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Pathogenicity of Fowl Adenovirus in Specific Pathogen Free Chicken Embryos
J. Comp. Path. 2012, Vol. 146, 223e229
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EXPERIMENTALLY INDUCED DISEASE
Pathogenicity of Fowl Adenovirus in Specific Pathogen Free Chicken Embryos W. Alemnesh*, M. Hair-Bejo†, I. Aini† and A. R. Omar† * Faculty of Veterinary Medicine, Gondar University, PO Box 196 Gondar, Ethiopia and † Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Summary Inclusion body hepatitis (IBH) associated with fowl adenovirus (FAdV) infection has a worldwide distribution. The aim of the present study was to determine the pathogenicity of Malaysian FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken embryos. FAdV (titre 105.8/ml) was inoculated into SPF embryonated chicken eggs (0.1 ml per egg) via the chorioallantoic membrane (CAM). There was 100% embryo mortality within 4e11 days post infection (dpi). The gross and microscopical lesions of the embryo were confined to the liver and were noted at 5, 7, 9 and 11 dpi. The liver was pale with multifocal areas of necrosis, fibrosis and haemorrhage. Microscopically, there was moderate to severe congestion and haemorrhage and severe and diffuse hepatocyte degeneration and necrosis, with intranuclear inclusion bodies (INIBs) and associated inflammation. Haemorrhage, congestion, degeneration, necrosis and hyperplasia of the CAM with INIBs were observed at 5, 7, 9 and 11 dpi. Varying degrees of congestion, haemorrhage, degeneration and necrosis were also observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius. Ultrastructurally, numerous viral particles in the nucleus of hepatocytes were recorded at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous INIBs were observed. The INIBs in the CAM were present either as fine granular filamentous structures or as large viral inclusions. FAdV (UPM04217) is therefore highly pathogenic to SPF chicken embryos and the embryonic liver should be used for isolation and propagation of the virus. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: chicken embryo; fowl adenovirus; pathogenicity
Introduction Inclusion body hepatitis (IBH) is an acute disease, mainly occurring in young broiler chickens (3e7 weeks of age) and caused by several serotypes of fowl adenovirus (FAdV; McFerran and Adair, 2003). FAdVs that infect mostly broiler chickens are classified in the genus Aviadenovirus under the family Adenoviridae, and are further classified into five species and 12 serotypes (Benko et al., 2000). Some of the FAdVs are ubiquitous in chickens, as demonstrated by serological surveys (Yates et al., 1976; Grimes et al., 1977a) and the high isolation rates of FAdVs from specimens taken from normal and sick birds (McFerran et al., 1971; Cowen et al., 1978). In contrast, certain serotypes of FAdVs have been more often associated with naturally occurring outbreaks of IBH and hydropericardium synCorrespondence to: M. Hair-Bejo (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2011.05.001
drome (McFerran et al., 1976; Abe et al., 1998). Natural outbreaks of IBH are characterized by a sudden onset of mortality, peaking after 3e4 days post infection (dpi), which usually returns to normal at 5 dpi, but occasionally continues for 2e3 weeks. Morbidity is low and sick chickens adopt a crouching position with ruffled feathers (McFerran et al., 1976). The main lesions of IBH are pale, friable and swollen livers. Petechial or ecchymotic haemorrhages may be present in the liver and skeletal muscles. Basophilic or eosinophilic intranuclear inclusion bodies (INIBs) are often observed in degenerate hepatocytes (Grimes et al., 1977a, b; Hair-Bejo, 2005). FAdVs can be transmitted vertically through the embryonated egg or horizontally, since they are present in all excretions and at high titre in the faeces. Fomites, personnel and transport can also be important contributors to the spread of the virus (McFerran and Adair, 2003). Isolation of FAdVs can be from faeces, Ó 2011 Elsevier Ltd. All rights reserved.
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the pharynx, the trachea and affected organs such as the liver and gizzard, but in cases of IBH, the specimen of choice for virus isolation is the liver (McFerran and Adair, 2003). Isolation of FAdV can be performed on chick embryo liver or kidney cell cultures or in embryonated eggs; however, not all FAdVs multiply in embryonated eggs and cause recognizable lesions (McFerran, 1991). Furthermore, the majority of FAdVs isolated in eggs have been of serotype 1 or 5 (McFerran, 1991). The chorioallantoic membrane (CAM) route of inoculation of embryonated eggs was found to be more sensitive for adenovirus virus isolation than inoculation via the allantoic cavity (Kawamura et al., 1964), although it has been shown that the yolk sac is also a sensitive route for isolating laboratory strains of adenovirus (Cowen, 1988). However, the role of most FAdV isolates as primary pathogens during field outbreaks has not been well defined, because FAdV can be isolated from sick and clinically normal chickens (McFerran et al., 1971; Cowen et al., 1978). The aim of the present study was to isolate and propagate FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken eggs in order to determine the pathogenicity of the virus in SPF chicken embryos.
Materials and Methods FAdV isolate UPM04217 was obtained from an outbreak of IBH on a commercial broiler farm in Perak, Malaysia in 2004. It induces typical histological lesions of IBH with basophilic and eosinophilic INIBs in the liver (Hair-Bejo, 2005). Non-enveloped icosahedral adenovirus particles (70e85 nm in diameter) are detected by transmission electron microscopy (TEM) (Hair-Bejo, 2005). The isolate was characterized as FAdV serotype 9 (Jason et al., 2007) and the samples of origin were shown to be free of infectious bursal disease virus (IBDV) and chicken anaemia virus (CAV) by real-time polymerase chain reaction (PCR) (Hairul Aini et al., 2008) and agarose gelbased PCR (Hasmah et al., 2004), respectively. The liver of infected chickens was stored at 20 C until used. The liver was frozen and thawed three times and then macerated with a sterile mortar and pestle to prepare a 1 in 2 (w/v) suspension in sterile phosphate buffered saline (PBS; pH 7.4, 0.1 M). The suspension was centrifuged at 2,000 g for 30 min for clarification. The supernatant was collected and passed through a 0.45 mm filter and treated with a commercial antibiotic and antimycotic preparation (GIBCO Laboratories, New York, NY, USA) at a 1 in 10 (v/v) dilution and incubated at 4 C for 1 h prior to inoculation.
