Inclusion body hepatitis as a primary disease in broilers in Saskatchewan, Canada

In recent years inclusion body hepatitis (IBH) has emerged as an economically important disease in Western Canada. Historically, infections with infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) have been known to suppress the immune system of broilers and make them more susceptible to a secondary disease such as IBH. Recently it has been reported that virulent adenoviruses are able to cause IBH as a primary disease in broilers without apparent involvement of IBDV and CAV. The objectives of this study were to examine the possible association of IBH with IBDV and CAV infections in Western Canada and to identify adenoviruses involved in outbreaks. Serum samples from 17 broiler-breeder flocks and their progeny were collected when broilers were hatched and then again from broilers at the time of slaughter, and these samples were tested for IBDV and CAV antibodies by enzyme-linked immunosorbent assay (ELISA). Based on the ELISA titers the antibody response to vaccination against IBDV and CAV was at an expected level in all broiler flocks. Therefore, IBH outbreaks in these flocks were not due to inadequate levels of antibodies against IBDV and CAV. Moreover, there was no correlation found between occurrences of IBH outbreaks in broilers and their IBDV or CAV titers at the time of processing. Viruses that were isolated from livers of birds suffering from IBH could be classified into four different genotypes. Their hexon gene loop 1 sequences showed high percentages of identity to FAdV-7, FAdV-8a, FAdV-8b, and FAdV-11. The results of this study could not demonstrate an association of IBH with IBDV and CAV infections, but they supported the hypothesis that IBH in broilers in Western Canada is a primary disease with no apparent immunosuppressive involvement.     

A primary epidemic of inclusion body hepatitis in broilers

Inclusion body hepatitis (IBH) was diagnosed in 15 broiler flocks supplied by one breeder in the South Island of New Zealand. The affected flocks suffered mortality up to 30%. Malaise and slightly increased mortality were noticed by growers from about day 12 post-hatch; mortality peaked in the fourth week, and, in most flocks, declined to normally accepted levels from day 33 on. Gross signs seen at necropsy usually included bone-marrow aplasia, atrophy of the bursa of Fabricius and the thymus, and swollen hemorrhagic livers with focal necrosis. Jaundice was seen in many surviving birds. In some flocks, there was also proventricular hemorrhage, mild tracheitis, and airsacculitis. Downgrading and condemnation rates were increased in all flocks. Eosinophilic intranuclear inclusion bodies were seen in hepatocytes of some affected birds. An adenovirus was isolated from a number of cases investigated. The disease in broilers was preceded by production drops associated with feed refusal and increased mortality in the breeder stock.     

Inclusion body hepatitis caused by fowl adenovirus in broiler chickens in Japan, 2009-2010

From January 2009 to June 2010, many broiler chicks suddenly died without clinical signs. The mortality rates were from 1.2% to 17.0% in affected flocks. Inclusion body hepatitis (IBH) was detected in 13 prefectures (northern, eastern, western, and southern areas) in Japan. The livers were enlarged and pale. The bursa of Fabricius and thymus had not atrophied. Multifocal necroses of hepatocytes with basophilic intranuclear inclusions were seen in the liver. Eosinophilic intranuclear inclusion bodies in hepatocytes were rare. Focal necrosis of acinar cells with basophilic intranuclear inclusions was found in the pancreas. Basophilic intranuclear inclusion bodies were detected in intact surface epithelial cells of gizzard and epithelial cells of the small intestine. The intranuclear inclusions of liver, pancreas, gizzard, and small intestine were stained positively for immunohistochemistry of fowl adenovirus (FAV) antigen. Ultrastructurally, basophilic intranuclear inclusions consisted of viral particles approximately 70 nm in diameter and arranged in a crystalline array. FAV was isolated from the liver of chickens affected with IBH. The serotype of most isolates was 2. This study suggests that IBH produced by FAV is epidemic in broiler chicks in Japan and that the present cases occurred as the primary disease without the association of infectious bursal disease virus or chicken anemia virus.     

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.     

