks (MacPherson et al., 1974; Christensen and Saifuddin, 1989) and experimental infections (Grimes et al., 1977; Fadly et al., 1980) suggest that IBD virus, or any other factor, is not necessary for IBH to occur following infection with fowl adenovirus (FAV). Hoffmann et al. ( 1975) suggested that infection with some strains of FAV causes lymphoid depletion of bursa, thymus and spleen which may have an effect on both humoral and cell-mediated immunity. Moreover, New Zealand is free from IBD (Howell, Bagust and Alexander, 1982; Jones, 1986) and the field outbreaks of IBH that occurred in 1987 (Christensen and Saifuddin, 1989) were unrelated to other identifiable immunosuppressive agents (i.e. chicken anaemia agent (CAA) and IBD virus). Lymphoid depletion of the bursa and thymus was apparent on histological examination of tissues obtained from affected chickens in these outbreaks (Christensen and Saifuddin, 1989). The purpose of this study was to investigate the potential of a virulent strain of FAV alone to induce lymphoid depletion in the bursa of Fabricius and other lymphoid organs and thus compromise the immune capabilities of the host. Materials
and Methods
Examination of Birds Naturally Infected with FA V Twenty dead or moribund chickens, from a 3-week-old broiler flock of 15 000 birds with sudden high mortality, were examined. At necropsy, liver, spleen, bursa, thymus, caecal tonsil, gizzard and femur were prepared for histological examination and 10 per cent suspensions of individual tissues were tested for FAV antigens by indirect ELISA (Saifuddin and Wilks, 1990a). A 10 per cent suspension of pooled liver tissue was inoculated onto monolayers of specific pathogen-free (SPF) (tested free of common chicken pathogens including fowl adenoviruses, infectious bursal disease virus and chicken anaemia agent) chicken kidney cells (CKC) for the isolation of FAV and into the Marek’s disease lymphoma (MDCC-MSBl) cell line (National Animal Health Laboratories, Tokyo, Japan) for the isolation of chicken anaemia agent (CAA). Blood samples were collected from the moribund birds to measure packed cell volume (PCV) and FAV neutralizing antibody titres. The virus isolated from the liver suspension was examined by electron microscopy (Philips 201C) and serotyped by neutralization tests with 12 prototype FAV antisera (Saifuddin and Wilks, 1990a).
Examination of Birds Experimentally Infected with FA V Eighteen 3-day-old SPF birds received oral inoculations of 0.25 ml ( IO4 TCID,,) of the FAV which was isolated from liver tissue obtained from naturally infected birds. This FAV had been isolated in primary monolayer cultures of CKC derived from the kidneys of SPF chickens and was cloned by three series of limiting dilutions. At 2, 3,4, 5, 7 and 9 days post-inoculation (p.i.), seven tissues (liver, spleen, bursa, thymus, caecal tonsil, gizzard and bone marrow) were collected from each of three birds which had died or had been killed. Portions of the selected tissues were fixed in neutral buffered 10 per cent formalin for histopathology and immunocytochemistry (Saifuddin, Wilks and Birtles, 1991) and 10 per cent suspensions of these tissues in PBS were tested for viral antigens by indirect ELISA (Saifuddin and Wilks, 1990a). ELISA results were expressed as ELISA specific absorbence (ESA) obtained by subtracting the absorbence with SPF tissue from that obtained with test tissue. The results of
Fowl
Adenovirus
Infection
287
immunocytochemistry were expressed as intense ( + + + ), less intense ( + + ), trace ( + ) and negative ( - ) development of brown to dark brown colour in the cytoplasm and nuclei of mononuclear cells. Humoral Immune Responses in Birds Challenged with FAV
Forty-six SPF birds (3-day-old) were individually marked by wing tags. Blood samples were collected from 10 birds selected at random to serve as the negative control for antibody to sheep red blood cells (SRBC). Each of the 46 SPF birds received an intramuscular (im.) inoculation of 0.05 ml of 10 per cent SRBC in PBS homogenized with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, U.S.A.). The 46 SPF birds were divided into two groups consisting of 20 and 26 birds. The 26 birds in one group each received 0.25 ml ( IO4TCID,,) of the isolated FAV orally and were housed separately in a positive pressure bubble isolator and supplied with autoclaved feed and water. The control group, consisting of 20 birds, was housed separately under similar conditions. All of the birds in the control group and the survivors in the virus inoculated group were bled at 2 weeks p.i. and re-inoculated i.m. with 0.05 ml of 10 per cent SRBC homogenized with incomplete Freund’s adjuvant (Difco Laboratories). After an additional 2 weeks, blood samples were collected from all birds. Serum samples were inactivated at 56°C for 30 min and tested in a haemagglutination (HA) assay using 0.5 per cent SRBC in PBS. Post-mortem examinations were conducted on birds that died and samples of liver, spleen, bursa, thymus, caecal tonsil and bone marrow were collected for histological examination. Haemagglutination
(HA)
Test
The HA tests were carried out in 96-well V-bottom microtitre plates (Linbro, Flow Laboratories, Virginia, U.S.A.). Two-fold dilutions (comprising 1 in 2 to 1 in 128 and 1 in 8 to 1 in 5 12) of 50 pl of each serum sample in PBS plus an equal volume (50 pl) of 0.5 per cent SRBC was added to each well. After a minimum of 1 h of incubation at room temperature (20 to 25°C) the results were recorded. The tests were run in triplicate and the HA titre was expressed as log base 2 of the highest dilution that caused complete agglutination of the added SRBC suspension. Results History of the Natural Infection The course of the disease was rapid: mortality peaked on the third day and remained high for 2 more days and was largely over by day 7. The predominant signs of the disease included reluctance to eat, ruffled feathers, occasionally white pasty droppings, marked weakness, depression and eventual prostration and death. Post-mortem Lesions In naturally infected birds, the liver was pale, swollen and often had multiple petechiae. Multiple haemorrhages were seen in the thymus, breast muscle and intestinal tract. Severe mucosal erosions were observed in the gizzard of a few birds. The bursa of Fabricius and thymus were smaller than in uninfected birds. The spleen was swollen in some birds. The bone marrow was markedly pale and the PCV ranged from 10 to 18 per cent.