The extracts from the liver homogenates were inoculated onto the CAM of SPF embryonated chicken eggs following standard embryo inoculation technique (Senne, 1989). The embryo was candled daily to determine mortality. Embryonic death within the first 24 h was considered to be due to non-specific causes. Embryos that died after 24 h were examined for the presence of gross lesions. The CAM from dead embryos was harvested under sterile conditions for further passages. The second passage of CAM homogenate was stored at 20 C. A portion of the CAM and liver were collected into 10% neutral buffered formalin and processed routinely for histopathological examination. CAM from the second passage was harvested and homogenized with an equal volume of PBS. After centrifugation at 2,000 g for 30 min, the supernatant was filtered and a drop of suspension was placed onto a 300 mesh carbon-coated grid and stained with 3% phosphotungstic acid. Samples were examined by TEM for the identification of the virus (Doane and Anderson, 1987; Hair-Bejo, 1993). Titration of the virus was carried out with serial tenfold dilutions of CAM homogenate inoculated into 9-day-old SPF embryonated chicken eggs via the CAM (Senne, 1989). The median embryo infective dose (EID50) of the FAdV isolate was analyzed using the formula of Reed and Muench (1938). The pathogenicity of the virus was determined in SPF embryonated chicken eggs. Sixty, 9-day-old SPF embryonated chicken eggs were inoculated (0.1 ml/ egg) with CAM homogenate containing 105.8 EID50/ ml FAdV of Malaysian isolates via the CAM for sacrifice (n ¼ 30 eggs) and for determination of the percentage mortality (n ¼ 30 eggs). Forty SPF embryonated chicken eggs were left uninoculated and acted as controls. The eggs were observed and candled twice daily for embryo mortality. Five embryos were sacrificed at 1, 3, 5, 7, 9 and 11 dpi and at 0, 3, 9 and 12 dpi from the sacrificed and control groups, respectively. On necropsy examination, gross changes were recorded and samples of liver, CAM, yolk sac, spleen, kidney, heart and bursa of Fabricius were collected for microscopical examination. The liver and CAM were also collected for electron microscopy. Samples of the organs were fixed in 10% neutral buffered formalin for 24 h, processed routinely and sections were stained with haematoxylin and eosin (HE; Bancroft et al., 1996). The severity of microscopical changes in the liver was scored subjectively as 0 (normal), 1 (mild), 2 (mild to moderate), 3 (moderate), 4 (moderate to severe) or 5 (severe). Small pieces of liver and CAM were fixed in 2.5% glutaraldehyde cacodylate (pH 7.2) at 4 C for 12 h and processed for TEM (Doane and Anderson, 1987; Hair-Bejo, 1993).
Pathogenicity of Fowl Adenovirus in Chicken Embryos
Results The FAdV isolate was passaged successfully in SPF embryonated eggs. Embryo mortality of 100% was recorded at 4e6 dpi (first passage), 3e4 dpi (second passage), 3e4 dpi (third passage) and 3 dpi (fourth passage). On necropsy examination there was congestion, haemorrhage and opacity of the CAM. The embryos displayed congestion and haemorrhage at 3e5 dpi and at 6 dpi the liver was swollen with focal necrosis and petechial haemorrhages. Microscopically, degeneration and necrosis of the liver, with eosinophilic and basophilic INIBs in degenerate hepatocytes, was observed at 5e6 dpi. INIBs were also found in the CAM at 5e6 dpi. Non-enveloped icosahedral adenovirus particles (70e85 nm in diameter) were observed in the liver and CAM samples examined by TEM. The virions were hexagonal in appearance and both empty and complete virions were observed. Embryo mortality of 100% within 4e11 dpi was recorded in the mortality group. The percentage mortality was 3% at 4 dpi, 7% at 5 dpi, 13% at 7 dpi, 27% at 8 dpi, 27% at 9 dpi, 17% at 10 dpi and 7% at 11 dpi. No mortality was recorded in the control group. No gross lesions were recorded at 1 dpi in the sacrificed group; however, at 3 and 5 dpi, the embryos were congested and haemorrhagic. Congestion and focal areas of necrosis in the liver and a mild to moderate thickening of the CAM at the inoculation site were also observed. At 7, 9 and 11 dpi, the infected embryos were smaller than those of the control group, but there were no obvious external lesions. However, the liver of these embryos was pale with multifocal areas of necrosis, fibrosis and haemorrhage (Fig. 1a). The CAM was opaque, friable and thickened (Fig. 1b). In addition, varying degrees of renal enlargement, congestion and haemorrhage were also
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observed commonly. Mild to moderate congestion was observed in the spleen, yolk sac and heart at 7 dpi, while at 9 and 11 dpi there was hydropericardium, congestion and haemorrhage in the yolk sac and multifocal necrosis and petechial haemorrhage of the spleen in some embryonated eggs. Microscopically, the liver parenchyma was normal at 1 dpi, while at 3 dpi there was mild to moderate degeneration and necrosis of the hepatocytes with congestion of the sinusoids (lesion score 1.6 0.5). At 5 dpi there was moderate congestion, haemorrhage, degeneration and focal necrosis, and infiltration of inflammatory cells was observed (lesion score 3.0 0.0). At 7 dpi there was moderate to severe congestion, haemorrhage and multifocal degeneration and necrosis with inflammatory infiltration (lesion score 4.8 0.4). Severe congestion, haemorrhage and extensive diffuse degeneration and necrosis of the hepatocytes with inflammatory infiltration and fibrosis were observed at 9 and 11 dpi (lesion score 5.0 0.0). Haemorrhage, congestion, degeneration and necrosis of the chorionic epithelium and hyperplasia of the chorionic epithelium of the CAM were also observed. Microscopical lesions observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius included haemorrhage, congestion, degeneration and necrosis and infiltration of inflammatory cells. No lesions were observed in embryos of the control groups. Two types of INIBs were seen in the hepatocytes: (1) basophilic inclusions surrounded by a clear halo in enlarged or normal nuclei and/or basophilic inclusions occupying the entire normal or enlarged nucleus (basophilic INIBs) and (2) eosinophilic INIBs (Fig. 2a). Eosinophilic inclusions were observed in degenerate hepatocytes in moderate number at 5 and 11 dpi, but were only occasionally noted at 7 and 9 dpi.
