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

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1 2005 Poultry Science Association, Inc. Inclusion Body Hepatitis in Young Broiler Breeders Associated with a Serotype 2 Adenovirus in Ontario, Canada C. Philippe,* H. Grgic, and É. Nagy,1 *Horizon Poultry, 90-10th Avenue, Box 1000, Hanover, Ontario, N4N 3T4, Canada; and Department of Pathobiology, University of Guelph, Guelph, Ontario, NIG 2W1, Canada Primary Audience: Veterinarians, Service People, Production Managers SUMMARY 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. Key words: fowl adenovirus, serotype 2, broiler breeder, inclusion body hepatitis, restriction fragment length polymorphism 2005 J. Appl. Poult. Res. 14: DESCRIPTION OF PROBLEM Group I avian adenoviruses are ubiquitous in domestic fowl and frequently of low virulence. They have often been isolated from asymptomatic chickens [1, 2]. The fowl adenoviruses (FAdV) have been classified into 11 serotypes, some of which have more often been associated with clinical disease. Serotype 8 viruses have been isolated from peracute inclusion body hepatitis (IBH) in Australia [3], and serotype 4 viruses have been involved in cases of hydropericardium syndrome in Asia and Latin America [4, 5]. Other serotypes have been involved more frequently in disease episodes in the last few years, leading to an increased interest in identifying these various isolates [6, 7], as is the case in Ontario, Canada [8]. Genomic characterization of the viral DNA by the polymerase chain reaction (PCR) or restriction fragment length polymorphism (RFLP) or both constitutes a valuable tool in the investigation of adenoviruses implicated in disease outbreaks [9, 10, 11]. Inclusion body hepatitis was first described in the US in 1963 by Helmbolt and Frazier [12]. 1 To whom correspondence should be addressed: enagy@ovc.uoguelph.ca.

2 PHILIPPE ET AL.: INCLUSION BODY HEPATITIS IN YOUNG BROILER BREEDERS 589 The disease occurs mainly in chickens 3 to 7 wk of age, but it has been reported in chickens less than 1 wk old [13]. A sudden onset and sharply increased mortality are seen with the disease. Mortality can reach up to 10% and comes back to normal levels after 5 to 10 d. Enlarged mottled liver, pale icteric skin, hemorrhages, swollen pale kidney, and pale bone marrow are present at necropsy, and intranuclear inclusion bodies are often seen at histopathology. Adenovirus is believed to act as a secondary pathogen, following infection with an immunosuppressive agent, such as infectious bursal disease virus or chicken infectious anemia virus [14]. However, adenovirus has been reported as the primary pathogen of IBH [15, 16]. This case report describes an IBH outbreak in 10-d-old broiler breeder pullets in southwestern Ontario, Canada. FIELD REPORT Case History, Gross Pathology, and Histopathology In July 2001 in southwestern Ontario, Canada, a 10-d-old broiler breeder flock experienced a sudden increase in mortality. From d 10 to 14, 208 pullets out of 9,750 died (2.1% mortality). Starting at 15 d of age, the mortality went back to normal (0.2% per week). The cockerels raised on the same premise in a different barn were not affected. Clinically, the affected pullets were lying on their sides, were weak and depressed, and some held their head back and their legs were stretched out. Some birds died very quickly with full crops and without any other clinical signs. Necropsy revealed friable, pale yellow-white livers scattered with hemorrhages. Spotted hemorrhages were seen on the abdominal walls and thighs and on the pale and swollen kidneys. Areas of congestion of ventriculus and spleen were present. The crops were full, and the thymus and bursa were grossly normal for this age. Histologically, necrotizing hepatitis with large basophilic intranuclear inclusion bodies in the hepatocytes compatible with inclusion body hepatitis was present. The bursas had histologically evident follicular lymphoid depletion. The Premises and Background of Flocks The affected pullet farm had 3 barns (1, 2, and 3) situated next to one another and was used to grow broiler breeder flocks from 0 to 20 wk of age. Each barn had 2 levels, a lower level cement floor and an upper level wooden floor. The barns were heated with propane, using tube heaters located all along the brooding side (vent side). All barns were equipped with a side-to-side ventilation system and automatic trough feeders and nipple drinker water systems. Water supply to all 3 barns came from 1 well located on the premise. The period between flocks for each barn was 4 wk. During this 4-wk downtime period, the barn was cleaned out and disinfected as follows. Litter