288
M. Saifuddin
and C. R. Wilks
In experimentally infected birds, the post-mortem changes in liver, spleen, bursa, thymus, intestinal tract and bone marrow were similar to those in naturally infected birds except that, in some of the severely affected birds, pinpoint white foci were observed on the swollen liver surface and atrophy of the bursa and thymus was more obvious. Neither haemorrhages in the muscles nor mucosal erosions of the gizzard were observed in experimentally infected birds. HistoFathology In naturally infected birds, liver lesions included multiple coalescing foci of vacuolated hepatocytes, necrosis and haemorrhage with eosinophilic INIBs in hepatocytes. A mixed infiltrate of lymphocytes and heterophils was present throughout the liver parenchyma. Varying degrees of cell depletion were observed in bone marrow. Lymphocytic depletion was evident in the bursa, thymus and spleen. The bursa had severely depleted follicles with a relative increase in connective tissues in a few birds. Occasional cysts were present in the medullary areas of the lymphoid follicles and INIBs were in the lining epithelial cells of the bursa. Splenic corpuscles were depleted of lymphoid cells and there was a relative increase in stroma and histiocytes. The cortex of the thymus was depleted of lymphocytes and haemorrhages were present throughout the medulla. Scattered, small focal haemorrhages were present in the lamina propria of the intestine in a few birds. Moderate depletion of lymphocytes was observed in the caecal tonsil. Extensive necrosis, severe haemorrhage and lymphocytic infiltrates were present in the submucosa of the gizzard. In experimental cases, similar liver lesions, including a large number of basophilic (large in size and filling the whole nucleus) and a few eosinophilic (medium sized surrounded by a clear halo) INIBs were observed in the hepatocytes. Changes in other tissues were similar to those in the naturally infected birds except for the gizzard, where no lesions were observed. The lymphocytic depletion in the bursa, spleen and thymus is shown in Figs 1,2 and 3, respectively. Isolation and Identijcation of Virus from Natural& Infected Birds A cytopathic effect (CPE) characterized by rounding of cells was observed by the second passage of liver suspensions inoculated onto CKC monolayers. Typical icosahedral, adenovirus particles (size 70 to 80 nm) were demonstrated in the infected cell culture suspension by electron microscopy. The virus was resistant to chloroform and it was grouped as serotype 8 by virus neutralization (VN) tests. No evidence of the growth of CAA was observed in the MDCCMSBI cell line which was inoculated with the same infected liver suspension after five passages. Variable amounts of FAV antigen were detected by ELISA in all of the tissues collected from naturally infected birds (data not shown). The VN antibody titres to serotype 8 FAV in the sera collected from naturally infected birds were 20 to 80. The antibody titres against other serotypes of FAV were < 10.
Fowl
Fig. 1. Lymphocytic x 6.
Adenovims
289
Infection
depletion in bursa from experimentally
infected chicken at 5 days post-infection.
Fig. 2.
Lymphocytic depletion in splenic corpuscles of spleen from experimentally days post-infection. HE X 12.
Fig. 3.
Lymphocytic HE x6.
Fig. 4.
Viral antigens in the lymphoid tissues of spleen detected by an avidin-biotin technique at 5 days post-infection. Haematoxylin counterstain X 6.
depletion in thymus from experimentally
HE
infected chicken at 5
infected chicken at 4 days post-infection. peroxidase complex
Replication of Virus in Lymphoid Tissues from Experimentally Infected Birds In the caecal tonsil, a high content of viral antigen was detected by ELISA from 2 to 9 days p.i. with a peak (ESA 2.03) at 5 days. Viral antigens were demonstrated in the bursa at 2 to 9 days and spleen at 3 to 7 days p.i. The level of viral antigens in the bursa and spleen peaked at 5 days p.i. In the thymus and bone marrow, the viral antigens were detected from 3 to 5 and 3 to 7 days p.i. with peaks (ESA 0.28 and 0.34, respectively) at 4 days p.i. The viral antigens detected by ELISA in different lymphoid tissues are shown in Table 1. Viral antigens were located by immunocytochemical staining in the lining epithelium of caecal tonsil at 2 to 9 days p.i., epithelial and follicular cells of
290
M. Saifuddin
Detection
of viral
antigens
and C. R. Wilks
Table 1 in lymphoid organs by ELISA after oral inoculation chickens with avian adenovirus
Times e.xamined
of 3-day-old
SPF
Days post-inoculation --
Caecal tonsil Bllrsa Thymus Spleen Bone marrow
?
3
4
5
7
9
1.77* 0.25 0.04 0.03 0.03
1.80 0.34 0.16 0.18 0.17
1.92 0.42 0.28 0.47 0.34
2.03 0.55 0.19 0.69 0.29
1.79 0.30 0.04 0.35 0.13
1.21 0.15 0.03 0.04 0.03
* Mean ELISA specific absorbence (ESA) value of three birds. ESA greater than 0.06 is considered positive. This represents more than three standard deviations above the mean ELISA absorbence value for 22 SPF chicken tissues.
Fig. 5.
Viral antigens in epithelial cells and lymphoid tissue of the caecal tonsil detected by an avidin-biotin peroxidase complex technique at 5 days post-infection. Haematoxylin counterst ain x 40.
