Experimental infection of specific-pathogen-free chickens with serotype-1 fowl adenovirus isolated from a broiler chicken with gizzard erosions

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.     

Pathogenicity of serotype 8 fowl adenovirus isolated from gizzard erosions of slaughtered broiler chickens

The pathogenicity of serotype 8 fowl adenovirus (FAV), isolated from gizzard erosions of slaughtered broiler chickens, was investigated. In experiment 1, 29 5-day-old specific-pathogen-free (SPF) chickens were inoculated with the isolates of serotype 8 FAV, M013 (group 1) or G0054 (group 2) strain, via an oral route. There were no clinical signs in any of chickens after inoculation, and mild gizzard erosions were observed macroscopically and microscopically in three inoculated chickens of group 2. FAV was recovered from gizzards and rectums but was not recovered from pancreas and livers from chickens in both inoculated groups. In experiment 2, 27 1-day-old SPF chickens were inoculated with the G0054 strain by intramuscular route. Five, 6, and 3 inoculated chickens died on days 3, 4, and 5 postinoculation (PI), respectively. Four, 3, 1, and 1 inoculated chickens became moribund with severe clinical signs such as ruffled feathers, severe depression and closed eyes from days 3 to 6 PI, respectively. Macroscopically, the common characteristic of the gross lesions of dead chickens and euthanized moribund chickens was discoloration of liver. FAV was recovered from the gizzard, liver, pancreas and rectum. Virus titers in the liver and pancreas were high until day 6 PI. Histologically, necrotizing hepatitis and pancreatitis with intranuclear inclusion bodies were observed in the inoculated chickens. These results indicate that some strains of serotype 8 FAV are able to reproduce not only gizzard erosion by oral inoculation but inclusion body hepatitis (IBH) by intramuscular inoculation.     

Adenoviral gizzard erosion in commercial broiler chickens

Pathologic and immunohistochemical changes caused by group I of the fowl adenovirus (FAV) serotype-1 99ZH strain, isolated from broiler chickens exhibiting gizzard erosion, were investigated in commercial broiler chickens. One hundred twenty-two chickens were inoculated with the strain by both oral and ocular routes at 1, 3, or 5 weeks of age and euthanatized for necropsy within 4-18 days of inoculation. Focal gizzard erosions were observed in the inoculated chickens of each age group. A histologically degenerative koilin layer, necrotic mucosa, intranuclear inclusion bodies in the glandular epithelial cells, inflammatory cell infiltrations in the lamina propria, submucosa, and a muscle layer were seen in the gizzards. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies. These findings were recognized regardless of their maternal antibody levels for FAV serotype-1. Gizzard lesions appeared later in the lower-dose-inoculated chickens than in the higher-dose-inoculated chickens. Numerous CD3-positive cells and IgY-positive plasma cells were seen in the gizzard lesions. In 5-week-old chickens the heterophil infiltrations in the lesions were milder than in younger chickens. Intranuclear inclusion bodies also were observed in the epithelial cells of the ileum or cecal tonsils of some chickens. Thus, this study shows that FAV-99ZH causes adenoviral gizzard erosion in broiler chickens without hepatic or pancreatic lesions and that cell infiltration is more severe than in dietary gizzard erosions.     

Pathogenicity of serotype 1 fowl adenovirus in commercial broiler chickens.

The pathogenicity of a serotype 1 fowl adenovirus (FAV-99ZH), isolated from broiler chickens exhibiting gizzard erosion, was investigated in commercial broiler chickens. Five-, 3-, and 1-wk-old commercial broiler chickens were inoculated with FAV-99ZH by both oral and ocular routes. In the 5-wk-old chickens (trial 1), none of which had the maternal antibody to FAV-99ZH, severe gizzard erosions were observed on days 5, 7, and 10 postinoculation (PI). Among the 3-wk-old chickens (trial 2), which were separated into a control group and three treatment groups according to their maternal antibody titer levels, some chickens showed clinical signs such as depression and anorexia. Compared with the control group, all the treatment groups showed decreased weight gain. One treatment group, moreover, showed significantly decreased (P < 0.05) weight gain on day 10 PI. Severe gizzard lesions, such as erosion or ulcers, were observed from day 4 PI in all treatment groups regardless of their maternal antibody levels. The 1-wk-old chickens (trial 3) were separated into a control group and two treatment groups according to their titer levels of the inoculated virus. In spite of high maternal antibody levels, severe gizzard lesions were observed in both treatment groups, which also showed decreased weight gain. One treatment group, inoculated with the higher dose, showed significantly decreased (P < 0.05) weight gain on days 10 and 14 PI compared with the control group. Fowl adenovirus was recovered mainly from gizzard and rectal (including feces) samples from inoculated chickens but was not recovered from liver samples in any of the trials or in any of the control chickens. Although the reproduced disease was similar to that described in a previous report of experimental infection of specific-pathogen-free (SPF) white leghorn chickens with fowl adenovirus, the pathogenicity of FAV-99ZH in commercial broiler chickens was more severe than that in the SPF white leghorn chickens. The results of the present study indicate that FAV-99ZH isolated from gizzard erosion had pathogenicity and produced severe lesions in the gizzards of broiler chickens and that FAV-99ZH could infect and produce illness in broiler chickens with maternal antibodies against this virus.     