Fig. 1. (a) Swollen and friable liver with multifocal areas of necrosis and petechial haemorrhage and hydropericardium at 7 dpi. (b) Opacity and thickening of the CAM at 9 dpi.
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Fig. 2. (a) Basophilic intranuclear inclusion body surrounded by a clear halo (large arrow). Basophilic (arrowhead) and eosinophilic (small arrow) INIBs occupy the entire nucleus of hepatocytes at 7 dpi. (b) Basophilic intranuclear inclusion body (arrow) in the chorionic epithelium at 7 dpi. HE. Bar, 20 mm.
Basophilic INIBs were more commonly observed in degenerate hepatocytes between 5 and 11 dpi, but were greatest in number at 7 and 9 dpi. Moderate numbers of eosinophilic INIBs were observed in the chorionic epithelium, but there were few basophilic INIBs (Fig. 2b). In addition, basophilic and eosinophilic INIBs were observed occasionally in the yolk sac, renal tubule and spleen at 5e11 dpi, but were never seen in the bursa of Fabricius and heart. Ultrastructural changes in the liver included margination of chromatin and slight cristolysis of mitochondria that was first observed at 3 dpi. At 5 dpi the nucleus was filled by inclusion bodies and finely granular and filamentous material. Congestion and infiltration of inflammatory cells, especially heterophils, were also observed. At 7 dpi and thereafter, numerous viral particles and inclusions were noted in the nucleus of hepatocytes (Fig. 3a). The FAdV particles were characterized as non-enveloped hexagonal with an electron-lucent and electron-dense core ranging from 70 to 85 nm in diameter (Fig. 3b). Blebbing and rupture of the nuclear membrane, markedly swollen mitochondria with cristolysis, dilation of rough endoplasmic reticulum, separation of the ribosome, proliferation of peroxisomes, vacuolation of cytoplasm and cell necrosis with infiltration of inflammatory cells were observed at 7, 9 and 11 dpi (Fig. 3c). The cytoplasm was usually markedly disrupted and viral particles were scattered or in clusters within homogeneous electron-lucent and electron-dense masses. Ultrastructural changes of the CAM at 3 dpi included margination of nucleoli to the periphery, necrosis of the nucleus leaving aggregates of chromatin
and vacuolation of the cytoplasm. At 5 dpi, inclusions and a loose granular structure of the nucleus and swelling of the mitochondria with loss of cristae were observed in the ectodermic epithelium. At 7 dpi and thereafter, large intranuclear viral inclusions (Fig. 3d) and loose granular or fibrillar structures filled the nucleus. Nuclear membrane blebbing was prominent and there was indentation (invagination) of the nuclear membrane with condensed and intensely electrondense margination of the chromatin. Swollen mitochondria with cristolysis and dilation of rough endoplasmic reticulum, separation of the ribosomes, proliferation of peroxisomes, vacuolation of the cytoplasm and indistinguishable cytoplasmic organelles were observed.
Discussion In the present study, a Malaysian isolate of FAdV was successfully inoculated into SPF embryonated chicken eggs via the CAM. The ultrastructural features of the virus were typical of adenovirus, with the characteristic non-enveloped, icosahedral, empty and complete virions with a diameter of 70e85 nm (Ganesh et al., 2002). A 100% cumulative mortality of embryos was recorded within 4e11 dpi and the highest mortality was recorded at 8 (27%) and 9 (27%) dpi. Therefore, 8e9 dpi would be the best time to harvest this particular FAdV isolate from SPF chicken embryonated eggs. An earlier study reported the infectivity of cell culture-propagated avian adenovirus of 11 different serotypes to embryonated eggs. In that investigation,
Pathogenicity of Fowl Adenovirus in Chicken Embryos
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Fig. 3. (a) Virus particles and inclusions in the nucleus of hepatocytes at 7 dpi. The nucleoli are found at the periphery of the nucleus and there is breaking of the nuclear membrane. TEM. Bar, 2 mm. (b) FAdV particles are non-enveloped and hexagonal with an electron-lucent and electron-dense core and range from 70 to 85 nm in diameter. TEM. Bar, 0.2 mm. (c) Clumping and condensation of chromatin, vacuolation of the cytoplasm, vesiculation of the rough endoplasmic reticulum, proliferation of peroxisomes, necrotic cells and cellular debris in the liver at 11 dpi. TEM. Bar, 2 mm. (d) Large intranuclear viral inclusion and margination of chromatin in a cell of the chorionic epithelium of the CAM at 9 dpi. TEM. Bar, 1 mm.