Fowl
Adenovirus
291
Infection
the bursa and in the cellular components in the white and red pulp of the spleen from 4 to 5 days p.i. (Fig. 4). Viral antigens were demonstrated in lymphoid aggregates of the caecal tonsil at 4 to 7 days (Fig. 5) and lymphoid aggregates of the thymus at 4 days p.i. A comparison of the amounts of viral antigen detected by ELISA and immunocytochemistry in various lymphoid tissues is illustrated in Table 2. High amounts of viral antigens were detected in the liver of experimentally infected birds by ELISA and immunocytochemistry 3 to 7 days p.i. (data not shown). Viral antigens were not detected in the gizzards. Antibody Response to SRBC Five of the 26 birds that had received an inoculation of virus died between 3 and 7 days p.i. with hepatic lesions and INIBs in the hepatocytes. After 2 weeks pi. the log. mean HA titre of anti-SRBC antibodies was 2.8 (range 0 to 5) in the control group and 1.5 (range 0 to 4) in the virus inoculated group (Table 3). In the control group, 17 of 20 (85 per cent) of the birds had detectable HA antibodies compared with only 13 of 21 (62 per cent) of the virus inoculated group. At 4 weeks following the first inoculation (2 weeks after the booster dose), the log. mean HA titres obtained were 6.5 and 5.3, respectively, in the control and the virus-infected groups of chickens. Antibody to SRBC was not detected by HA test in any of the 10 negative control sera. While there was a trend towards lower antibody titres in the virus inoculated group, statistical analysis did not show this to be significant (P>O*O2). Discussion The effects of both natural and experimental infection of chickens with FAV have been demonstrated by ELISA, immunocytochemistry and histology, on various lymphoid organs in this study. Natural IBH infection was associated with high mortality, characteristic hepatic changes, INIBs and depletion of lymphocytes in the bursa, thymus and spleen. The bursa and thymus were grossly atrophied, the bone marrow had
Comparison tissues
T&us examined Caecal tonsil Bllrsa Thymus SplK3l Bone marrow
of viral following
antigens detected oral administration
by
Table 2 ELISA and by immunocytochemistry in various of avian adenovirus to J-day-old SPF chickens
Mean peak ESA (dayof peak) 2.03 0.55 0.28 0.69 0.34
(5) (5) (4) (5) (4)
Immunocytochemist~v (intensity of colour) ++ ++ + +++ NT
ESA=ELISA specific absorbence (value > 0.06 considered positive); + + + = intense dark brown many cells; + + = moderately intense staining; + =faint staining in few cells; NT= not tested.
stain in
292
M. Saifuddin
Humoral inoculated
and C. R. Wilks
Table 3 SPF chickens antibody response to sheep RBC (SRBC) in J-day-old intramuscularly with SRBC at the same time as oral administration adenovirus
that were of avian
Birds infected with IBH virus (weeks post-inoculation)
Uninfected birds (weks post-inoculation) 2
I
2’ 4 2 5 3 4 0 3 4 3 2 4 0 2 4 2 3 0 3 3
6 7
LMT = 2.8
LMT = 6.5
8 7 6 5 7 8 7 6 6 5 6 8 6 6 5 7 7
4 2 3 2 0 3 2 4 0 0 3 2 2 0 2 0 0 3 0 3 0 2 LMT= 1.5
6 5 5 5 6 4 7 6 z 6 5 3 4 5 6 4 5 7 6 LMT = 5.3
* Reciprocal of the highest dilution ofserum which completely agglutinated 50 ~1 of 0.5 per cent sheep RBC and expressed as log. base 2 titre. LMT = Log. mean titre.
reduced cell mass and the PCV was low. Variable amounts of FAV antigens were detected by indirect ELISA (Saifuddin and Wilks, 1990a) in all lymphoid tissues examined and in liver. Serotype 8 FAV was isolated from the liver, but there was no evidence of CAA in the same tissue. Recently, severe outbreaks of IBH, causing up to 30 per cent mortality in several broiler flocks in New Zealand, associated with lymphocytic depletion in the bursa and thymus have been reported (Christensen and Saifuddin, 1989). These outbreaks were not associated with IBD virus infection or any other identifiable immunosuppressive factors (Christensen and Saifuddin, 1989). The serotype 8 FAV isolated from the infected liver tissue of birds in these outbreaks was found to be highly pathogenic for conventionally raised as well as for SPF chickens (Saifuddin and Wilks, 1990b). The severity of the clinical signs observed in any particular outbreak of an infectious disease, such as IBH, will depend on the pathogenicity of the infecting strain of virus as well as the presence of other agents or factors which may lower the individual’s ability to control the infection. It has been demonstrated that the mortality rate was dramatically increased (up to 40 per cent) in some broiler chicken flocks in Australia when IBH was associated with diseases such as coccidiosis, colibacillosis, paratyphoid or respiratory disease
Fowl
Adenovirus
Infection
293
complex (Reece, Barr, Grix, Forsyth, Condron and Hindmarsh, 1986). The importance of strain variation has been emphasized by Rivas and Fabricant (1988), who suggested that there is a direct relationship between Marek’s disease virus strains of higher pathogenicity and more profound immunodepression. Although the presence of bursal changes in many outbreaks of IBH has been claimed to be due to IBD virus infection (Rosenberger et al., 1975; Fadly et al., 1976)) there are several reports of the involvement of lymphoid organs including bursa, thymus, spleen and caecal tonsil in naturally occurring IBH infections without any evidence of IBD or any other immunosuppressive factors (MacPherson et al., 1974; Pettit and Carlson, 1972; Itakura, Yasuba and Goto, 1974). SPF birds infected with the FAV isolated from the naturally infected birds in this study showed severe lymphocytic depletion in the bursa, thymus and spleen in addition to typical hepatic lesions with a large number of INIBs. Significant amounts of viral antigens were detected by ELISA in all of the lymphoid organs including bursa, thymus, spleen, caecal tonsil and bone marrow. Viral antigens were localized by immunocytochemistry in lymphocytes in the white and red pulp of the spleen, in the lymphoid follicles of the bursa, in caecal tonsil and in the cortical thymocytes. Our contention that FAV was the cause of the compromised immune system is supported by the fact that this FAV had been isolated in CKC cultures derived from SPF birds shown to be free of IBD virus and CAA and further had been cloned three times using the limiting dilution method before inoculation. In addition to the morphological and antigenic evidence of viral involvement and damage to lymphoid tissues, the results of this study also indicate that birds infected with a pathogenic strain of FAV have a decreased ability to mount an antibody response to unrelated antigens. In the IBH infected group, the mean HA titres were lower than in the non-infected control group both at 2 and 4 weeks p.i. Thus the findings from this study support the hypothesis that a virulent strain of FAV alone does compromise the immune capacity of the host. Acknowledgments
We express our gratitude to Dr D. A. Barr, Veterinary Research Institute, Victoria, and Dr J. B. McFerran, Veterinary Research Laboratories, Belfast, for supplying the reference strains of fowl adenoviruses, and Dr J. C. Murphy, Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, U.S.A. for his constructive criticism in the preparation of this manuscript. References
Christensen, N. H. and Saifuddin, Md. (1989). A primary epidemic of inclusion body hepatitis in broilers. Avian Diseases, 33, 622-630. Fadly, A. M., Riegle, B. J., Nazerian, K. and Stephens, E. A. (1980). Some observations on an adenovirus isolated from specific pathogen free chickens. Poultry Science, 59, 2 1-27.