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.     

Pathogenicity by parenteral injection of fowl adenovirus isolated from gizzard erosion and resistance to reinfection in adenoviral gizzard erosion in chickens

The pathogenicity of a serotype-1 fowl adenovirus (FAV-99ZH), which causes adenoviral gizzard erosion by oral inoculation in chickens, was investigated in specific pathogen-free white leghorn chickens. In trial 1, 14 chickens were inoculated intravenously with the virus at 21 days of age and euthanatized for necropsy within 1-14 days of inoculation. Gizzard erosion was grossly observed from day 7 postinoculation (PI), and histologically, FAV-99ZH antigen-positive, basophilic intranuclear inclusion bodies were seen in the gizzard lesions from day 7 to 11 PI. Necrotizing pancreatitis, and cholecystitis and cholangitis associated with the inclusions were observed from day 3 to 14 PI (pancreatitis) and from day 5 to 9 PI (cholecystitis and cholangitis), respectively. The inclusions were also observed in the epithelial cells of the cecal tonsils from day 3 to 5 PI. The virus was recovered from samples of the lesions. It was revealed that FAV-99ZH causes not only gizzard erosion but also pancreatitis, cholecystitis, and cholangitis by intravenous inoculation in chickens. In trial 2, 10 chickens were inoculated orally with the virus twice, at 13 and 36 days of age, and euthanatized for necropsy within 4-17 days after reinfection. Macroscopically, focal gizzard lesions were observed; however, neither necrosis nor inclusions were observed by microscopy. Moreover, FAV was not recovered from the gizzard or rectum of any of the chickens at necropsy. This suggests that the gizzard lesions occurred as a result of the primary infection, and that the chickens were able to resist reinfection.     

Comparison of the polymerase chain reaction-restriction fragment length polymorphism pattern of the fiber gene and pathogenicity of serotype-1 fowl adenovirus isolates from gizzard erosions and from feces of clinically healthy chickens in Japan.

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.     

Experimental gizzard erosions in specific-pathogen-free chicks by serotype 1 group I avian adenoviruses from broilers

The pathogenicity of serotype 1 group I avian adenovirus (GIAAV) strains isolated from gizzard erosions for 1-wk-old specific-pathogen-free (SPF) chickens was investigated. In Experiment 1, SPF chickens inoculated orally with a serotype 1 strain (Tokushima2000/GE) of GIAAV isolated from gizzard erosion in broilers were euthanatized and necropsied from 3 to 12 days after inoculation. Clinically, there were no significant signs in the chickens inoculated with Tokushima strain. The gizzards were found macroscopically to be dilated with liquefied contents. The koilin layer of the gizzard exhibited multifocal white lesions with occasional erosions. Gross lesions of the koilin layer (white foci and erosion) appeared at 3 days after inoculation and persisted until 12 days after inoculation. The gross lesions were most severe at 9 days after inoculation. Histologically, multifocal degeneration, necrosis, and loss of the glandular epithelial cells with intranuclear inclusions, liquefactive change of koilin substance, and erosive lesions of koilin layers and surface epithelium of the gizzards were observed. Macrophages increased in the focal areas of the lamina propriabelow the liquefactive koilin substances. Immunohistochemically, intranudear inclusions were positive against GIAAV antigen. Histologic lesions of gizzards were most severe at 7 and 9 days after inoculation, but the frequency of intranuclear inclusions was greatest at 5 days after inoculation. In Experiment 2, the pathogenicities of four strains of serotype 1 GIAAV for 1-wk-old SPF chickens were compared. Three strains isolated from gizzard erosion had similar pathogenicities but one strain from a normal chicken was not pathogenic for chickens. These results suggest that gizzard erosions by serotype 1 GIAAV infection may occur in broiler chickens in Japan.     