Chicken anemia virus and fowl adenoviruses: association to induce the inclusion body hepatitis/ hydropericardium syndrome

The effects of a simultaneous and/or a subsequent coinfection with chicken anemia virus (CAV) isolate 10343 and fowl adenovirus (FAV) isolate 341 in specific-pathogen-free light chickens were evaluated. The simultaneous coinfection was conducted by the intramuscular route, whereas the subsequent coinfection trial considered FAVs administered orally. In trial 1, 20-day-old chickens simultaneously coinfected with CAV (10343) and FAV (341) intramuscularly (i.m.) showed 55% mortality and characteristic signs and lesions of inclusion body hepatitis/hydropericardium (IBH/HPS). In contrast, birds singly infected with FAV i.m. showed 10% mortality due to IBH/HPS. Trial 2 showed that birds receiving FAV 341 orally at day 7 post-CAV intramuscular infection (group A) developed a mild form of IBH/HPS with presence of inclusion bodies (INIBs) in 60% of the group and virus-neutralizing antibodies against FAV 341. Group B (FAV orally 14 days after CAV) showed significant decreased weight gain, nonspecific microscopic lesions in the liver, spleen, bursa, and thymus, and an antibody response against FAV 341. However, no INIBs could be detected in the hepatocytes of these chickens. Group C (FAV orally 35 days after CAV) showed nonspecific histopathologic changes in the liver and no antibody response to FAV. The oral single infection with FAV isolate 341 induced neither mortality nor macroscopic lesions of IBH/HPS in the birds. The present results corroborate previous reports on pathogenicity of Chilean FAV isolates, which suggest that synergism with other viruses or prior immunosuppression is necessary to produce IBH/HPS in chickens. These results also suggest that the susceptibility of chickens to FAV oral infection resulting in IBH/HPS varies throughout the course of CAV infection.     

The fowl adenovirus (Fadv-11) outbreak in Iranian broiler chicken farms: The first full genome characterization and phylogenetic analysis

Fowl adenoviruses D and E (FAdV-D and E) can cause inclusion body hepatitis (IBH) in commercial chicken flocks. Recently, IBH outbreaks have been increasingly reported in different regions of Iran, particularly in broiler farms. The present study was conducted to perform, for the first time, a complete genome characterization of a FAdV isolate from an IBH outbreak in Iran. Briefly, liver samples were collected from affected broiler flocks and following viral DNA extraction and confirming by PCR technique; one positive sample was selected from an affected flock to conduct a complete genome sequencing. The current FAdV, named "Fowl_Adenovirus_D_isolate_iran/UT-Kiaee_2018", was placed into FAdV-11 serotype (D species). According to the complete genome sequence analysis, UT-Kiaee had high homology with Chinese and Canadian FAdV. The partial sequence of the hexon gene revealed that UT-Kiaee shared 100% identity with previous Iranian FAdVs. The present study was the first to report full genome FAdV in Iran and complete the puzzle of molecular epidemiology of FAdV in Iran through determining the possible origin of Iranian FAdvs, which are the causative agents of recent IBH outbreaks in Iran.     

Induction of hydropericardium in one-day-old specific-pathogen-free chicks by adenoviruses from inclusion body hepatitis

The pathogenicities of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) strains of adenovirus for specific-pathogen-free (SPF) chicks were compared. One-day-old SPF chicks inoculated intramuscularly with the DPI-2 (serotype 2), S-PL1 (serotype 2), TR630 (serotype 8), and Saga97 (serotype 8) strains from IBH and with the LVP-1 strain (serotype 4) from HPS exhibited the mortality, liver enlargement, and hydropericardium characteristic of gross change found in HPS. The chicks inoculated with the IBH and HPS strains exhibited similar histologic and immunohistochemical changes. Neither mortality nor pathologic changes occurred in 3-wk-old SPF chicks inoculated with IBH strains, although HPS strain induced HPS lesions in them. This study indicates that IBH strains of adenovirus can also reproduce HPS lesions and mortality in 1-day-old SPF chicks and that IBH and HPS strains may have similar pathogenicities except for their different virulence for older chickens.     