Fadly, A. M., Winterfield, R. W. and Olander, H. J. (1976). Role of bursa of Fabricius in the pathogenicity of inclusion body hepatitis and infectious bursal disease viruses. Avian Diseases, 20, 467477.
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and C. R. Wilks
Grimes, T. M., King, D. J., Kleven, S. H. and Fletcher, 0. J. (1977). Involvement of a type-8 avian adenovirus in the etiology of inclusion body hepatitis. Avian Diseases, 21, 26-38. Hoffmann, R., Wessling, E., Dorn, P. and Dangschat, H. (1975). Lesions in chickens with spontaneous or experimental infectious hepato-myelopoietic disease (inclusion body hepatitis) in Germany. Avian Diseases, 19, 224-236. Howell, J., MacDonald, D. W. and Christian, R. G. (1970). Inclusion body hepatitis in chickens. Canadian Veterinary Journal, 11, 99-10 1. Howell, L. J., Bagust, T. J. and Alexander, A. M. (1982). Serological investigations of infectious bursal disease and reticuloendotheliosis virus in New Zealand chickens. New Zealand Veterinary Journal, 30, 128. Itakura, C., Yasuba, M. and Goto, M. (1974). Histopathological studies on inclusion body hepatitis in broiler chickens. Japanese Journal of Veterinary Science, 36, 329-340. Jones, B. A. H. (1986). Infectious bursal disease serology in New Zealand poultry flocks. New Zealand Veterinary Journal, 34, 36. MacPherson, I., McDougall, J. S. and Laursen-Jones, A. P. (1974). Inclusion body hepatitis in a broiler integration. Veterinary Record, 95, 286-289. McFerran, J. B. (1981). Adenoviruses of vertebrate animals. In Comparative diagnosis of viral diseases, Vol. III. E. Kurstak and C. Kurstak, Eds, Academic Press, New York, pp. 101-165. Pettit, J. R. and Carlson, H. C. (1972). Inclusion body hepatitis in broiler chickens. Avian Diseases, 16, 858-863. Reece, R. L., Barr, D. A., Grix, D. C., Forsyth, W. M., Condron, R. J. and Hindmarsh, M. (1986). Observations on naturally occurring inclusion body hepatitis in Victorian chickens. Australian Veterinary Journal, 63, 201-202. Rivas, A. L. and Fabricant, J. (1988). Indications of immunodepression in chickens infected with various strains of Marek’s disease virus. Avian Diseases, 32, 1-8. Rosenberger, J. K., Klopp, S., Eckroade, R. J. and Krauss, W. C. (1975). The role of the infectious bursal agent and several avian adenoviruses in the hemorrhagic-aplastic-anemia syndrome and gangrenous dermatitis. Avian Diseases, 19, 7 17-729. Saifuddin, Md. and Wilks, C. R. (1990a). Development of an enzyme-linked immunosorbent assay to detect and quantify adenovirus in chicken tissues. Avian Diseases, 34, 239-245. Saifuddin, Md. and Wilks, C. R. (1990b). Reproduction of inclusion body hepatitis in conventionally raised chickens inoculated with a New Zealand isolate of avian adenovirus. New Zealand Veterinary Journal, 38, 62-65. Saifuddin, Md., Wilks, C. R. and Birtles, M. J. (1991). Development of an immunocytochemical procedure to detect adenoviral antigens in chicken tissues. Journal of Veterinary Diagnostic Investigation, 3, 3 13-3 18. Received, March 30th, 1992 Accepted, June 19th, 1992
1
Effects
of Fowl
Adenovirus Infection System of Chickens M. Saifuddin
on the Immune
and C. R. Wilks
Department of Veterinary Pathology and Public Health, Massey Universip, .New Zealand
Palmerston North,
Summary
A virulent strain of serotype 8 fowl adenovirus (FAV) was isolated from an outbreak of inclusion body hepatitis (IBH) in broiler flocks. Post-mortem changes included characteristic liver lesions with intranuclear inclusion bodies in the hepatocytes and severe lymphocytic depletion in the bursa, thymus and spleen. The packed cell volume was reduced by 50 per cent or more and varying amounts of cell depletion were observed in the bone marrow. Typical IBH was reproduced in specific pathogen-free chickens inoculated orally with the FAV isolated from the natural infection. There was severe depletion of lymphocytes in the bursa, thymus and spleen of the experimentally infected birds and FAV antigens were detected by ELISA and immunocytochemical staining in various lymphoid tissues. Humoral antibody responses against sheep red blood cells, detected by the haemagglutination test, were decreased in the chickens infected with FAV. These findings suggest that the damage caused by replication of this virulent strain of FAV in lymphoid tissues compromises the immunological capabilities of infected chickens.
