Isolation And Identification Of Fowl Adenovirus Serotype 4 And Evaluation Of Immunization Effect Of Inactivated Vaccine

Since 2015,fowl adenovirus serotype 4（FAV-4） disease has occurred successively in poultry in Hebei,Shandong,Jiangsu and other places in China.With the epidemic of this disease,it has occurred in all parts of the country at present.Due to the lack of vaccine prevention and control and effective treatment drugs,the disease has resulted in rapid epidemic speed,wide range,high mortality,and great losses to the aquaculture industry.Clinic autopsy of dead poultry infected with FAV-4 clearly showed a large amount of pale yellow fluid in the pericardium and enlarged and necrotic liver,fowl adenovirus could be detected from the liver of dead chickens in the laboratory.The virus can be transmitted horizontally or vertically through chicken embryos,so it brings great difficulties for prevention and control.In this study,a strain of virus was isolated and cultured from liver tissue of dead chickens suspected of pericardial effusion-hepatitis syndrome in Qiyang County,Yongzhou City,Hunan Province by inoculating chicken liver cancer（LMH） passage cells.In the experiment,a series of studies were carried out,such as PCR detection,serotyping,animal regression test,viral content determination,specificity identification,immunogenicity test and physical and chemical characteristics test.The results showed that the nucleic acid fragments with the size of 2000 bp amplified by PCR were in accordance with the expected size of amplified fragments;the nucleotide sequence homology with the nucleotide sequence of fowl adenovirus serotype 4（FAV-4） strains registered in Gen Bank was 94.4%;the virus fluid of the isolates could be neutralized by group I serotype 4 FAV-specific antisera;the virus content was 10^7.34 TCID₅₀/0.1 ml;It can cause 90%-100% of SPF chickens at 21,42 and 56 days of age,and pathologically dissected chickens show typical pathological changes of pericardial effusion-hepatitis syndrome.The virus liquid isolated and identified as fowl adenovirus was diluted into three titers of 10^7.0TCID₅₀/0.1 ml,10^6.0TCID₅₀/0.1 ml and 10^5.0TCID₅₀/0.1 ml,inactivation with 1‰formaldehyde at the final concentration and emulsified into an oil emulsion inactivated vaccine.The safety test of the finished product of the vaccine showed that the isolate inactivated vaccine had good safety.Through a series of animal tests,including the immunogenicity study of the inactivated isolate vaccine,the immune duration test,etc.The agar amplification titer of immunized chicken flock antibody was the highest at 1:2,and the immunogenicity was good.The protection period of SPF chickens at different ages after single immunization was 2-4 months.The test proved that the isolated virus was group Ⅰ fowl adenovirus,named HN strain,and could be used as a candidate strain for high-quality vaccine.