was removed from the barn and taken away from the premise. The lower and upper level floors were first pressure washed and sprayed with formaldehyde-type disinfectant and let to dry; the lower and upper level floors were then fogged with a quaternary ammonia-based disinfectant. The drinker lines were flushed with chlorine after barn disinfection. A rodenticide was placed in bait stations. There was 7-d period between the end of the barn cleanout and disinfection and the next flock s placement for each barn. The pullets of the affected flock, flock A, were housed in barn 1, while the cockerels of flock A were placed in barn 2. At the time of flock A placement, barn 3 was empty. Day-old broiler breeders (flock B) from a different primary breeder supplier and owned by another Ontario company were placed 2 wk later in barn 3. Flock B did not experience IBH. When flock B was 7 wk old, litter from barn 1 was transferred to barn 2 and to barn 3 to ensure flock exposure to this particular adenovirus prior to lay: 10, 20-L buckets filled with litter from the brooding area in barn 1 were spread on each floor in barn 3. Five of these buckets were spread in the cockerel pen in barn 2. The company s broiler breeder flocks are always started on fresh wood shavings. Flock A was vaccinated at the primary breeder hatchery with a commercial Marek s disease vaccine. Coccidiosis vaccination was performed at d 1 at the pullet farm using vaccine pucks distributed uniformly in the brooding area. Day-old chick box paper swabs were tested by classical enrichment method at the Animal Health Laboratory (AHL) [17] and were negative for Salmonella. Day-old sera (20 sera from pullets and 10 sera from cockerels) tested negative for Mycoplasma gallisepticum

3 590 JAPR: Field Report TABLE 1. Serology results of grandparent flock Enzyme-linked Agar gel immunosorbent precipitation assay (ELISA) (number (number Mean positive/number positive/number ELISA Age (weeks) tested) tested) titer 33 5/12 ND /20 20/20 6, /20 20/20 8, /20 19/20 8,375 1 ND = not determined. and Mycoplasma synoviae with the plate agglutination test also performed at AHL. Broiler breeder hens (flock C), located on a different premise, had been raised in barn 1 prior to flock A pullets. In July 2001, just after the IBH outbreak at the pullet farm (barn 1), 10, 29-wkold flock C hens were submitted to AHL for testing. Serum samples, oviduct, kidneys, and cecal tonsils were collected for serology, virus isolation, and detection by PCR. No adenovirus was isolated after 3 passages in hepatoma cells, and the PCR results were also negative done on the samples and third passage cells for fowl adenovirus. Serology results based on agar gel precipitation test (AGPT) conducted by AHL indicated that broiler breeder flock C was positive for adenovirus. Follow-up serology results, using AGPT for flock C, were as follows: 11/15, 13/15, 10/ 15, and 11/15 were positive for adenovirus at 37, 40, 46, and 50 wk, respectively. Grandparent flocks from the primary breeder company were routinely vaccinated twice during the rearing period against adenovirus with an autogenous-inactivated vaccine containing an FAdV serotype 6 strain. Periodic serologic monitoring, using AGPT and a locally produced enzyme-linked immunosorbent assay (ELISA) us- ing an FAdV serotype 6 strain as an antigen, was performed by the company before the onset of egg production and during the production cycle. This strategy has been practiced for many years to induce immunity in grandparent flocks under stringent biosecurity practices, to reduce the risk of adenovirus infection during egg production, and to provide maternal immunity to day-old progeny. The primary breeder indicated that at the time of egg contribution to the pullet stock order, the grandparent flock (female line) was just over 40 wk old. The grandparent flock serological results (Table 1) indicated the presence of antibodies against adenovirus. No other IBH cases were reported in broiler breeder placements coming from this grandparent flock during the life of the flock. Virology and DNA Analysis Virus isolation was done from liver samples with lesions from clinically sick or dead chickens. The liver samples were homogenized and centrifuged at 3,000 rpm for 10 min at 4 C. The supernatant was passed through a 0.22-µm filter and then inoculated onto chicken hepatoma cells (cell line) [18]. Cytopathic effect consisting of rounding, clumping of cells and detachment from TABLE 2. Results of virus neutralization assay, using 100 plaque-forming units (PFU) of the Ontario field isolate of fowl adenovirus and a panel of known antibodies Plaque reduction (%) Serotype Ab Fold dilution of antibody (Ab).

