Isolation And Identification And Study On Pathogenicity Of Fowl Adenovirus Group ? In Hubei Province

“Hydrocardia-hepatitis syndrome”(HHS)is an emerging disease of poultry caused by serotype 4 of aviadenovirus group(Fowl adenovirus serotbe4,FAV4).The disease affects birds between 3-6 weeks of age due to significantly high/spiking mortality in young broiler birds that may reach up to 80%.Clinically,the HPS is characterized by typical hydropericardium,anemia and necrotic hepatitis.The disease can spread both vertically and horizontally/laterally and has been reported the India,canada,Japan,Russia and many others countries.In recent years,this epidemic has occurred in the Chinese provinces of jiangsu,henan,shandong and fujian provinces in 817,three yellow chicken and local varieties of young chickens.The role of group I adenoviuses as pathogens is not well defiend,so its immune pathways have been unable to determine.Fowl Adenovirus-I can cause severe immune suppression which results in poor or failure of the immune effects to the development of poultry industry in China has brought no small blow.In this study,the avian adenovirus was detected by the PCR method from the disease in the poultry area of hubei province,virus isolation and hexon gene sequencing.It is to investigate areas of hubei province I group of fowl adenovirus serotype distribution and infection situation.A serum isolate of type 4 was selected for artificial infection test to understand the pathogen characteristics of isolated strains in Hubei area.Using immunohistochemical method to explore the FAV-I distribution inside the infected chickens.The main results obtained are as follows: 1.Isolation and identification of group I avian adenovirus isolates from Hubei ProvinceThe twelve pericardial hydro-hepatitis syndromes of the chicken from in Hubei province.Using PCR methood to amplify hexon gen.The suquence alignment analysis,the nucleotide homology between 12 isolates and 12 different serotypes of avian adenoviruses in Genbank shared 31.1%~98.3%,the Avian virus homology with FAV-4 and FAV-10 in Fowl Adenovirus-I is closest,the virus shared 98.1%~98.3% identity with FAV-4 in group I.The genetic analysis showed that 12 isolates were closest to Fowl Adenovirus-I and the 12 isolates with FAV-4 belong to the same branch,It was further confirmed that 12 isolates belonged to group I avian adenovirus serotype 4.Epidemic materials supernatant to inoculation the chicken embryo hepatocytes(CEL),it was that the cells contracted and the cell membrane atrophy,the boundaries of the cells were blurred and the refraction was enhanced.It was that occur in the process are Consistent with those of the avian adenovirus on the cell.The TCID50 of 12 isolates were determined to be between 104.75-107.5.2.Study on pathogenicity of FAVI/HBtuanfeng/151023In this study,a serum type 4 FAVI/HBtuanfeng/151023 isolate was selected for artificial infection of 33-day-old Nick white feather laying hens.Dividing them into three groups with a random number table: intramuscular group,oral groups and control group.At the same time,the intramuscular injection group and the oral group were given 10 chickens in each group,Intramuscular group by subcutaneous injection,oral group through oral route to inoculation 0.2mL?(0.2×107.5TCID50)virus liquid plume.The control group was inoculated with 0.2 mL sterilized saline.Observeing the of chicken every day.The chickens of different experimental and control groups were killed at 3d,5d,and 7d after infection.The organize organs were collected.Using 4% paraformaldehyde to complete the fixation and stained by conventional H.E.Staining.Then the pathological changes of these organs were observed.The results showed that the isolates had strong pathogenicity,the infection rate was 100%,the chicken appeared depression,loss of appetite,drowsiness and other symptoms after inoculation.Detection of the disease can be seen in the chest there are obvious pericardial effusion,liver is covered with large and small bleeding spots,the edge of the liver has white necrosis,Severe pulmonary edema.The histopathological changes were characterized by fatty degeneration extensive hemorrhage and inflammatory cells infiltration in the liver,karyopyknosis,cell necrosis and basophilia inclusion body were observed in liver tissue from sick chicken.3.Using the immunohistochemistry detection of chiken group I avian adenovirusTo obtain the primary antibody,New Zealand rabbits were immunized with the enrichmented FAVI/HBtuanfeng/151023 to obtain FAV-I specific antibody.Rabbits were immunized by formaldehyde inactivation.Using the indirect AGP to detect serum antibody titer,the titer was up to 1:64.Using immunohistochemical technique was used to detect the tissues and organs of different groups of infected chickens.The results show: the positive signals of 3d,liver,heart,kidney,spleen,thymus and duodenum were found in the chickens after infection,FAV-I invades the chicken's organs for the liver,spleen,kidney and intestine.Antigen staining of these argans gradually enhanced as time went by.Immune organs showed an alternately antigen staining during the experiment.The rest of the tissues and organs showed weak or moderate antigen staining.To conclude,In this study,12 isolates isolated from Hubei poultry belonged to type I avian adenovirus serotype 4.The FAVI/HBtuanfeng/151023 strain was infected by oral and intramuscular injection,and the chickens died after the disease,it has a strong pathogenicity.Using immunohistochemical methods to detect FAV-I,the liver,spleen,kidney and intestine are the target organs of the avian adenovirus.The method has good specificity.It was can localize the antigen in the subcellular level as well as diagnose FAV-I.Which provides useful scientific data for elucidating the pathogenesis of FAV-?.