embryo mortality occurred at 3e9 dpi in the embryos inoculated via the CAM and at 3e13 dpi in embryos inoculated via the yolk sac (Cowen, 1988). Infecting chicken embryos with titres of up to 105 plaque forming units of field isolate serotype 4 FAdV via the yolk sac resulted in 100% mortality within 5e10 dpi (Mazaheri et al., 1998). This variation in the time of death may be related to the route of inoculation, the virulence and titre of the virus inoculum and the age of the embryos (Cowen, 1988; Mendelson et al., 1995; Mazaheri et al., 1998). Gross lesions observed in the liver, kidney, spleen and heart of the embryos substantiate the findings of earlier reports (Cowen, 1988; Mazaheri et al., 1998). In addition, thickening of the CAM with opaque lesions and congestion and haemorrhage of the yolk sac were also frequent findings in the present study. The major microscopical lesions were observed in the liver, ranging from mild to severe and occurring
at 3e11 dpi. Basophilic INIBs were observed most frequently in the hepatocytes (Gallina et al., 1973; Cowen, 1988), although eosinophilic INIBs were common in a field outbreak of IBH in chickens (Grimes et al., 1977a, b; Hair-Bejo, 2005). Moderate numbers of eosinophilic INIBs were observed in the hepatocytes at 5 and 11 dpi, but the number of basophilic INIBs was greater at 7 and 9 dpi. This finding was compatible with the results of the ultrastructural examination of the liver where numerous viral particles were observed in the nucleus of hepatocytes at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous material was observed. These observations suggest that basophilic INIBs consist of adenovirus particles, while the eosinophilic inclusions contain only fibrillar granular material and filaments, which may represent a degenerative process or an early stage in the formation of virus (Itakura et al., 1977; Riddell, 1987; Weissenbock and Fuchs, 1995).
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Higher numbers of eosinophilic INIBs were observed in the ectoderm of the CAM, where the number of basophilic INIBs was low throughout the experiment. This observation correlated with ultrastructural studies of the CAM in which it was shown that most of the nuclei of the ectoderm were filled by fine granular and filamentous structures, but only some nuclei contained large viral inclusions. The ultrastructural changes in the nucleus, nuclear membrane and cytoplasmic organelles of the hepatocytes and cells of the ectoderm of the CAM may be related to viral multiplication and release of viral particles into the cytoplasm of the cells. Adenoviruses bind to host cell receptors via their penton fibres and enter cells by endocytosis via clathrin-coated pits. The outer capsid is then removed and the core, comprising the viral genome with its associated histones, enters the nucleus where mRNA transcription, viral DNA replication and assembly of new virions occur. Maturation also occurs in the nucleus. Each infected cell yields thousands of virions that may infect other cells and these are released by cell lysis (Murphy et al., 1999). FAdV first replicated in the CAM, leading to primary viraemia and allowing virus to reach the liver. Massive replication of FAdV occurred in the liver, with resulting cellular necrosis or lysis and spread of virus infection into neighboring cells and causing secondary viraemia, during which virus disseminated into other susceptible tissues. Mortality, gross lesions and histological and ultrastructural changes observed in the embryonic tissues were all closely correlated. The mortality of the embryo was greatest at 8 and 9 dpi and gross lesions and histological and ultrastructural changes were recorded from 7 to 11 dpi. It is therefore concluded that the UPM04217 FAdV isolate is able to adapt, and is pathogenic to, SPF chicken embryos. Embryonic liver is the best organ to be used for isolation and propagation of the virus.
Acknowledgments This work was funded by the Ministry of Science, Technology and Innovations, Malaysia, under ScienceFund grant 5450195 and by the Nuffic project for an MVSc programme scholarship.
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Bancroft JD, Stevens A, Tuner DR (1996) Theory and Practice of Histological Techniques, 4th Edit, Churchill Livingstone, New York. pp. 23e112. Benko M, Harrach B, Russel WC (2000) Family Adenoviridae. In: Virus Taxonomy, Classification, and Nomenclature of Viruses: Seventh Report of the International Committee on Taxonomy of Viruses, MH Van Regenmortel, DHL Bishop, EB Carstens, MK Estes, S Lemon et al., Eds., Academic Press, San Diego, pp. 227e238. Cowen BS (1988) Chicken embryo propagation of type I avian adenoviruses. Avian Diseases, 32, 347e352. Cowen B, Mitchell GB, Calnek BW (1978) An adenovirus survey of poultry flocks during the growing and laying periods. Avian Diseases, 22, 115e121. Doane FW, Anderson N (1987) Electron Microscopy in Diagnostic Virology. Cambridge University Press, Cambridge. pp. 14e46. Gallina AM, Winterfield RW, Fadly AM (1973) Adenovirus infection and disease. II. Histopathology of natural and experimental disease. Avian Diseases, 17, 343e353. Ganesh K, Raghavan R, Gowda RNS, Satyanarayana ML, Suryanarayana VVS (2002) Purification and characterization of the aetiological agent of hydropericardium hepatitis syndrome from infected liver tissues of broiler chickens. Tropical Animal Health and Production, 34, 7e17. Grimes TM, Culver DH, King DJ (1977a) Virus-neutralizing antibody titers against 8 avian adenovirus serotypes in breeder hens in Georgia by a microneutralization procedure. Avian Diseases, 21, 220e229. Grimes TM, King DJ, Kleven SH, Fletcher OJ (1977b) Involvement of type-8 avian adenovirus in the etiology of inclusion body hepatitis. Avian Diseases, 21, 26e38. Hair-Bejo M (1993) Infectious bursal disease in broilers: pathological changes and virus detection. Journal of Veterinary Malaysia, 5, 49e51. Hair-Bejo M (2005) Inclusion body hepatitis in a flock of commercial broiler chickens. Journal of Veterinary Malaysia, 17, 23e26. Hairul Aini H, Omar AR, Hair-Bejo M, Aini I (2008) Comparison of SYBR Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus. Microbiological Research, 163, 556e563. Hasmah S, Omar AR, Wan KF, Hair-Bejo M, Aini I (2004) Genetic diversity of chicken anemia virus following cell culture passaging in MSB-1 cells. Acta Virologica, 48, 85e89. Itakura C, Matsushita S, Goto M (1977) Fine structure of inclusion bodies in hepatic cells of chickens naturally affected with inclusion body hepatitis. Avian Pathology, 6, 19e32. Jason PS, Hair-Bejo M, Omar AR, Aini I (2007) Molecular characterization of fowl adenovirus isolated from an outbreak of inclusion body hepatitis in broiler chickens in Malaysia. In: Proceedings of the 19th Congress of the Veterinary Association of Malaysia, Kuala Lumpur, Malaysia, pp. 56e58. Kawamura H, Shimizu F, Tsubahara H (1964) Avian adenovirus: its properties and serological classification. National Institute of Animal Health Quarterly (Tokyo), 4, 183e193.