Introduction Inclusion body hepatitis (IBH) is an acute infectious disease of young chickens normally of 5 to 7 weeks of age (Howell, MacDonald and Christian, 1970; MacPherson, McDougall and Laursen-Jones, 1974). It is caused by several serotypes of Group I avian adenovirus including serotype 8 (McFerran, 198 1) . Natural outbreaks are characterized by a sudden onset of sharply increased flock mortality (10 to 30 per cent), a short clinical course, anaemia and necrotic hepatitis. Although hepatitis with intranuclear inclusion bodies (INIBs) in the hepatocytes is characteristic of IBH (Howell et al., 1970; Pettit and Carlson, 1972)) the involvement of bone marrow and lymphoid organs, particularly the bursa, thymus and spleen, has also been reported by several workers (MacPherson et al., 1974; Fadly, Riegle, Nazerian and Stephens, 1980). Similar lesions in lymphoid organs following experimental infection have also been observed (Hoffmann, Wessling, Dorn and Dangschat, 1975; Grimes, King, Kleven and Fletcher, 1977; Fadly et al., 1980). It has been suggested that concurrent infection with infectious bursal disease (IBD) virus enhances the pathogenicity of adenovirus and that the bursal Address for correspondence: Dr Mohammed Saifuddin, Department of Immunology/Microbiology, Medical School, 1653 West Congress Parkway, Chicago, IL 60612, U.S.A. 0021p9975/92/070285 + 10 $08,00/O
Rush
@ 1992 Academic Press Limited
286
M. Saifuddin
and C. R. Wilks
lesions described in natural outbreaks of IBH may have been due to IBD virus infection (Rosenberger, Klopp, Eckroade and Krauss, 1975; Fadly, Winterfield and Olander, 1976). However, results from both natural outbreaks (MacPherson et al., 1974; Christensen and Saifuddin, 1989) and experimental infections (Grimes et al., 1977; Fadly et al., 1980) suggest that IBD virus, or any other factor, is not necessary for IBH to occur following infection with fowl adenovirus (FAV). Hoffmann et al. ( 1975) suggested that infection with some strains of FAV causes lymphoid depletion of bursa, thymus and spleen which may have an effect on both humoral and cell-mediated immunity. Moreover, New Zealand is free from IBD (Howell, Bagust and Alexander, 1982; Jones, 1986) and the field outbreaks of IBH that occurred in 1987 (Christensen and Saifuddin, 1989) were unrelated to other identifiable immunosuppressive agents (i.e. chicken anaemia agent (CAA) and IBD virus). Lymphoid depletion of the bursa and thymus was apparent on histological examination of tissues obtained from affected chickens in these outbreaks (Christensen and Saifuddin, 1989). The purpose of this study was to investigate the potential of a virulent strain of FAV alone to induce lymphoid depletion in the bursa of Fabricius and other lymphoid organs and thus compromise the immune capabilities of the host. Materials
and Methods
Examination of Birds Naturally Infected with FA V Twenty dead or moribund chickens, from a 3-week-old broiler flock of 15 000 birds with sudden high mortality, were examined. At necropsy, liver, spleen, bursa, thymus, caecal tonsil, gizzard and femur were prepared for histological examination and 10 per cent suspensions of individual tissues were tested for FAV antigens by indirect ELISA (Saifuddin and Wilks, 1990a). A 10 per cent suspension of pooled liver tissue was inoculated onto monolayers of specific pathogen-free (SPF) (tested free of common chicken pathogens including fowl adenoviruses, infectious bursal disease virus and chicken anaemia agent) chicken kidney cells (CKC) for the isolation of FAV and into the Marek’s disease lymphoma (MDCC-MSBl) cell line (National Animal Health Laboratories, Tokyo, Japan) for the isolation of chicken anaemia agent (CAA). Blood samples were collected from the moribund birds to measure packed cell volume (PCV) and FAV neutralizing antibody titres. The virus isolated from the liver suspension was examined by electron microscopy (Philips 201C) and serotyped by neutralization tests with 12 prototype FAV antisera (Saifuddin and Wilks, 1990a).
Examination of Birds Experimentally Infected with FA V Eighteen 3-day-old SPF birds received oral inoculations of 0.25 ml ( IO4 TCID,,) of the FAV which was isolated from liver tissue obtained from naturally infected birds. This FAV had been isolated in primary monolayer cultures of CKC derived from the kidneys of SPF chickens and was cloned by three series of limiting dilutions. At 2, 3,4, 5, 7 and 9 days post-inoculation (p.i.), seven tissues (liver, spleen, bursa, thymus, caecal tonsil, gizzard and bone marrow) were collected from each of three birds which had died or had been killed. Portions of the selected tissues were fixed in neutral buffered 10 per cent formalin for histopathology and immunocytochemistry (Saifuddin, Wilks and Birtles, 1991) and 10 per cent suspensions of these tissues in PBS were tested for viral antigens by indirect ELISA (Saifuddin and Wilks, 1990a). ELISA results were expressed as ELISA specific absorbence (ESA) obtained by subtracting the absorbence with SPF tissue from that obtained with test tissue. The results of
Fowl
Adenovirus
Infection
287
immunocytochemistry were expressed as intense ( + + + ), less intense ( + + ), trace ( + ) and negative ( - ) development of brown to dark brown colour in the cytoplasm and nuclei of mononuclear cells. Humoral Immune Responses in Birds Challenged with FAV
Forty-six SPF birds (3-day-old) were individually marked by wing tags. Blood samples were collected from 10 birds selected at random to serve as the negative control for antibody to sheep red blood cells (SRBC). Each of the 46 SPF birds received an intramuscular (im.) inoculation of 0.05 ml of 10 per cent SRBC in PBS homogenized with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, U.S.A.). The 46 SPF birds were divided into two groups consisting of 20 and 26 birds. The 26 birds in one group each received 0.25 ml ( IO4TCID,,) of the isolated FAV orally and were housed separately in a positive pressure bubble isolator and supplied with autoclaved feed and water. The control group, consisting of 20 birds, was housed separately under similar conditions. All of the birds in the control group and the survivors in the virus inoculated group were bled at 2 weeks p.i. and re-inoculated i.m. with 0.05 ml of 10 per cent SRBC homogenized with incomplete Freund’s adjuvant (Difco Laboratories). After an additional 2 weeks, blood samples were collected from all birds. Serum samples were inactivated at 56°C for 30 min and tested in a haemagglutination (HA) assay using 0.5 per cent SRBC in PBS. Post-mortem examinations were conducted on birds that died and samples of liver, spleen, bursa, thymus, caecal tonsil and bone marrow were collected for histological examination. Haemagglutination
(HA)
Test
The HA tests were carried out in 96-well V-bottom microtitre plates (Linbro, Flow Laboratories, Virginia, U.S.A.). Two-fold dilutions (comprising 1 in 2 to 1 in 128 and 1 in 8 to 1 in 5 12) of 50 pl of each serum sample in PBS plus an equal volume (50 pl) of 0.5 per cent SRBC was added to each well. After a minimum of 1 h of incubation at room temperature (20 to 25°C) the results were recorded. The tests were run in triplicate and the HA titre was expressed as log base 2 of the highest dilution that caused complete agglutination of the added SRBC suspension. Results History of the Natural Infection The course of the disease was rapid: mortality peaked on the third day and remained high for 2 more days and was largely over by day 7. The predominant signs of the disease included reluctance to eat, ruffled feathers, occasionally white pasty droppings, marked weakness, depression and eventual prostration and death. Post-mortem Lesions In naturally infected birds, the liver was pale, swollen and often had multiple petechiae. Multiple haemorrhages were seen in the thymus, breast muscle and intestinal tract. Severe mucosal erosions were observed in the gizzard of a few birds. The bursa of Fabricius and thymus were smaller than in uninfected birds. The spleen was swollen in some birds. The bone marrow was markedly pale and the PCV ranged from 10 to 18 per cent.