Serologic monitoring of a broiler breeder flock previously affected by inclusion body hepatitis and testing of the progeny for vertical transmission of fowl adenoviruses

The increasing number of clinical cases of inclusion body hepatitis (IBH) associated with fowl adenoviruses (FAdVs) is a growing concern in different parts of the world, including Canada. After an outbreak of IBH in a 10-d-old pullet broiler breeder flock, we serologically monitored the flock from 8 to 46 wk of age, using the agar gel precipitation test (AGPT) offered by diagnostic laboratories and an FAdV group-specific enzyme-linked immunosorbemt assay (ELISA) developed earlier. In addition, we tested 1-d-old progeny for possible vertical transmission of FAdV when the breeder flock approached the peak of egg production by performing virus isolation and polymerase chain reaction (PCR) procedures on target organs. As in previous studies comparing the 2 tests, ELISA was more sensitive than AGPT. With ELISA, a few birds had weakly positive results at 8 wk of age, and all the birds had strongly positive results from 12 wk of age until the end of the study. This group-specific ELISA is therefore a sensitive and practical way to monitor FAdV antibodies in commercial flocks. None of the 1-d-old chicks tested were positive by PCR, nor was FAdV isolated from the same tissues, indicating an absence of transmission of infectious virus to the progeny. The lack of virus production and transmission could be due to the presence of high antibody titers in the layers.     

Inclusion Body Hepatitis in Young Broiler Breeders Associated with a Serotype 2 Adenovirus in Ontario,Canada

In July 2001 in southwestern Ontario, a young broiler breeder pullet flock experienced 2.1% mortality from 10 to 14 d of age. Mortality prior to and after this period was normal. Clinical signs included depression, lateral recumbency, and death with feed present in the crop. Necropsy lesions included friable, pale yellow-white livers scattered with hemorrhages. Histological examination revealed necrotizing hepatitis with large basophilic intranuclear inclusion bodies in the hepatocytes compatible with inclusion body hepatitis (IBH). An adenovirus from livers was isolated in chicken hepatoma cells. Virus neutralization and DNA analysis based on restriction fragment length polymorphism performed on this isolate were consistent with group I fowl adenovirus serotype 2. The origin of the adenovirus involved in this outbreak of IBH could not be established with certainty.     

Serological identification of avian adenoviruses isolated from cases of inclusion body hepatitis in Victoria, Australia

Sixteen avian adenoviruses isolated from 12 cases of inclusion body hepatitis (IBH), 3 cases of respiratory disease, and a case of ruptured tendons were compared using antisera raised against 9 fowl adenovirus prototype strains. Eleven isolates from livers of birds with IBH were classified into 4 different serological groups: 1) YR36 (type 7)-related; 2) HVI (type 8)-related; 3) Variants--type 6-,7-, and S-related; and 4) Type 50--not closely related to any of the prototype antisera tested. These results indicate that, as in other countries, IBH in Victoria is associated with several serologically distinct adenoviruses. The other five adenovirus isolates were found to be related to CELO (type 1).     

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.     

Pathogenicity of quail's inclusion body hepatitis virus (avian adenovirus-1) for Japanese quails and broiler chicks

Quail (Coturnix coturnix japonica) and broiler (Gallus domesticus) chicks were inoculated experimentally with IBH virus (avian adenovirus-1) derived from quails to determine its pathogenicity. Quail chicks were inoculated by the intraperitoneal route at 3, 4, 5, 6 or 7 weeks of age. Lesions were encountered most frequently in the liver, kidneys and lungs. These included pale, swollen and mottled liver, swollen nephrotic kidneys, and congested and pneumonic lungs. The lesions were severe in birds inoculated at 5 weeks of age. Large basophilic intranuclear inclusion bodies were seen in hepatocytes and occasionally in the renal epithelium. The results showed that this isolate is pathogenic for quails above 3 weeks of age. Broiler chicks were inoculated at 4 weeks of age by the intraperitoneal route. The lesions produced in these chicks were similar to those of adenovirus-induced inclusion body hepatitis. Viral antigen was also demonstrated by dot-ELISA in suspensions of liver tissue from both quail and broiler chicks.Abbreviations AAF amnio-allantoic fluid - AAV avian adenovirus - DPI days post inoculation - EID₅₀ dose infective for 50% of embryos - ELISA enzyme-linked immunosorbent assay - IBH inclusion body hepatitis - INIBs intranuclear inclusion bodies - NAF normal allantoic fluid     

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis

Objective Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. Methods Twenty‐six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high‐resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. Results FAdV‐8b and FAdV‐11 were identified in 13 cases each. In one case, FAdV‐1 was also identified. Cross‐neutralisation was observed between the FAdV‐11 field strain and the reference FAdV‐2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV‐8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV‐11 field strain had the highest identity to FAdV‐11 (93.2%) and FAdV‐2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. Conclusion These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species.     