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Mazaheri A, Prusas C, Voß M, Hess M (1998) Some strains of serotype 4 adenoviruses cause inclusion body hepatitis and hydropericardium syndrome in chickens. Avian Pathology, 27, 269e276. McFerran JB (1991) Adenovirus (group I) infections of chickens. In: Diseases of Poultry, 9th Edit., BW Calnek, HJ Barnes, CW Beard, WM Reid, HW Yoder Jr., Eds., Iowa State Press, Ames, pp. 553e563. McFerran JB, Adair BM (2003) Group I adenovirus infections. In: Diseases of Poultry, 11th Edit., YM Saif, HJ Barnes, JR Glisson, AM Fadly, LR McDougald et al., Eds., Iowa State Press, Ames, pp. 214e227. McFerran JB, Gordon WAM, Taylor SM, McParland PJ (1971) Isolation of viruses from 94 flocks of fowl with respiratory disease. Research in Veterinary Science, 12, 565e569. McFerran JB, McCracken RM, Connor TJ, Evants RT (1976) Isolation of viruses from clinical outbreaks of inclusion body hepatitis. Avian Pathology, 5, 315e324. Mendelson C, Nothelfer HB, Monreal G (1995) Identification and characterization of an avian adenovirus isolated from a ‘spiking mortality syndrome’ field outbreak in broilers on the Delmarva Peninsula, USA. Avian Pathology, 24, 693e706. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ (1999) Veterinary Virology, 3rd Edit, Academic Press, New York, pp. 327e335.
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Reed LJ, Muench H (1938) A simple method of estimating fifty percent end points. American Journal of Hygiene, 27, 493e497. Riddell C (1987) Avian Histopathology. American Association of Avian Pathologists, Kennett Square, Pennsylvania, pp. 7e17. Senne DA (1989) Virus propagation in embryonating eggs. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3rd Edit., HG Purchage, LH Arp, CH Domermuth, JE Pearson, Eds., American Association of Avian Pathologists, Kennett Square, Pennsylvania, pp. 176e181. Weissenbock H, Fuchs A (1995) Histological and ultrastructural characterization of hepatic intranuclear inclusion bodies in psittacine birds and pigeons. Avian Pathology, 24, 507e521. Yates VJ, Rhee YO, Fry DE, Al Mishad AM, McCormick KJ (1976) The presence of avian adenoviruses and adenovirus associated viruses in healthy chickens. Avian Diseases, 20, 146e152.
February 3rd, 2011 ½ Received, Accepted, May 4th, 2011
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Pathogenicity of Fowl Adenovirus in Specific Pathogen Free Chicken Embryos W. Alemnesh*, M. Hair-Bejo†, I. Aini† and A. R. Omar† * Faculty of Veterinary Medicine, Gondar University, PO Box 196 Gondar, Ethiopia and † Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Summary Inclusion body hepatitis (IBH) associated with fowl adenovirus (FAdV) infection has a worldwide distribution. The aim of the present study was to determine the pathogenicity of Malaysian FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken embryos. FAdV (titre 105.8/ml) was inoculated into SPF embryonated chicken eggs (0.1 ml per egg) via the chorioallantoic membrane (CAM). There was 100% embryo mortality within 4e11 days post infection (dpi). The gross and microscopical lesions of the embryo were confined to the liver and were noted at 5, 7, 9 and 11 dpi. The liver was pale with multifocal areas of necrosis, fibrosis and haemorrhage. Microscopically, there was moderate to severe congestion and haemorrhage and severe and diffuse hepatocyte degeneration and necrosis, with intranuclear inclusion bodies (INIBs) and associated inflammation. Haemorrhage, congestion, degeneration, necrosis and hyperplasia of the CAM with INIBs were observed at 5, 7, 9 and 11 dpi. Varying degrees of congestion, haemorrhage, degeneration and necrosis were also observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius. Ultrastructurally, numerous viral particles in the nucleus of hepatocytes were recorded at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous INIBs were observed. The INIBs in the CAM were present either as fine granular filamentous structures or as large viral inclusions. FAdV (UPM04217) is therefore highly pathogenic to SPF chicken embryos and the embryonic liver should be used for isolation and propagation of the virus. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: chicken embryo; fowl adenovirus; pathogenicity
Introduction Inclusion body hepatitis (IBH) is an acute disease, mainly occurring in young broiler chickens (3e7 weeks of age) and caused by several serotypes of fowl adenovirus (FAdV; McFerran and Adair, 2003). FAdVs that infect mostly broiler chickens are classified in the genus Aviadenovirus under the family Adenoviridae, and are further classified into five species and 12 serotypes (Benko et al., 2000). Some of the FAdVs are ubiquitous in chickens, as demonstrated by serological surveys (Yates et al., 1976; Grimes et al., 1977a) and the high isolation rates of FAdVs from specimens taken from normal and sick birds (McFerran et al., 1971; Cowen et al., 1978). In contrast, certain serotypes of FAdVs have been more often associated with naturally occurring outbreaks of IBH and hydropericardium synCorrespondence to: M. Hair-Bejo (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2011.05.001
drome (McFerran et al., 1976; Abe et al., 1998). Natural outbreaks of IBH are characterized by a sudden onset of mortality, peaking after 3e4 days post infection (dpi), which usually returns to normal at 5 dpi, but occasionally continues for 2e3 weeks. Morbidity is low and sick chickens adopt a crouching position with ruffled feathers (McFerran et al., 1976). The main lesions of IBH are pale, friable and swollen livers. Petechial or ecchymotic haemorrhages may be present in the liver and skeletal muscles. Basophilic or eosinophilic intranuclear inclusion bodies (INIBs) are often observed in degenerate hepatocytes (Grimes et al., 1977a, b; Hair-Bejo, 2005). FAdVs can be transmitted vertically through the embryonated egg or horizontally, since they are present in all excretions and at high titre in the faeces. Fomites, personnel and transport can also be important contributors to the spread of the virus (McFerran and Adair, 2003). Isolation of FAdVs can be from faeces, Ó 2011 Elsevier Ltd. All rights reserved.