288
M. Saifuddin
and C. R. Wilks
In experimentally infected birds, the post-mortem changes in liver, spleen, bursa, thymus, intestinal tract and bone marrow were similar to those in naturally infected birds except that, in some of the severely affected birds, pinpoint white foci were observed on the swollen liver surface and atrophy of the bursa and thymus was more obvious. Neither haemorrhages in the muscles nor mucosal erosions of the gizzard were observed in experimentally infected birds. HistoFathology In naturally infected birds, liver lesions included multiple coalescing foci of vacuolated hepatocytes, necrosis and haemorrhage with eosinophilic INIBs in hepatocytes. A mixed infiltrate of lymphocytes and heterophils was present throughout the liver parenchyma. Varying degrees of cell depletion were observed in bone marrow. Lymphocytic depletion was evident in the bursa, thymus and spleen. The bursa had severely depleted follicles with a relative increase in connective tissues in a few birds. Occasional cysts were present in the medullary areas of the lymphoid follicles and INIBs were in the lining epithelial cells of the bursa. Splenic corpuscles were depleted of lymphoid cells and there was a relative increase in stroma and histiocytes. The cortex of the thymus was depleted of lymphocytes and haemorrhages were present throughout the medulla. Scattered, small focal haemorrhages were present in the lamina propria of the intestine in a few birds. Moderate depletion of lymphocytes was observed in the caecal tonsil. Extensive necrosis, severe haemorrhage and lymphocytic infiltrates were present in the submucosa of the gizzard. In experimental cases, similar liver lesions, including a large number of basophilic (large in size and filling the whole nucleus) and a few eosinophilic (medium sized surrounded by a clear halo) INIBs were observed in the hepatocytes. Changes in other tissues were similar to those in the naturally infected birds except for the gizzard, where no lesions were observed. The lymphocytic depletion in the bursa, spleen and thymus is shown in Figs 1,2 and 3, respectively. Isolation and Identijcation of Virus from Natural& Infected Birds A cytopathic effect (CPE) characterized by rounding of cells was observed by the second passage of liver suspensions inoculated onto CKC monolayers. Typical icosahedral, adenovirus particles (size 70 to 80 nm) were demonstrated in the infected cell culture suspension by electron microscopy. The virus was resistant to chloroform and it was grouped as serotype 8 by virus neutralization (VN) tests. No evidence of the growth of CAA was observed in the MDCCMSBI cell line which was inoculated with the same infected liver suspension after five passages. Variable amounts of FAV antigen were detected by ELISA in all of the tissues collected from naturally infected birds (data not shown). The VN antibody titres to serotype 8 FAV in the sera collected from naturally infected birds were 20 to 80. The antibody titres against other serotypes of FAV were < 10.
Fowl
Fig. 1. Lymphocytic x 6.
Adenovims
289
Infection
depletion in bursa from experimentally
infected chicken at 5 days post-infection.
Fig. 2.
Lymphocytic depletion in splenic corpuscles of spleen from experimentally days post-infection. HE X 12.
Fig. 3.
Lymphocytic HE x6.
Fig. 4.
Viral antigens in the lymphoid tissues of spleen detected by an avidin-biotin technique at 5 days post-infection. Haematoxylin counterstain X 6.
depletion in thymus from experimentally
HE
infected chicken at 5
infected chicken at 4 days post-infection. peroxidase complex
Replication of Virus in Lymphoid Tissues from Experimentally Infected Birds In the caecal tonsil, a high content of viral antigen was detected by ELISA from 2 to 9 days p.i. with a peak (ESA 2.03) at 5 days. Viral antigens were demonstrated in the bursa at 2 to 9 days and spleen at 3 to 7 days p.i. The level of viral antigens in the bursa and spleen peaked at 5 days p.i. In the thymus and bone marrow, the viral antigens were detected from 3 to 5 and 3 to 7 days p.i. with peaks (ESA 0.28 and 0.34, respectively) at 4 days p.i. The viral antigens detected by ELISA in different lymphoid tissues are shown in Table 1. Viral antigens were located by immunocytochemical staining in the lining epithelium of caecal tonsil at 2 to 9 days p.i., epithelial and follicular cells of
290
M. Saifuddin
Detection
of viral
antigens
and C. R. Wilks
Table 1 in lymphoid organs by ELISA after oral inoculation chickens with avian adenovirus
Times e.xamined
of 3-day-old
SPF
Days post-inoculation --
Caecal tonsil Bllrsa Thymus Spleen Bone marrow
?