High incidence of glomerulonephritis associated with inclusion body hepatitis in broiler chickens: routine histopathology and histomorphometric studies

During the routine histologic evaluation of an outbreak of inclusion body hepatitis (IBH) in Mississippi broilers, a high incidence of renal enlargement and glomerulonephropathy was observed in the birds presenting classic hepatic pathology. Characteristic intranuclear adenoviral inclusion bodies were demonstrated in the livers of these birds, and fowl adenovirus was identified by viral isolation and by PCR. The glomerular lesions were consistent with proliferative or membranoproliferative forms of glomerulonephritis. Histomorphometric evaluations were performed to generate a more quantitative analysis of altered glomerular size and cellularity, to detect statistically significant borderline changes, and to get a clearer insight into the incidence of the glomerular alterations. Marked increases in both the average glomerular size (area) and the total glomerular cellularity were observed for the affected glomeruli relative to normal controls. The average glomerular area values for normal glomeruli in the peripheral subcapsular cortical and central cortical kidney regions were 1791 microm2 and 5302 microm2, respectively. In contrast, glomerular measurements for kidneys exhibiting glomerulonephritis by routine histopathology, had average values for the two regions of 4429 microm2 and 11,063 microm2. The average glomerular cell counts for the two regions in controls were 44 and 107 cells/ glomeruli, while averages for birds with glomerulonephritis were 85 and 193 cells/glomeruli. The proportion of IBH-associated glomeruli greater than two standard deviations above the mean glomerular size of the normal controls was 52% for the central region and 62% for the peripheral region.     

Economic effects of clinical chicken anemia agent infection on profitable broiler production.

An outbreak of anemia dermatitis syndrome caused by chicken anemia agent (CAA) occurred in 15 broiler flocks. An average of 29% of chickens in these flocks were derived from a common breeder flock. The breeder flock had no antibody to CAA at 20 weeks of age but had seroconverted by 31 weeks. Diseased broiler flocks were derived from eggs laid by the breeder flock between 25 and 30 weeks of age. CAA infection in the breeder flock was subclinical, with no apparent effects on mortality or performance. A strategic program of therapeutic and/or prophylactic antibiotic therapy was begun in affected broiler flocks as soon as the disease was diagnosed. Nevertheless, when the cost of therapy was taken into account, affected broiler flocks had a net income 17.3% to 19.6% lower than normal flocks. Average bird weights were 3.3% to 3.5% lower in affected flocks than in unaffected flocks, and affected flocks had a significantly greater proportion of lighter birds. Average mortality in affected flocks was 2.0% to 2.3% higher than in normal flocks, with peak mortality occurring in the third week of life. There was no apparent effect on feed-conversion ratio.     

Incidence of histomonosis in turkeys in France since the bans of dimetridazole and nifursol

Between April 2003 and March 2005, 113 outbreaks of histomonosis were recorded in standard turkey farms in France, and 15 cases were recorded in turkey breeding centres. Most of the cases were in north-west France, the principal farming area for turkeys. The majority of the cases occurred during the hottest months, from April to September. Large numbers of cases occurred among birds from four to eight weeks of age, but there were some cases in three-week-old birds and some in birds up to 17 weeks of age. In most of the standard turkey flocks the mortality was less than 10 per cent, but it was above 30 per cent in nearly 20 per cent of the outbreaks. In the breeding flocks, the average mortality was 60.2 per cent. The size of the flocks, the sex of the birds and the age at which the first clinical signs appeared did not seem to influence the mortality.     

Studies on effect of lighting on "Sudden death syndrome" in broiler chickens.