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the pharynx, the trachea and affected organs such as the liver and gizzard, but in cases of IBH, the specimen of choice for virus isolation is the liver (McFerran and Adair, 2003). Isolation of FAdV can be performed on chick embryo liver or kidney cell cultures or in embryonated eggs; however, not all FAdVs multiply in embryonated eggs and cause recognizable lesions (McFerran, 1991). Furthermore, the majority of FAdVs isolated in eggs have been of serotype 1 or 5 (McFerran, 1991). The chorioallantoic membrane (CAM) route of inoculation of embryonated eggs was found to be more sensitive for adenovirus virus isolation than inoculation via the allantoic cavity (Kawamura et al., 1964), although it has been shown that the yolk sac is also a sensitive route for isolating laboratory strains of adenovirus (Cowen, 1988). However, the role of most FAdV isolates as primary pathogens during field outbreaks has not been well defined, because FAdV can be isolated from sick and clinically normal chickens (McFerran et al., 1971; Cowen et al., 1978). The aim of the present study was to isolate and propagate FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken eggs in order to determine the pathogenicity of the virus in SPF chicken embryos.
Materials and Methods FAdV isolate UPM04217 was obtained from an outbreak of IBH on a commercial broiler farm in Perak, Malaysia in 2004. It induces typical histological lesions of IBH with basophilic and eosinophilic INIBs in the liver (Hair-Bejo, 2005). Non-enveloped icosahedral adenovirus particles (70e85 nm in diameter) are detected by transmission electron microscopy (TEM) (Hair-Bejo, 2005). The isolate was characterized as FAdV serotype 9 (Jason et al., 2007) and the samples of origin were shown to be free of infectious bursal disease virus (IBDV) and chicken anaemia virus (CAV) by real-time polymerase chain reaction (PCR) (Hairul Aini et al., 2008) and agarose gelbased PCR (Hasmah et al., 2004), respectively. The liver of infected chickens was stored at 20 C until used. The liver was frozen and thawed three times and then macerated with a sterile mortar and pestle to prepare a 1 in 2 (w/v) suspension in sterile phosphate buffered saline (PBS; pH 7.4, 0.1 M). The suspension was centrifuged at 2,000 g for 30 min for clarification. The supernatant was collected and passed through a 0.45 mm filter and treated with a commercial antibiotic and antimycotic preparation (GIBCO Laboratories, New York, NY, USA) at a 1 in 10 (v/v) dilution and incubated at 4 C for 1 h prior to inoculation.
The extracts from the liver homogenates were inoculated onto the CAM of SPF embryonated chicken eggs following standard embryo inoculation technique (Senne, 1989). The embryo was candled daily to determine mortality. Embryonic death within the first 24 h was considered to be due to non-specific causes. Embryos that died after 24 h were examined for the presence of gross lesions. The CAM from dead embryos was harvested under sterile conditions for further passages. The second passage of CAM homogenate was stored at 20 C. A portion of the CAM and liver were collected into 10% neutral buffered formalin and processed routinely for histopathological examination. CAM from the second passage was harvested and homogenized with an equal volume of PBS. After centrifugation at 2,000 g for 30 min, the supernatant was filtered and a drop of suspension was placed onto a 300 mesh carbon-coated grid and stained with 3% phosphotungstic acid. Samples were examined by TEM for the identification of the virus (Doane and Anderson, 1987; Hair-Bejo, 1993). Titration of the virus was carried out with serial tenfold dilutions of CAM homogenate inoculated into 9-day-old SPF embryonated chicken eggs via the CAM (Senne, 1989). The median embryo infective dose (EID50) of the FAdV isolate was analyzed using the formula of Reed and Muench (1938). The pathogenicity of the virus was determined in SPF embryonated chicken eggs. Sixty, 9-day-old SPF embryonated chicken eggs were inoculated (0.1 ml/ egg) with CAM homogenate containing 105.8 EID50/ ml FAdV of Malaysian isolates via the CAM for sacrifice (n ¼ 30 eggs) and for determination of the percentage mortality (n ¼ 30 eggs). Forty SPF embryonated chicken eggs were left uninoculated and acted as controls. The eggs were observed and candled twice daily for embryo mortality. Five embryos were sacrificed at 1, 3, 5, 7, 9 and 11 dpi and at 0, 3, 9 and 12 dpi from the sacrificed and control groups, respectively. On necropsy examination, gross changes were recorded and samples of liver, CAM, yolk sac, spleen, kidney, heart and bursa of Fabricius were collected for microscopical examination. The liver and CAM were also collected for electron microscopy. Samples of the organs were fixed in 10% neutral buffered formalin for 24 h, processed routinely and sections were stained with haematoxylin and eosin (HE; Bancroft et al., 1996). The severity of microscopical changes in the liver was scored subjectively as 0 (normal), 1 (mild), 2 (mild to moderate), 3 (moderate), 4 (moderate to severe) or 5 (severe). Small pieces of liver and CAM were fixed in 2.5% glutaraldehyde cacodylate (pH 7.2) at 4 C for 12 h and processed for TEM (Doane and Anderson, 1987; Hair-Bejo, 1993).