3
4
5
7
9
1.77* 0.25 0.04 0.03 0.03
1.80 0.34 0.16 0.18 0.17
1.92 0.42 0.28 0.47 0.34
2.03 0.55 0.19 0.69 0.29
1.79 0.30 0.04 0.35 0.13
1.21 0.15 0.03 0.04 0.03
* Mean ELISA specific absorbence (ESA) value of three birds. ESA greater than 0.06 is considered positive. This represents more than three standard deviations above the mean ELISA absorbence value for 22 SPF chicken tissues.
Fig. 5.
Viral antigens in epithelial cells and lymphoid tissue of the caecal tonsil detected by an avidin-biotin peroxidase complex technique at 5 days post-infection. Haematoxylin counterst ain x 40.
Fowl
Adenovirus
291
Infection
the bursa and in the cellular components in the white and red pulp of the spleen from 4 to 5 days p.i. (Fig. 4). Viral antigens were demonstrated in lymphoid aggregates of the caecal tonsil at 4 to 7 days (Fig. 5) and lymphoid aggregates of the thymus at 4 days p.i. A comparison of the amounts of viral antigen detected by ELISA and immunocytochemistry in various lymphoid tissues is illustrated in Table 2. High amounts of viral antigens were detected in the liver of experimentally infected birds by ELISA and immunocytochemistry 3 to 7 days p.i. (data not shown). Viral antigens were not detected in the gizzards. Antibody Response to SRBC Five of the 26 birds that had received an inoculation of virus died between 3 and 7 days p.i. with hepatic lesions and INIBs in the hepatocytes. After 2 weeks pi. the log. mean HA titre of anti-SRBC antibodies was 2.8 (range 0 to 5) in the control group and 1.5 (range 0 to 4) in the virus inoculated group (Table 3). In the control group, 17 of 20 (85 per cent) of the birds had detectable HA antibodies compared with only 13 of 21 (62 per cent) of the virus inoculated group. At 4 weeks following the first inoculation (2 weeks after the booster dose), the log. mean HA titres obtained were 6.5 and 5.3, respectively, in the control and the virus-infected groups of chickens. Antibody to SRBC was not detected by HA test in any of the 10 negative control sera. While there was a trend towards lower antibody titres in the virus inoculated group, statistical analysis did not show this to be significant (P>O*O2). Discussion The effects of both natural and experimental infection of chickens with FAV have been demonstrated by ELISA, immunocytochemistry and histology, on various lymphoid organs in this study. Natural IBH infection was associated with high mortality, characteristic hepatic changes, INIBs and depletion of lymphocytes in the bursa, thymus and spleen. The bursa and thymus were grossly atrophied, the bone marrow had
Comparison tissues
T&us examined Caecal tonsil Bllrsa Thymus SplK3l Bone marrow
of viral following
antigens detected oral administration
by
Table 2 ELISA and by immunocytochemistry in various of avian adenovirus to J-day-old SPF chickens
Mean peak ESA (dayof peak) 2.03 0.55 0.28 0.69 0.34
(5) (5) (4) (5) (4)
Immunocytochemist~v (intensity of colour) ++ ++ + +++ NT
ESA=ELISA specific absorbence (value > 0.06 considered positive); + + + = intense dark brown many cells; + + = moderately intense staining; + =faint staining in few cells; NT= not tested.
stain in
292
M. Saifuddin
Humoral inoculated
and C. R. Wilks
Table 3 SPF chickens antibody response to sheep RBC (SRBC) in J-day-old intramuscularly with SRBC at the same time as oral administration adenovirus
that were of avian
Birds infected with IBH virus (weeks post-inoculation)
Uninfected birds (weks post-inoculation) 2
I
2’ 4 2 5 3 4 0 3 4 3 2 4 0 2 4 2 3 0 3 3
6 7
LMT = 2.8
LMT = 6.5
8 7 6 5 7 8 7 6 6 5 6 8 6 6 5 7 7
4 2 3 2 0 3 2 4 0 0 3 2 2 0 2 0 0 3 0 3 0 2 LMT= 1.5
6 5 5 5 6 4 7 6 z 6 5 3 4 5 6 4 5 7 6 LMT = 5.3
* Reciprocal of the highest dilution ofserum which completely agglutinated 50 ~1 of 0.5 per cent sheep RBC and expressed as log. base 2 titre. LMT = Log. mean titre.
