Preparation Of Inactivated Avian Adenovirus Type 4 Vaccine(JH Strain)

In recent years,hepatitis-pericardial effusion syndromes caused by fowl adenovirus serotype 4(FAdV-4)has occurred frequently in China,with a mortality rate of 20%～80%,causing great economic losses to the poultry industry.In this study,based on the isolation and identification of the FAdV-4,the biological characteristics of FAdV-4 isolates were systematically studied,and FAdV-4 JH strain with good immunogenicity was screened out as the inactivated vaccine candidate,and the large-scale suspension culture,inactivation process,emulsification process and safety and effectiveness studies have been carried out.Finally,a safe and efficient inactivated FAdV-4 vaccine was prepared.Providing the technical support and product guarantee for the effective prevention and control of FAdV-4.1.Screening of fowl adenovirus serotype 4 inactivated vaccine candidate strainIn this study,five strains of FAdV-4 were isolated and identified from chickens suspected to be infected with fowl adenovirus,designated JH,SD,GX,TJ and WF respectively.The pathogenicity of 5 isolates in SPF chickens at 21 days of age was evaluated.The results showed that after 2×105.0 TCID50 inoculation,the mortality of each strain was 100%,100%,90%,80%and 80%,respectively.The pathological changes of the dead chickens were pericardial effusion and inclusion body hepatitis,and the histological changes were basophilic intranuclear inclusion bodies in liver cells.Immunogenicity test was conducted on the 5 isolates after inactivation.The results showed that the neutralizing antibody level induced by JH strain was much higher than that of the other 4 isolates,which demonstrated the JH strain had better immunogenicity.The whole genome sequence of JH strain and the amino acid sequence of Fiber 1 and Fiber 2,which are the main serotype protective antigen,were analyzed with the evolutionary tree of 30 representative strains of fowl adenovirus serotype 4 reported in NCBI.The results showed that the sequence of JH strain and domestic fowl adenovirus serotype 4 had high homology.In addition,JH strains was suitable for cell culture and got a high virus tirer.These data demonstrated that JH strain was a suitable vaccine candidate for the prevention and control of fowl adenovirus caused by serotype 4 fowl adenovirus in China.Purity detection of JH strain demonstrated that JH strain could meet the requirements as a kind of seed of the vaccine.The virulence,immunogenicity and protection efficiency detections show that JH strains could provide good protection for immunized chikens,and immunization group showed no without viral shedding within 14 days after immunization.Therefore,JH strain was finally selected as a vaccine candidate for subsequent culture process and factory production research.2.Study on culture technology and factory of Fowl adenovirus serotype 4In order to obtain high quality inactivated vaccine,this study firstly studied the culture process of JH strain.The results showed that the speed of the bioreactor was 40 rpm,and the dissolved oxygen value was not less than 50%,which could ensure the oxygen-containing conditions for cell growth.The initial number of cells inoculated with 200 g paper carrier was(1±0.2)×109 cells,containing 10%fetal bovine serum DMEM/F-12 medium,and pH was maintained at 7.0-7.2.The JH strain was inoculated with MOI of 0.1,the serum content of maintenance solution was 2%,and the virus titer could reach 109.0TCID50/mL in 94～98 h after infection.The preparation process of oil emulsion inactivated vaccine was further studied,and the results showed that the final antigen content of vaccine was 107.0TCID50/mL with 0.1%formaldehyde solution inactivated at 37□ for 24 h,the ratio of water phase to oil phase was 1:3,and the emulsification shear speed was 3000-4000 rpm.In order to evaluate the quality of the vaccine,SPF chickens of different days were selected for safety test in this study.Three batches of vaccine were inoculated subcutaneously or intramuscular by single dose,single dose repetition and overdose.The results showed that the safety of different batches of vaccine was up to 100%,and the production performance of chickens was not affected.The efficacy test was conducted by challenge protection test and neutralization antibody detection method.SPF chickens at 21 days were immunized subcutaneously.Meanwhile,JH strain,SD strain and GX strain were used for viral challenge protection test.The results showed that both subcutaneous and intramuscular injection of the neck could produce high levels of neutralizing antibodies and provide high protection for the chickens after 21 days of immunization,while all the chickens in the control group died.The results also showed that the duration of immunization was 21 days and the duration of efficacy was 5 months.The successful development of this vaccine provided a reliable guarantee for the prevention and control of FAdV-4.