Detection,Isolation,Identification And Antibody Preparation Of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4(FAd V-4)is the main pathogen causing Hydroperi-cardium-hepatitis syndrome(HHS).Since 2014,poultry in Guangdong,Henan,Hunan,Hubei,Yunnan and other regions in China have reported outbreaks of the above diseases,which have cause economic losses to the poultry industry in many places.The disease mainly infects 3-5week old chickens,5-10 week old breeds of ducks and geese,and has no obvious symptoms in the early stage of infection.After one week,symptoms such as depression,weak legs,white or pale green faeces.Dissection showed obvious yellowish transparent pericardial effusion and liver swelling and bleeding.In this experiment,a nest-PCR method for detection of Fowl adenovirus serotype 4 was established to detect suspected cases,further isolating the virus from confirmed cases.The isolated and con-centrated virus was used to prepare oil emulsion inactivated vaccine to further prepare egg yolk antibody.The main contents of the test are as follows:1 Establishment and Application of FAd V-4 Nested PCR Detection Method For quickly and sensitively of suspected cases of FAd V-4,this experiment designed two specific primers based on the Fowl adenovirus Hexon gene sequence publis-hed on Gen Bank.Through the optimization of reaction conditions,a nested PCR meth-od for detection of Fowl adenovirus serotype 4(FAd V-4)was established.Using this method to detect the standard strain of FAd V-4,the size of the fragment amplified by two-step PCR was 663 bp and 593 bp.The sensitivity,specificity and nucleotide sequence of amplified fragments of the established nested PCR method were determin-ed.The results show that the sensitivity of this method is 1000 times higher than that of conventional PCR detection methods,and it cannot amplify GPV,MDPV,AIV,DHAV and other viruses.The nucleotide sequences of amplified DNA fragments were more than 99% similar to the reported FAd V-4 sequences.The established nested PCR method can detect FAd V-4 fromsuspected materials,and its sensitivity is higher than universal primer PCR of FAd V-4;And this method can detect viruses from anal swabs of laying hens and ducks.The above results show that the established FAd V-4 nested PCR detection method has high specificity and sensitivity,and can be used for the detection of FAd V-4 in clinical materials and animal anal swabs.2 FAdV-4 isolation and identification A FAd V-4 disease material treatment solution that was positive by nested PCR,7-day-old chicken embryos were inoculated with yolk sac.A single band of 600 bp was detected in the collected allantoic fluid,and the nucleotide sequence of 595 bp was cloned and sequenced.By using Blast software in Genebank,Nucleotide sequence of cloned fragment is more than 96% similar with FAd V-4L160962(MF981081.1)and other strains,and in the same branch.The isolated strain was pure and free from bacter-ial and exogenous virus contamination.The median lethal dose(ELD50)of 7-day-old chicken embryos inoculated through allantoic cavity was 10-2.16/0.2ml.One-day-old ducklings and 14-day-old geese were inoculated through the leg muscles.Some of the ducklings occurrence of disease on the 17 th day after vaccination and died,the main manifestations were sudden fall to the ground,convulsion and death.Pericardial effusion,liver swelling and bleeding were observed by anatomy.Goslings have no obvious symptoms and no deaths;Culled at different times after inoculation,a small amount of pericardial effusion was found in the test group,there were no obvious gross lesions in other organs,the tissues of the heart,liver,spleen and pancreas were positive for FAd V-4by nested PCR.Serum aspartate aminotransferase(AST),alkaline phospha-tase(ALP),alanine aminotransferase(ALT),lactate dehydrogenase(LDH)and other activities increased significantly at 3 and 5 dpi after inoculation.3 Preparation of Chicken Anti-FAd V-4 Antibody Concentrate the preserved allantoic fluid of FAd V-4 virus,preparation of water-in-oil emulsion antigen after formaldehyde inactivation.Laying hens are immunized 3 times at 15-day intervals,the yolk was collected 15 days after the third immunization to prepare yolk antibodies.Using FAd V-4 strain as the test virus,neutralization test of chi-cken embryo was carried out by the method of immobilized virus dilution antibody,determination of neutralization titer of chicken FAd V-4 yolk antibody.The results of three repeated experiments were 1:380,1:348,and 1:363,with an average of approximately 1:364.