Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis from broiler chickens in Egypt

Tropical Animal Health and Production - A case-control study was performed to assess prescence of inclusion body hepatitis (IBH) caused by fowl adenoviruses (FAdVs) at Kafr EL-Shiekh Governorate,...     

Inclusion body hepatitis in broiler chickens in Iraq

Eighteen outbreaks of inclusion body hepatitis (IBH) were identified in broiler chickens in Baghdad in 1977 and 1978. The disease was seen mainly in 4-to-6-week-old broiler chickens. The mortality rate did not exceed 1% in any outbreak investigated. The most common gross findings were stellate or punctiform hemorrhagic areas in markedly fatty livers. Noticed in many cases were enlarged and pale kidneys, hemorrhagic lesions in the skeletal muscles, and pale fattened bone marrow. Histological examination of tissues revealed fat droplets and intranuclear inclusion bodies in degenerated liver cells. Eosinophilic inclusion bodies were seen in all cases. Only five cases had basophilic inclusions along with the eosinophilic ones. The etiologic agent was isolated on chorioallantoic membrane (CAM) of 12-day-old embryonated chicken eggs. The disease was produced experimentally in 4-week-old chicks using infected CAM suspension.     

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.     

Characterisation of avian adenoviruses associated with inclusion body hepatitis

Eleven avian adenoviruses were isolated in monolayer cultures of specific pathogen free chicken kidney cells which were inoculated with suspensions of liver, intestine or bursa obtained from 15 broiler flocks experiencing outbreaks of inclusion body hepatitis (10 isolates) and from five unaffected flocks (one isolate). Of the 11 isolates obtained, nine were identified by virus neutralisation tests as serotype 8, one as serotype 1 and one as serotype 12. Adeno-associated viruses were only observed in combination with adenoviral particles of the serotype 12 isolate which was derived from a relatively mild outbreak of inclusion body hepatitis. Only the serotype 1 isolate, obtained from the unaffected broiler flock, consistently caused the death of embryos with marked pathological changes. All of the isolates produced basophilic intranuclear inclusion bodies surrounded by clear halos in chicken kidney cell cultures. DNA preparations, obtained from six strains of serotype 8 avian adenovirus (two New Zealand isolates, three Australian isolates and the reference strain HVI) after digestion with the restriction enzymes EcoRI and BamHI, gave electrophoretic patterns showing the New Zealand isolates to be similar to one another and to strain HVI, but quite distinct from the Australian isolates.     

Pathological studies in broiler chicks fed aflatoxin or ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence

Day-old broiler chicks found negative for maternal antibodies against inclusion body hepatitis (IBH) virus by agar gel precipitation test and viral antigen in cloacal swabs by dot enzyme immunoassay were divided into 6 groups of 20 chicks each. Group A was fed aflatoxin B₁ at 1.25 ppm from 3 to 38 days of age; group O was fed ochratoxin A at 0.5 ppm from 3 to 38 days of age; group V was inoculated with 1 ml of IBH virus of titre log₁₀ 6.5 EID₅₀ per 0.2 ml. Groups AV and OV were given aflatoxin B₁ and ochratoxin A, respectively, and also infected with the virus. Group C served as control. There was mild enlargement and paleness of the liver up to 18 days post inoculation in group V; there were no lesions in group A; and there was gradual enlargement of the kidneys from 10 days post feeding of mycotoxin onwards in group O. In the combined groups AV and OV the gross lesions were slightly more severe. In group V, varying degrees of degenerative histopathological changes, congestion and haemorrhages were seen particularly in the liver, followed by the kidneys, bursa, spleen, myocardium and lungs, along with intranuclear inclusion bodies in the hepatocytes, mostly in the early stages of infection. Similar microscopic changes, but without inclusion bodies, were seen in groups A and O and the changes were pronounced in the later stages. In group O, the kidney lesions were more pronounced than the liver lesions. In the concurrently infected groups, AV and OV, the changes were similar but slightly more marked than in the corresponding individual groups. Inclusion bodies in hepatocytes were more frequent, more prominent and appeared earlier in the concurrent groups.Abbreviations AGPT agar gel precipitation test - DPF days post feeding of the mycotoxin - DPI days post inoculation - EIA enzyme immunoassay - EID₅₀ dose at which 50% of embryos will be infected - IBH inclusion body hepatitis - PAU Punjab Agricultural University     