Broiler chicken flocks were studied to determine the mortality from sudden death syndrome occurring in the flocks. The difference in the incidence of the syndrome in pullets and cockerels, and the age at which the most birds are affected were also studied. The weight of sudden death syndrome birds was compared with the flock average and the effect of continuous lighting as opposed to intermittent lighting was examined.The results suggest that; continuous lighting produces more sudden death syndrome deaths than intermittent lighting; that the incidence of sudden death syndrome is higher in cockerels than pullets; that the highest death rate occurred during the third and fourth weeks of life, and that sudden death syndrome birds on the average were heavier than the flock average.     

Characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in Chile.

Three fowl adenovirus (FAV) isolates (341, 344, and 215) obtained during 1996-97 from field outbreaks of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) affecting broilers and broiler breeders in Chile were characterized by virus neutralization tests (VNTs) and restriction enzyme analysis of a DNA fragment. Furthermore, the pathologic characteristics of one of these FAV isolates (FAV 341) was studied in experimentally infected chickens. The VNTs conducted with isolates 341 and 344 against reference strains and antisera belonging to each of 12 FAV serotypes demonstrated a close antigenic relationship with strain KR5 of the FAV serotype 4. Polymerase chain reaction using the primers H3/H4 and subsequent HpaII digestion was used for serotype identification of isolates 341 and 215. The length of the PCR products and the restriction profiles of isolates 341, 215, and the reference strain KR5 (FAV4) were identical. The present results confirmed the classification of all three isolates as FAV4. The pathogenicity test with 1000 mean tissue infectious dose of isolate 341 inoculated intramuscularly in 20-day-old specific-pathogen-free chickens resulted in the death of 9% (two birds) six days postinoculation (PI). Both birds showed characteristic IBH/HPS gross and microscopic lesions; the remaining birds, sacrificed at day 10 PI, showed less severe lesions. On the basis of epidemiologic and experimental data of the virulence of Chilean FAV isolates, and the pathogenicity results with isolate 341, we speculate that Chilean FAV strains may require an association with other agents (immunosuppressive agents) to induce IBH/HPS outbreaks in the field.     

Studies on Effect of Lighting on “Sudden Death Syndrome” in Broiler Chickens

Broiler chicken flocks were studied to determine the mortality from sudden death syndrome occurring in the flocks. The difference in the incidence of the syndrome in pullets and cockerels, and the age at which the most birds are affected were also studied. The weight of sudden death syndrome birds was compared with the flock average and the effect of continuous lighting as opposed to intermittent lighting was examined.

The results suggest that; continuous lighting produces more sudden death syndrome deaths than intermittent lighting; that the incidence of sudden death syndrome is higher in cockerels than pullets; that the highest death rate occurred during the third and fourth weeks of life, and that sudden death syndrome birds on the average were heavier than the flock average.

     

Emergence of fowl aviadenovirus C-4 in a backyard chicken flock in California

Fowl aviadenovirus (FAdV) species D and E are associated with inclusion body hepatitis (IBH); species C, serotype 4 (hereafter, FAdV4) is associated with hepatitis–hydropericardium syndrome (HHS) in young chickens. Outbreaks of HHS have led to significant losses in the poultry industry in several countries, predominantly in China. In April 2020, FAdV4 was detected in a remote backyard flock in California. In a mixed flock of chickens of various breeds and ages (6 mo to 2 y old), 7 of 30 were found dead within a week without premonitory signs. One additional bird died after the flock was relocated to fresh pasture, bringing the total mortality to 8 of 30 (27%). Postmortem examination of 3 birds revealed good body condition scores and active laying. One chicken had subtle hemorrhages throughout the liver, and the other 2 had diffusely dark mahogany livers. On histopathology, 2 chickens had hepatic necrosis with hepatocytes containing large, mostly basophilic, intranuclear inclusion bodies, identified by electron microscopy as 82.2-nm diameter adenoviral particles. Virus isolation and genomic sequencing performed on a liver sample revealed strains with 99.9% homology to FAdV4 isolates reported from China. To our knowledge, FAdV4 has not been reported in the United States to date. Furthermore, the chickens affected here were all adults and exhibited a variation of serotype 4 disease in which IBH was present but not hydropericardium.