4 PHILIPPE ET AL.: INCLUSION BODY HEPATITIS IN YOUNG BROILER BREEDERS 591 FIGURE 1. Ethidium-bromide-stained agarose gels of restriction endonuclease digested DNA extracted from fowl adenoviruses (FAdV). Panels A and B are BamHI and EcoRI digestions, respectively. Lane 1: FAdV-9, strain A- 2A; lane 2: FAdV-1, CELO virus; lane 3: 1998 Ontario field isolate; lane 4: 2001 Ontario field isolate; lane 5: isolate from this outbreak; lane 6: 2001 Quebec field isolate; lane M: 1-kb DNA ladder. the surface was present by d 3 postinoculation. A plaque reduction assay, using a panel of antibodies against FAdV serotypes, was performed as we described earlier [19], and the data are presented in Table 2. Based on the results, the isolated virus was identified as serotype 2. When 7-d-old agent-free chickens were inoculated with the isolated virus, we were able to reproduce the disease and reisolate the same virus. For DNA analysis, fowl adenoviruses were propagated in chicken hepatoma cells and harvested at maximum cytopathic effect. Virus purification was done as described [20]. The DNA was extracted by phenol and chloroform after digestion with 0.5% final concentration of sodium dodecyl sulfate and 500 µg/ml of proteinase K for 2 h at 56 C and overnight at room temperature [20]. The DNA samples were digested with EcoRI and BamHI restriction enzymes [21], according to the manufacturer s recommendation. The DNA fragments were separated in 0.8% agarose gels [20]. The RFLP patterns of the isolate from the present case for both BamHI (panel A) and EcoRI (panel B) shown in Figure 1, lanes 5, were very similar to that published for strain 685, a serotype 2 virus [22], corroborating the virus neutralization test. Based on the RFLP analysis, the isolate from this case can be grouped with viruses of genotype D [22]. Source of the Virus Group I adenoviruses are widely distributed throughout the world. Viruses have been isolated from both clinically sick and healthy chickens and other species of birds, such as turkeys, geese, ducks, pigeons, gulls, and psittacines. Fowl ade-

5 592 novirus infections are often subclinical because most chicks hatch with maternal antibodies and many strains are of low virulence. However, losses incurred due to adenovirus infections (peracute IBH and hydropericardium syndrome) have increased significantly in the last 10 yr. In North America, even though peracute IBH and hydropericardium syndrome have not been reported, IBH cases have been reported more frequently compared with previous years [6]. Increased biosecurity for broiler breeder flocks to address food safety issues may reduce the chances of exposure to infectious agents. Potentially, this may result in limited immunity and passive transfer of maternal antibodies to broiler progeny. New diagnostic tests, such as PCR, RFLP, and ELISA are now readily available, allowing better detection and characterization of adenoviruses in poultry flocks than 5 to 10 yr ago. The IBH outbreaks are most often reported in chickens around 2 to 3 wk of age, and that has been seen in Ontario in the recent surge of outbreaks [23], although there have been reports in chickens as young as 7dofageandin1-dold turkeys [13, 24, 25]. The case herein occurred in chickens at 10 d of age. The IBH outbreaks are usually associated with serotype 6, 7, and 8. The virus isolated from this outbreak was serotype 2, as determined by virus neutralization and RFLP analysis. The adenovirus involved in this IBH outbreak in Ontario may have come from several potential sources, but we were unable to conclusively establish its origin. No other IBH case was reported to the primary breeder company from the same grandparent flock (female line). The absence of reported cases, however, does not fully exclude the possibility of a