Haematological and biochemical studies in broiler chicks fed ochratoxin and inoculated with inclusion body hepatitis virus,singly and in concurrence

Day-old broiler chicks, which had been shown to be negative for maternal antibodies against inclusion body hepatitis (IBH) virus and for viral antigen in cloacal swabs, were divided into four groups of 20 chicks each. One group was fed ochratoxin-A at 0.5 ppm from 3 to 38 days of age, another group was inoculated with 1 ml of IBH virus containing 10^6.5 EID₅₀ per 0.2 ml. A third group was given both ochratoxin-A and infected with IBH virus. The fourth group served as the control. Anaemia was observed in all three treated groups but the changes were more pronounced in the combined group. The biochemical changes also suggested a cumulative damaging effect by ochratoxin-A and IBH virus.     

Vertical transmission of avian adenovirus associated with inclusion body hepatitis

Using an indirect enzyme-linked immunsorbent assay, avian adenoviral antigens were detected in the yolk and albumen of eggs derived from broiler breeder chickens which were known to be infected with a strain of virus capable of causing inclusion body hepatitis. Viral antigens were detected in egg yolk (16/60) more frequently than in the albumen (5/60). Direct detection of viral antigens in eggs strongly supports the hypothesis that transovarian transmission of inclusion body hepatitis virus occurs if infection is present in breeder flocks.     

Genetic characterization, pathogenicity, and protection studies with an avian adenovirus isolate associated with inclusion body hepatitis

An avian adenovirus (AAV) was isolated from liver samples of two 2-wk-old broiler-breeder flocks obtained from grandparents vaccinated at 10 and 17 wks of age with an autogenous inactivated vaccine containing the European AAV 8 (8565 strain) and 11 (1047 strain) serotypes (AAV8/11 vaccine). Affected broiler-breeders exhibited clinical signs and macroscopic and microscopic lesions associated with inclusion body hepatitis (IBH). The isolated adenovirus, identified as Stanford, was molecularly characterized as European serotype 9. The pathogenicity of the Stanford strain was confirmed after inoculation of specific-pathogen-free (SPF) chickens at 1-7 days of age, causing 100% and 20% mortality, respectively. The level of protection against IBH was evaluated in two broiler-breeder progenies from AAV 8/11-vaccinated grandparent flocks and a commercial broiler flock by challenge at 1 or 7 days of age with the AAV 8 and 11 serotypes and/or the Stanford strain. The broiler-breeder progenies and the commercial broiler flock exhibited protection against IBH after challenge. No significant differences in mean body weights were observed at 3 wk of age in any of the evaluated groups. We conclude that broiler-breeder progenies from 30- to 50-wk-old grandparents vaccinated with the AAV 8/11 vaccine were adequately protected against challenge with the AAV 8 and 11 serotypes and the Stanford strain.     

Structure of inclusion bodies and electron microscopic virus detection in naturally occurring inclusion body hepatitis in chickens]

Electron microscopy was used to examine the liver of chickens with spontaneous inclusion body hepatitis. Eosinophilic inclusion bodies only were established from two flocks, mainly amphophilic from one flock, and primarily basophilic from another two flocks. Eosinophilic inclusion bodies were predominant in broiler chickens with dystrophic fatty degeneration of the liver, while basophile inclusion bodies were recorded primarily from parental or laying-hen chickens with reduced metabolic stress of the liver and more focal necrosis. The eosinophilic inclusion bodies consitsed of a filamentous matrix, with virus particles not safely detectable. The amphophilic inclusion bodies contained parvovirus particles, most likely adenoassociated virus, while the basophilic inclusion bodies inclused parvoviruses or adenoviruses (in flock NO. IV) or adenoviruses only (in flock No. V) in an amorphous chromatin matrix. The presence of parvoviruses in field material was taken to suggest a possible role of those pathogens in inclusion body hepatitis.     