Characterization of monoclonal antibodies against fowl adenovirus serotype 1 (FAV1) isolated from gizzard erosion

Six clones of monoclonal antibodies (Mabs) to fowl adenovirus (FAV) serotype 1 were produced. All Mabs reacted positively by enzyme-linked immunosorbent assay. Three Mabs recognized the putative 100-kD hexon protein and reacted to serotype 1 specifically by western blot analysis but did not react to other FAV serotypes (2, 3, 4, 5, 6, 7, and 8a). These Mabs will be useful for immunodiagnosis of FAV serotype 1 infection in chickens with gizzard erosion and in further research studies involving the genomes and proteins of FAV serotype 1.     

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.     

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.     

Vertical induction of the inclusion body hepatitis/hydropericardium syndrome with fowl adenovirus and chicken anemia virus

The hypothesis that fowl adenovirus (FAV) and chicken anemia virus (CAV), transmitted vertically and simultaneously, induce the inclusion body hepatitis (IBH)/hydropericardium (HP) syndrome in progeny chickens was tested. Thus, 35-wk-old light brown layer breeders, showing absence of antibodies against FAV and variable titers against CAV, were intramuscularly singly infected with the FAV serotype 4 isolate 341 or dually infected with CAV (isolate 10343) and FAV. All hens (groups A [FAV alone], B [FAV + CAV], and C [noninfected]) were clinically healthy throughout the experimental period. Both infectious viruses FAV and CAV were isolated from progenies obtained as early as 5 days after infection of their breeders. Hematocrit, serum proteins, and aspartate-aminotransferase values showed a few statistical differences between the progeny groups. Most of these differences were detected in the progeny chickens of group B. However, almost all values met reference values for the species. The pathologic findings showed that progeny chickens obtained from both singly and dually infected breeders developed macroscopic and histopathologic changes of IBH/HP. The pathologic findings shown by progeny chickens of group A (FAV) were not expected because neither synergism nor prior immunodepression by CAV was concurrent. Chickens of group B (CAV + FAV) also developed IBH/HP. Although not many differences in the evaluated parameters between groups A and B were statistically significant, most pathologic findings of group B indicated a more severe manifestation of the disease. However, because FAV alone did reproduce the syndrome, the results shown by group B would not allow a definitive confirmation of the hypothesis that the association of FAV and CAV is necessary for the successful induction of the IBH/HP syndrome in chickens when transmitted vertically.     

Putative host cell receptor for fowl adenovirus detected in gizzard

This work was done to identify a fowl adenovirus (FAV) binding protein in the gizzard, a known target organ for certain strains of FAV serotype 1. By using a virus overlay protein binding assay (VOPBA), a putative FAV binding protein of approximately 200 kDa expressed in the gizzard was detected.     

Histomonas meleagridis in chickens: attempted transmission in the absence of vectors

The progress and transmission of blackhead disease in chickens was studied in battery cages and floor pens in the absence of vectors. Two-week-old chicks were inoculated intracloacally with Histomonas meleagridis and allowed to commingle with others in floor pens. There was no confirmed transmission of blackhead to other birds in the pen, whether stocked at 10% or 25% with infected birds. A second experiment evaluated the effects of feed restriction of chickens on spread of blackhead within floor pens. Inoculated seeder birds had severe cecal lesions of blackhead at necropsy, regardless of feed restriction. Uninoculated birds did not develop lesions by the time of necropsy at 42 days of age, regardless of whether full-fed or limited by skip-day feeding. Chickens inoculated intracloacally with H. meleagridis and placed in battery cages became infected and had cecal lesions of blackhead, but few liver lesions. Chickens allowed to commingle with the inoculated birds in batteries had no lesions of histomoniasis at necropsy 2 wk postinoculation. Coccidial oocysts from turkeys (Eimeria adenoeides) were inoculated along with H. meleagridis from cultures to test the effects of sporozoite penetration in the ceca on progress of blackhead disease. Histomoniasis was not worsened by the interaction with sporozoites, as shown by unchanged severity of cecal lesions, the number of birds showing liver lesions, or the overall number of positive birds. Overall, blackhead infections showed no inclination to spread from bird to bird under conditions of these studies, in contrast to what has been reported for turkeys. These results suggest that the dynamics of blackhead transmission in chickens differs significantly from that of turkeys, where transmission from bird to bird is rapid and effective in the absence of vectors.     

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.     

Experimental infection of slaughter pigs with classical swine fever virus: transmission of the virus, course of the disease and antibody response.