vertical transmission. The hepatitis in flock A occurred at a very young age (10 d old), and AGPT results indicated that the grandparent flock, which was vaccinated with an FAdV serotype 6 strain-based vaccine, was seropositive when the eggs were laid. However, other serotypes of FAdV might have been present in the flock not causing clinical disease. Furthermore, a low mortality, such as the 2.1% in this case, may not always be considered as a significant problem nor submitted to the laboratory for diagnosis or reported back to the primary breeder. The FAdV serotype 2 viruses have not been frequently detected in commercial poultry in the JAPR: Field Report US nor in Ontario. From the RFLP study of more than 50 fowl adenoviruses [8] isolated in Ontario, only 1 isolate, dated from 1998 and from a different premise, showed a DNA pattern typical of serotype 2 viruses (Figure 1, panel A, lane 3). There was 1 additional isolate from a neighboring province (Quebec) with this type of DNA pattern (Figure 1, lanes 6). All other adenoviruses isolated in Ontario in 2001 gave different DNA patterns (Figure 1). We were not able to establish any relationships among the premises of this case and the other 2 cases for which the FAdV isolates had similar DNA patterns. In addition, there was a 3-yr period between the 2 Ontario isolates, and although there was only half a year difference between the Quebec and Ontario isolations, there was a long geographical distance of about 500 km. The premises themselves could be a possible source for the virus. Flock C, which was raised in the infected premise prior to flock A, as well as the premise itself, was investigated. Flock C was serologically positive for adenovirus from 29 wk on with the AGPT test [17], indicating that these birds were exposed during the rearing phase, and that adenovirus strains were present in the area. Virus isolation, performed on a small number (10 live hens) of 29-wk-old birds from flock C, was negative for adenovirus, suggesting they did not have active infection at that time and were not shedding virus. After flock C, the barns were washed and disinfected thoroughly, and a 7-d clean downtime was observed. However, adenoviruses are very hardy viruses [16] and may have survived the cleaning and disinfection process between the flocks. Alternatively, adenovirus may have been present in material (feed, litter) or tools brought into the barn postcleaning. Personnel (clothing, shoes) via contact with other domestic poultry cannot be entirely excluded either as a possible source. Poultry flocks in North America are commonly fed chicken by-products, as was the case for Flock A. One can speculate that adenoviruses could survive the heat treatment at the rendering plant. If this were the case, birds fed with chicken by-products could become infected with adenoviruses through the feed. However, this is an unlikely explanation for the IBH outbreak described in this report.

6 PHILIPPE ET AL.: INCLUSION BODY HEPATITIS IN YOUNG BROILER BREEDERS 593 CONCLUSIONS AND APPLICATIONS 1. Serotype 2 fowl adenovirus was identified as the causative agent of IBH in a young broiler breeder flock in southwestern Ontario, Canada. 2. This report provides further evidence of the role of fowl adenoviruses as agents causing clinical disease and moderate mortality in meat-type chickens and is a reminder of the importance of accurate diagnosis in every instance. 