reduced cell mass and the PCV was low. Variable amounts of FAV antigens were detected by indirect ELISA (Saifuddin and Wilks, 1990a) in all lymphoid tissues examined and in liver. Serotype 8 FAV was isolated from the liver, but there was no evidence of CAA in the same tissue. Recently, severe outbreaks of IBH, causing up to 30 per cent mortality in several broiler flocks in New Zealand, associated with lymphocytic depletion in the bursa and thymus have been reported (Christensen and Saifuddin, 1989). These outbreaks were not associated with IBD virus infection or any other identifiable immunosuppressive factors (Christensen and Saifuddin, 1989). The serotype 8 FAV isolated from the infected liver tissue of birds in these outbreaks was found to be highly pathogenic for conventionally raised as well as for SPF chickens (Saifuddin and Wilks, 1990b). The severity of the clinical signs observed in any particular outbreak of an infectious disease, such as IBH, will depend on the pathogenicity of the infecting strain of virus as well as the presence of other agents or factors which may lower the individual’s ability to control the infection. It has been demonstrated that the mortality rate was dramatically increased (up to 40 per cent) in some broiler chicken flocks in Australia when IBH was associated with diseases such as coccidiosis, colibacillosis, paratyphoid or respiratory disease
Fowl
Adenovirus
Infection
293
complex (Reece, Barr, Grix, Forsyth, Condron and Hindmarsh, 1986). The importance of strain variation has been emphasized by Rivas and Fabricant (1988), who suggested that there is a direct relationship between Marek’s disease virus strains of higher pathogenicity and more profound immunodepression. Although the presence of bursal changes in many outbreaks of IBH has been claimed to be due to IBD virus infection (Rosenberger et al., 1975; Fadly et al., 1976)) there are several reports of the involvement of lymphoid organs including bursa, thymus, spleen and caecal tonsil in naturally occurring IBH infections without any evidence of IBD or any other immunosuppressive factors (MacPherson et al., 1974; Pettit and Carlson, 1972; Itakura, Yasuba and Goto, 1974). SPF birds infected with the FAV isolated from the naturally infected birds in this study showed severe lymphocytic depletion in the bursa, thymus and spleen in addition to typical hepatic lesions with a large number of INIBs. Significant amounts of viral antigens were detected by ELISA in all of the lymphoid organs including bursa, thymus, spleen, caecal tonsil and bone marrow. Viral antigens were localized by immunocytochemistry in lymphocytes in the white and red pulp of the spleen, in the lymphoid follicles of the bursa, in caecal tonsil and in the cortical thymocytes. Our contention that FAV was the cause of the compromised immune system is supported by the fact that this FAV had been isolated in CKC cultures derived from SPF birds shown to be free of IBD virus and CAA and further had been cloned three times using the limiting dilution method before inoculation. In addition to the morphological and antigenic evidence of viral involvement and damage to lymphoid tissues, the results of this study also indicate that birds infected with a pathogenic strain of FAV have a decreased ability to mount an antibody response to unrelated antigens. In the IBH infected group, the mean HA titres were lower than in the non-infected control group both at 2 and 4 weeks p.i. Thus the findings from this study support the hypothesis that a virulent strain of FAV alone does compromise the immune capacity of the host. Acknowledgments
We express our gratitude to Dr D. A. Barr, Veterinary Research Institute, Victoria, and Dr J. B. McFerran, Veterinary Research Laboratories, Belfast, for supplying the reference strains of fowl adenoviruses, and Dr J. C. Murphy, Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, U.S.A. for his constructive criticism in the preparation of this manuscript. References
Christensen, N. H. and Saifuddin, Md. (1989). A primary epidemic of inclusion body hepatitis in broilers. Avian Diseases, 33, 622-630. Fadly, A. M., Riegle, B. J., Nazerian, K. and Stephens, E. A. (1980). Some observations on an adenovirus isolated from specific pathogen free chickens. Poultry Science, 59, 2 1-27.
Fadly, A. M., Winterfield, R. W. and Olander, H. J. (1976). Role of bursa of Fabricius in the pathogenicity of inclusion body hepatitis and infectious bursal disease viruses. Avian Diseases, 20, 467477.
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and C. R. Wilks
Grimes, T. M., King, D. J., Kleven, S. H. and Fletcher, 0. J. (1977). Involvement of a type-8 avian adenovirus in the etiology of inclusion body hepatitis. Avian Diseases, 21, 26-38. Hoffmann, R., Wessling, E., Dorn, P. and Dangschat, H. (1975). Lesions in chickens with spontaneous or experimental infectious hepato-myelopoietic disease (inclusion body hepatitis) in Germany. Avian Diseases, 19, 224-236. Howell, J., MacDonald, D. W. and Christian, R. G. (1970). Inclusion body hepatitis in chickens. Canadian Veterinary Journal, 11, 99-10 1. Howell, L. J., Bagust, T. J. and Alexander, A. M. (1982). Serological investigations of infectious bursal disease and reticuloendotheliosis virus in New Zealand chickens. New Zealand Veterinary Journal, 30, 128. Itakura, C., Yasuba, M. and Goto, M. (1974). Histopathological studies on inclusion body hepatitis in broiler chickens. Japanese Journal of Veterinary Science, 36, 329-340. Jones, B. A. H. (1986). Infectious bursal disease serology in New Zealand poultry flocks. New Zealand Veterinary Journal, 34, 36. MacPherson, I., McDougall, J. S. and Laursen-Jones, A. P. (1974). Inclusion body hepatitis in a broiler integration. Veterinary Record, 95, 286-289. McFerran, J. B. (1981). Adenoviruses of vertebrate animals. In Comparative diagnosis of viral diseases, Vol. III. E. Kurstak and C. Kurstak, Eds, Academic Press, New York, pp. 101-165. Pettit, J. R. and Carlson, H. C. (1972). Inclusion body hepatitis in broiler chickens. Avian Diseases, 16, 858-863. Reece, R. L., Barr, D. A., Grix, D. C., Forsyth, W. M., Condron, R. J. and Hindmarsh, M. (1986). Observations on naturally occurring inclusion body hepatitis in Victorian chickens. Australian Veterinary Journal, 63, 201-202. Rivas, A. L. and Fabricant, J. (1988). Indications of immunodepression in chickens infected with various strains of Marek’s disease virus. Avian Diseases, 32, 1-8. Rosenberger, J. K., Klopp, S., Eckroade, R. J. and Krauss, W. C. (1975). The role of the infectious bursal agent and several avian adenoviruses in the hemorrhagic-aplastic-anemia syndrome and gangrenous dermatitis. Avian Diseases, 19, 7 17-729. Saifuddin, Md. and Wilks, C. R. (1990a). Development of an enzyme-linked immunosorbent assay to detect and quantify adenovirus in chicken tissues. Avian Diseases, 34, 239-245. Saifuddin, Md. and Wilks, C. R. (1990b). Reproduction of inclusion body hepatitis in conventionally raised chickens inoculated with a New Zealand isolate of avian adenovirus. New Zealand Veterinary Journal, 38, 62-65. Saifuddin, Md., Wilks, C. R. and Birtles, M. J. (1991). Development of an immunocytochemical procedure to detect adenoviral antigens in chicken tissues. Journal of Veterinary Diagnostic Investigation, 3, 3 13-3 18. Received, March 30th, 1992 Accepted, June 19th, 1992
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