The spread of classical swine fever virus was investigated in an isolation unit containing four pens, each containing six slaughter pigs. One pig in the middle pen of three adjacent pens was inoculated intramuscularly and intranasally with the virus. The fourth pen was located in a separate compartment. The pens were visited in a strict order to study, first, the effect of indirect contact via contaminated clothing and footwear on the spread of the virus to adjacent pens and, secondly, the airborne transmission of the virus between compartments. The pigs were examined and blood samples were taken every other day for 62 days for virological and serological analyses. The virus was highly contagious for the five pigs that were in direct contact with the inoculated pig, but spread to the other pens only after all the pigs in the originally infected pen had become viraemic. The spread of the virus was promoted by contaminated clothing and footwear, but airborne transmission contributed considerably to the spread of the virus within the pighouse. The first clinical signs observed after the virus was introduced into a pen were decreased feed intake, increased mean rectal temperature and apathy. Neither the clinical course of the infection, nor the pattern of seroconversion observed over time, was affected by the differences in the intensity of contact with the virus between the pigs in the different pens.     

An experimental infection (II) to investigate the importance of indirect classical swine fever virus transmission by excretions and secretions of infected weaner pigs

An experiment was set up to investigate the role of excretions and secretions in the indirect transmission of classical swine fever virus (CSFV). In five small pens, 10 weaner pigs (two pigs per pen) were housed and inoculated with CSFV. Experimental infection was successful in all pigs. The infected pigs were kept in the pens for a period of 15 days after which the pens were depopulated and pigs were killed. At the moment of depopulation, all inoculated pigs were visibly clinically diseased and had high fever. Ten hours later the same pens were repopulated with five pairs of susceptible pigs. From inoculation onwards and especially between depopulation and restocking, the pens were neither cleaned nor disinfected. Four days post-repopulation, three of the susceptible pigs were detected positive on virus isolation. A fourth pig was detected positive 2 days later. Later on, the remaining pigs also became infected, most probably due to contact and between pen infections. It can be concluded that transmission of the virus via excretions and secretions succeeded in four of 10 pigs. This result indicates that transmission of CSFV via excretions and secretions can be of importance in a late, clinical stage of disease.     

Immunosuppressive effect of a highly virulent infectious bursal disease virus isolated in Japan.

The immunosuppressive effect of infectious bursal disease virus (IBDV) on vaccination against Newcastle disease (ND) was compared among 2-, 3-, and 4-week-old chickens inoculated with the highly virulent IBDV field isolate 90-11 and the reference serotype 1 strain GBF-1. In all age groups, isolate 90-11 severely suppressed antibody response to ND vaccination and protective vaccinal immunity against ND. In contrast, chickens inoculated with strain GBF-1 and vaccinated with ND vaccine were well protected from the ND virus challenge. The mitogenic response to phytohemagglutinin of splenic lymphocytes from chickens inoculated with isolate 90-11 or strain GBF-1 was significantly lower than that of uninoculated controls. There was no difference between the two inoculated groups in responsiveness, although lymphocyte depletion in the thymus was more severe in chickens inoculated with isolate 90-11 than in chickens inoculated with strain GBF-1.     

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.     

FTA liver impressions as DNA template for detecting and genotyping fowl adenovirus

The feasibility of using liver impressions on Flinders Technology Associates (FTA filter paper for the collection, inactivation, and molecular analysis of fowl adenovirus (FAV) was evaluated. FAV I European Union (EU) serotype 1 spotted on FTA was shown to be inactivated using specific-pathogen-free (SPF) primary chicken embryo liver cell culture as indicated by absence of cytopathic effect. Sensitivity of the polymerase chain reaction (PCR) test using tenfold dilutions of allantoic fluid from 100 to 10-4 for the detection of adenovirus serotype 1 on FTA cards was determined to be 0.0005 mean tissue culture infectious dose per FTA spot. The stability of the DNA from liver impressions on the FTA was found to be 198 days when stored at -20 degrees C. In a trial, inclusion body hepatitis (IBH) was experimentally reproduced in SPF chickens inoculated with FAV I EU serogroup 1, 4, 8, or 11, which presented weakness, pallor, depression, dehydration, and mortality within 6 days after inoculation. PCR performed on FTA liver impressions from the inoculated birds was able to detect all four viruses, and the nucleotide sequence analysis of the amplified PCR products (1219 bp of the hexone gene) revealed the expected serotypes. In addition to the trial, 55 clinical samples were analyzed from liver impressions on FTA cards, and FAV was detected in 11 of 55 (20%). Sequencing analysis showed that the viruses were EU serotypes 4, 5, 9, and 10. The results demonstrate that FTA filter paper inactivates the FAV I and maintains the DNA template for molecular analysis.