Pathogenicity of Fowl Adenovirus in Chicken Embryos
Results The FAdV isolate was passaged successfully in SPF embryonated eggs. Embryo mortality of 100% was recorded at 4e6 dpi (first passage), 3e4 dpi (second passage), 3e4 dpi (third passage) and 3 dpi (fourth passage). On necropsy examination there was congestion, haemorrhage and opacity of the CAM. The embryos displayed congestion and haemorrhage at 3e5 dpi and at 6 dpi the liver was swollen with focal necrosis and petechial haemorrhages. Microscopically, degeneration and necrosis of the liver, with eosinophilic and basophilic INIBs in degenerate hepatocytes, was observed at 5e6 dpi. INIBs were also found in the CAM at 5e6 dpi. Non-enveloped icosahedral adenovirus particles (70e85 nm in diameter) were observed in the liver and CAM samples examined by TEM. The virions were hexagonal in appearance and both empty and complete virions were observed. Embryo mortality of 100% within 4e11 dpi was recorded in the mortality group. The percentage mortality was 3% at 4 dpi, 7% at 5 dpi, 13% at 7 dpi, 27% at 8 dpi, 27% at 9 dpi, 17% at 10 dpi and 7% at 11 dpi. No mortality was recorded in the control group. No gross lesions were recorded at 1 dpi in the sacrificed group; however, at 3 and 5 dpi, the embryos were congested and haemorrhagic. Congestion and focal areas of necrosis in the liver and a mild to moderate thickening of the CAM at the inoculation site were also observed. At 7, 9 and 11 dpi, the infected embryos were smaller than those of the control group, but there were no obvious external lesions. However, the liver of these embryos was pale with multifocal areas of necrosis, fibrosis and haemorrhage (Fig. 1a). The CAM was opaque, friable and thickened (Fig. 1b). In addition, varying degrees of renal enlargement, congestion and haemorrhage were also
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observed commonly. Mild to moderate congestion was observed in the spleen, yolk sac and heart at 7 dpi, while at 9 and 11 dpi there was hydropericardium, congestion and haemorrhage in the yolk sac and multifocal necrosis and petechial haemorrhage of the spleen in some embryonated eggs. Microscopically, the liver parenchyma was normal at 1 dpi, while at 3 dpi there was mild to moderate degeneration and necrosis of the hepatocytes with congestion of the sinusoids (lesion score 1.6 0.5). At 5 dpi there was moderate congestion, haemorrhage, degeneration and focal necrosis, and infiltration of inflammatory cells was observed (lesion score 3.0 0.0). At 7 dpi there was moderate to severe congestion, haemorrhage and multifocal degeneration and necrosis with inflammatory infiltration (lesion score 4.8 0.4). Severe congestion, haemorrhage and extensive diffuse degeneration and necrosis of the hepatocytes with inflammatory infiltration and fibrosis were observed at 9 and 11 dpi (lesion score 5.0 0.0). Haemorrhage, congestion, degeneration and necrosis of the chorionic epithelium and hyperplasia of the chorionic epithelium of the CAM were also observed. Microscopical lesions observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius included haemorrhage, congestion, degeneration and necrosis and infiltration of inflammatory cells. No lesions were observed in embryos of the control groups. Two types of INIBs were seen in the hepatocytes: (1) basophilic inclusions surrounded by a clear halo in enlarged or normal nuclei and/or basophilic inclusions occupying the entire normal or enlarged nucleus (basophilic INIBs) and (2) eosinophilic INIBs (Fig. 2a). Eosinophilic inclusions were observed in degenerate hepatocytes in moderate number at 5 and 11 dpi, but were only occasionally noted at 7 and 9 dpi.
Fig. 1. (a) Swollen and friable liver with multifocal areas of necrosis and petechial haemorrhage and hydropericardium at 7 dpi. (b) Opacity and thickening of the CAM at 9 dpi.
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Fig. 2. (a) Basophilic intranuclear inclusion body surrounded by a clear halo (large arrow). Basophilic (arrowhead) and eosinophilic (small arrow) INIBs occupy the entire nucleus of hepatocytes at 7 dpi. (b) Basophilic intranuclear inclusion body (arrow) in the chorionic epithelium at 7 dpi. HE. Bar, 20 mm.
Basophilic INIBs were more commonly observed in degenerate hepatocytes between 5 and 11 dpi, but were greatest in number at 7 and 9 dpi. Moderate numbers of eosinophilic INIBs were observed in the chorionic epithelium, but there were few basophilic INIBs (Fig. 2b). In addition, basophilic and eosinophilic INIBs were observed occasionally in the yolk sac, renal tubule and spleen at 5e11 dpi, but were never seen in the bursa of Fabricius and heart. Ultrastructural changes in the liver included margination of chromatin and slight cristolysis of mitochondria that was first observed at 3 dpi. At 5 dpi the nucleus was filled by inclusion bodies and finely granular and filamentous material. Congestion and infiltration of inflammatory cells, especially heterophils, were also observed. At 7 dpi and thereafter, numerous viral particles and inclusions were noted in the nucleus of hepatocytes (Fig. 3a). The FAdV particles were characterized as non-enveloped hexagonal with an electron-lucent and electron-dense core ranging from 70 to 85 nm in diameter (Fig. 3b). Blebbing and rupture of the nuclear membrane, markedly swollen mitochondria with cristolysis, dilation of rough endoplasmic reticulum, separation of the ribosome, proliferation of peroxisomes, vacuolation of cytoplasm and cell necrosis with infiltration of inflammatory cells were observed at 7, 9 and 11 dpi (Fig. 3c). The cytoplasm was usually markedly disrupted and viral particles were scattered or in clusters within homogeneous electron-lucent and electron-dense masses. Ultrastructural changes of the CAM at 3 dpi included margination of nucleoli to the periphery, necrosis of the nucleus leaving aggregates of chromatin
and vacuolation of the cytoplasm. At 5 dpi, inclusions and a loose granular structure of the nucleus and swelling of the mitochondria with loss of cristae were observed in the ectodermic epithelium. At 7 dpi and thereafter, large intranuclear viral inclusions (Fig. 3d) and loose granular or fibrillar structures filled the nucleus. Nuclear membrane blebbing was prominent and there was indentation (invagination) of the nuclear membrane with condensed and intensely electrondense margination of the chromatin. Swollen mitochondria with cristolysis and dilation of rough endoplasmic reticulum, separation of the ribosomes, proliferation of peroxisomes, vacuolation of the cytoplasm and indistinguishable cytoplasmic organelles were observed.