Fowl adenoviruses isolated from chickens with inclusion body hepatitis in Japan, 2009-2010

Nine fowl adenoviruses (FAdVs) isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2009 to 2010 were characterized serologically and genetically. These isolates were all neutralized by antisera against the SR-48 strain (FAdV-2). Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all isolates were almost identical except one isolate in 2009. This suggests a common ancestor for the FAdVs obtained from chickens with IBH in Japan in 2010.     

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.     

Further characterization of an avian adenovirus associated with inclusion body hepatitis in bobwhite quails

An adenovirus (isolate 1452) associated with inclusion body hepatitis of bobwhite quails (Colinus virginianus) was characterized as a group I, serotype 1 avian adenovirus and was indistinguishable from quail bronchitis virus. Bobwhite quails were inoculated via the intratracheal or intraperitoneal route with 10(6) mean tissue-culture infective dose of isolate 1452 at 1, 3, 6, or 9 weeks of age. Lesions produced by either route of inoculation were similar to those of quail bronchitis and included necrotizing tracheitis, proliferative and necrotizing bronchitis and pneumonia, and multifocal necrotizing hepatitis, necrotizing splenitis with or without hyperplasia of splenic macrophages, and lymphoid necrosis and atrophy of the bursa of Fabricius. Basophilic intranuclear viral inclusions were present in respiratory mucosal epithelium, hepatocytes and occasionally bile duct epithelium, and the mucosal epithelium overlying follicles of the bursa. Results indicate that isolate 1452 is a field isolate of quail bronchitis virus and that inclusion body hepatitis of bobwhite quails is a manifestation of quail bronchitis.     

Reproduction of hydropericardium syndrome in three-week-old cyclophosphamide-treated specific-pathogen-free chickens by adenoviruses from inclusion body hepatitis

The pathogenicity of serotype 8 group I avian adenovirus (GIAAV) strains (TR630 and Saga97 strains) from inclusion body hepatitis (IBH) against cyclophosphamide (CY)-treated 3-wk-old specific-pathogen-free (SPF) chickens was examined. SPF chickens were inoculated intramuscularly with 10(7) plaque-forming units of viruses. Both strains from IBH could produce hydropericardium and mortality in CY-treated chickens as hydropericardium syndrome (HPS) that serotype 4 GIAAV strains cause, although they could not induce either hydropericardium or mortality in nontreated chickens. Histologically, hepatocytic necrosis with intranuclear inclusions, pancreatic acinar necrosis with intranuclear inclusions, and epicardial edema were seen in CY-treated chickens inoculated with GIAAV from IBH. Immunohistochemically, these inclusions were positive against GIAAV antigen. There were neither histologic lesions nor positive reactions against GIAAV antigen in nontreated chickens inoculated with GIAAV from IBH. From the present findings, pathogenic characteristics of IBH strains and HPS strains in the chickens were essentially the same.     

Reproduction of inclusion body hepatitis in conventionally raised chickens inoculated with a New Zealand isolate of avian adenovirus

Conventionally raised chickens were inoculated with a local isolate of serotype 8 of avian adenovirus by an oral or intraperitoneal route, or were exposed to the infection by contact. Fatal hepatitis, resembling inclusion body hepatitis, occurred in 30% and 45% of the birds inoculated by the oral and intraperitoneal routes respectively, and severe growth depression was recorded in survivors and in birds in contact. Birds which had maternally derived virus neutralising antibody titres of 64 or greater at the time of viral exposure did not succumb to fatal infection, but their growth rates were significantly depressed.