3. This report emphasizes the need for the development of improved control strategies. There is also a need for future development in molecular methods that could help to identify the origins of adenovirus strains in IBH. REFERENCES AND NOTES 1. Yates, V. J., Y. O. Rhee, D. E. Fry, A. M. Al Mishad, and K. J. McCormick The presence of avian adenoviruses and adenovirus associated viruses in healthy chickens. Avian Dis. 20: Cowen, B., B. W. Calnek, and S. B. Hitchner Broad antigenicity exhibited by some isolates of avian adenovirus. Am. J. Vet. Res. 38: Christensen, N. H., and M. Saifuddin A primary epidemic of inclusion body hepatitis in broilers. Avian Dis. 33: Anjum, A. D., M. A. Sabri, and Z. Iqbal Hydropericardium syndrome in broiler chickens in Pakistan. Vet. Rec. 124: Cowen, B. S Inclusion body hepatitis anaemia and hydropericardium syndrome: aetiology and control. Worlds Poult. Sci. J. 48: El-Attrache, J., and P. Villegas Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis. Avian Dis. 45: McFerran, J. B., and J. A. Smyth Avian adenoviruses. Rev. Sci. Tech. 19: Nagy, E University of Guelph, Guelph, Ontario, Canada. Personal communication. 9. Erny, K. M., D. A. Barr, and K. J. Fahey Molecular characterization of highly virulent fowl adenoviruses associated with outbreaks of inclusion body hepatitis. Avian Pathol. 20: Jiang, P., D. Ojkic, T. Tuboly, P. Huber, and E. Nagy Application of the polymerase chain reaction to detect fowl adenoviruses. Can. J. Vet. Res. 63: Meulemans, G., M. Boschmans, T. P. van den Berg, and M. Decaesstecker Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathol. 30: Helmbolt, C. F., and M. N. Frazier Avian hepatic inclusion bodies of unknown significance. Avian Dis. 7: Pilkington, P., T. Brown, P. Villegas, B. McMurray, R. K. Page, G. N. Rowland, and S. G. Thayer Adenovirus-induced inclusion body hepatitis in four-day-old broiler breeders. Avian Dis. 41: Toro, H., C. Gonzales, L. Cerda, M. Hess, E. Reyes, and C. Geissea Chicken anemia virus and fowl adenoviruses: Association to induce the inclusion body hepatitis/hydropericardium syndrome. Avian Dis. 44: McFerran, J. B., R. M. McCracken, T. J. Connor, and R. T. Evans Isolation of viruses from clinical outbreaks of inclusion body hepatitis. Avian Pathol. 5: McFerran, J. B., and B. M. Adair Group I adenovirus infections. Pages in Diseases of Poultry. 11th edition. Y. M. Saif, ed. Iowa State Press, Ames. 17. Animal Health Lab. University of Guelph. Guelph, Ontario, CA. 18. Alexander, H. S., P. Huber, J. Cao, P. J. Krell, and É. Nagy Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line. J. Virol. Methods 74: Ojkic, D., and É. Nagy Antibody response and virus distribution in chickens inoculated with wild-type and recombinant fowl adenoviruses. Vaccine 22: Ojkic, D., and É. Nagy The long repeat region is dispensable of fowl adenovirus replication in vitro. Virology 283: Invitrogen Life Technologies, Burlington, Ontario, CA. 22. Zsák, L., and J. Kisary Grouping of fowl adenoviruses based upon the restriction patterns of DNA generated by BamHI and HindIII. Intervirology 22: Ojkic, D., B. Binnington, and E. Martin Inclusion body hepatitis. AHL Newslett. 7: Goodwin, M. A., D. L. Hill, M. A. Dekich, and M. R. Putman Multisystemic adenovirus infection in broiler chicks with hypoglycemia and spiking mortality. Avian Dis. 37: Guy, J. S., and H. J. Barnes Characterization of an avian adenovirus associated with inclusion body hepatitis in day-old turkeys. Avian Dis. 41: Acknowledgments We gratefully acknowledge the contribution of A. Gregorio Rosales and Eric Jensen (Aviagen, Department of Veterinary Services, Huntsville, Alabama) to this work and appreciate the invaluable discussions. We also thank the excellent technical help of Alisha Campbell. This work was supported by the Ontario Ministry of Agriculture and Food and the Poultry Industry Council (Canada).

INCLUSION BODY HEPATITIS AND HYDROPERICARDIUM SYNDROME (ADENOVIRUS INFECTIONS)

INCLUSION BODY HEPATITIS AND HYDROPERICARDIUM SYNDROME (ADENOVIRUS INFECTIONS) AVIAN ADENOVIRUSES (CHICKEN ADENOVIRUSES, FADV) Adenoviruses are common in poultry. Many replicate in healthy birds without