Discussion In the present study, a Malaysian isolate of FAdV was successfully inoculated into SPF embryonated chicken eggs via the CAM. The ultrastructural features of the virus were typical of adenovirus, with the characteristic non-enveloped, icosahedral, empty and complete virions with a diameter of 70e85 nm (Ganesh et al., 2002). A 100% cumulative mortality of embryos was recorded within 4e11 dpi and the highest mortality was recorded at 8 (27%) and 9 (27%) dpi. Therefore, 8e9 dpi would be the best time to harvest this particular FAdV isolate from SPF chicken embryonated eggs. An earlier study reported the infectivity of cell culture-propagated avian adenovirus of 11 different serotypes to embryonated eggs. In that investigation,
Pathogenicity of Fowl Adenovirus in Chicken Embryos
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Fig. 3. (a) Virus particles and inclusions in the nucleus of hepatocytes at 7 dpi. The nucleoli are found at the periphery of the nucleus and there is breaking of the nuclear membrane. TEM. Bar, 2 mm. (b) FAdV particles are non-enveloped and hexagonal with an electron-lucent and electron-dense core and range from 70 to 85 nm in diameter. TEM. Bar, 0.2 mm. (c) Clumping and condensation of chromatin, vacuolation of the cytoplasm, vesiculation of the rough endoplasmic reticulum, proliferation of peroxisomes, necrotic cells and cellular debris in the liver at 11 dpi. TEM. Bar, 2 mm. (d) Large intranuclear viral inclusion and margination of chromatin in a cell of the chorionic epithelium of the CAM at 9 dpi. TEM. Bar, 1 mm.
embryo mortality occurred at 3e9 dpi in the embryos inoculated via the CAM and at 3e13 dpi in embryos inoculated via the yolk sac (Cowen, 1988). Infecting chicken embryos with titres of up to 105 plaque forming units of field isolate serotype 4 FAdV via the yolk sac resulted in 100% mortality within 5e10 dpi (Mazaheri et al., 1998). This variation in the time of death may be related to the route of inoculation, the virulence and titre of the virus inoculum and the age of the embryos (Cowen, 1988; Mendelson et al., 1995; Mazaheri et al., 1998). Gross lesions observed in the liver, kidney, spleen and heart of the embryos substantiate the findings of earlier reports (Cowen, 1988; Mazaheri et al., 1998). In addition, thickening of the CAM with opaque lesions and congestion and haemorrhage of the yolk sac were also frequent findings in the present study. The major microscopical lesions were observed in the liver, ranging from mild to severe and occurring
at 3e11 dpi. Basophilic INIBs were observed most frequently in the hepatocytes (Gallina et al., 1973; Cowen, 1988), although eosinophilic INIBs were common in a field outbreak of IBH in chickens (Grimes et al., 1977a, b; Hair-Bejo, 2005). Moderate numbers of eosinophilic INIBs were observed in the hepatocytes at 5 and 11 dpi, but the number of basophilic INIBs was greater at 7 and 9 dpi. This finding was compatible with the results of the ultrastructural examination of the liver where numerous viral particles were observed in the nucleus of hepatocytes at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous material was observed. These observations suggest that basophilic INIBs consist of adenovirus particles, while the eosinophilic inclusions contain only fibrillar granular material and filaments, which may represent a degenerative process or an early stage in the formation of virus (Itakura et al., 1977; Riddell, 1987; Weissenbock and Fuchs, 1995).
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Higher numbers of eosinophilic INIBs were observed in the ectoderm of the CAM, where the number of basophilic INIBs was low throughout the experiment. This observation correlated with ultrastructural studies of the CAM in which it was shown that most of the nuclei of the ectoderm were filled by fine granular and filamentous structures, but only some nuclei contained large viral inclusions. The ultrastructural changes in the nucleus, nuclear membrane and cytoplasmic organelles of the hepatocytes and cells of the ectoderm of the CAM may be related to viral multiplication and release of viral particles into the cytoplasm of the cells. Adenoviruses bind to host cell receptors via their penton fibres and enter cells by endocytosis via clathrin-coated pits. The outer capsid is then removed and the core, comprising the viral genome with its associated histones, enters the nucleus where mRNA transcription, viral DNA replication and assembly of new virions occur. Maturation also occurs in the nucleus. Each infected cell yields thousands of virions that may infect other cells and these are released by cell lysis (Murphy et al., 1999). FAdV first replicated in the CAM, leading to primary viraemia and allowing virus to reach the liver. Massive replication of FAdV occurred in the liver, with resulting cellular necrosis or lysis and spread of virus infection into neighboring cells and causing secondary viraemia, during which virus disseminated into other susceptible tissues. Mortality, gross lesions and histological and ultrastructural changes observed in the embryonic tissues were all closely correlated. The mortality of the embryo was greatest at 8 and 9 dpi and gross lesions and histological and ultrastructural changes were recorded from 7 to 11 dpi. It is therefore concluded that the UPM04217 FAdV isolate is able to adapt, and is pathogenic to, SPF chicken embryos. Embryonic liver is the best organ to be used for isolation and propagation of the virus.
Acknowledgments This work was funded by the Ministry of Science, Technology and Innovations, Malaysia, under ScienceFund grant 5450195 and by the Nuffic project for an MVSc programme scholarship.
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February 3rd, 2011 ½ Received, Accepted, May 4th, 2011