Study On Immune Efficacy Of Infectious Bursal Disease Virus–Fowl Adenovirus Serotype 4 Recombinant Bivalent Inactivated Vaccine

Infectious bursal disease(IBD)and hepatitis-hydropericardium syndrome(HHS)are two diseases which seriously endanger development of the poultry industry,caused by infectious bursal disease virus and fowl adenovirus serotype 4(FAd V-4),respectively.Especially the newly emerged IBDV variant has caused severe atrophy of the bursa of Fabricius,leading to the reduced protection efficacy of the currently existing vaccines.IBDV and FAd V-4 co-infection causes more serious consequences.The traditional IBD-HHS bivalent vaccines have high production cost and complicated production process.Therefore,it is of great significance to develop a new and efficient vaccine that can prevent and control both IBD and HHS at the same time.The immunogenicity and immune efficacy of a bivalent inactivated vaccine prepared using a recombinant FAd V-4 expressing the VP2 gene of IBDV variant strain,r FADV-4-IBDV-VP2,were evaluated in this thesis.The following aspects are included:1.Propagation and Identification of the recombinant FAd V-4 expressing IBDV VP2 protein for inactivated vaccine preparationTo prepare a novel,cheap and highly efficient IBD-HHS bivalent inactivated vaccine,the team constructed a recombinant FAd V-4 virus r FAd V4-IBDV/VP2 expressing the VP2 protein of an IBDV variant strain previously.The present study began with re-evaluation of the in vitro replication capacity of the recombinant virus.Polymerase chain reaction(PCR),sequencing and Western blot were used for further identification of the virus stocks for vaccine preparation.The results showed that the recombinant virus has good replication capacity on Leghorn male hepatocellular(LMH)cells.The IBDV VP2 gene was inserted at the correct position with the favorable direction,and the VP2 protein was expressed at a high level.The purity test of the recombinant virus indicated that the virus met the requirements for vaccine preparation.2.Prokaryotic expression of VP2 protein of the IBDV variant strain and establishment of IBDV indirect ELISA methodTo evaluate the antibody response against IBDV variant strain induced by the inactivated r FAd V4-IBDV/VP2 vaccine and perform epidemiological investigation of the IBDV variant strain,an indirect ELISA method for detecting the antibody agasint IBDV variant strain was established.The VP2 gene of the IBDV variant strain was optimized according to the preferred codons of E.coli,and cloned into a prokaryotic expression vector p ET32 a.The constructed plasmid was transferred into E.coli for protein expression.Using the purified VP2 protein as a coating antigen and after optimization of the experimental conditions an indirect ELISA method for detecting anti-IBDV variant strain antibody was successfully established.The test results showed that the method can detect anti-IBDV variant strain specific antibodies,and had no cross-reaction with other common avian viruses such as Newcastle disease virus,Infectious bronchitis virus,FAd V-4,H5,H7 and H9 subtype avian influenza viruses.The coefficients of variation of the established ELISA method were 0.32%-5.66% and 1.92%-4.81% within the same sample batch and among different sample batches,respectively,indicating that the established ELISA method had a credible repeatability.The clinical tests of the established ELISA method showed that there were different degrees of IBDV positive in the farms in the tested areas,and the average positive rate was 63.67%.The established ELISA method can be used for the detection of anti-IBDV antibodies,and provided a method for the evaluation of the immunogenicity of IBD vaccines and the epidemiological investigation of IBDV.3.Evaluation of immunogenicity and immune efficacy of the r FAd V4-IBDV/VP2oil-emulsion inactivated vaccineTo evaluate the immunogenicity and immune efficacy of the r FAd V4-IBDV/VP2oil-emulsion inactivated vaccine,2-week-old specific pathogen free(SPF)chickens were intramuscularly injected with the r FAd V4-IBDV/VP2 oil-emulsion inactivated vaccine.The anti-IBDV antibody level was detected by the constructed IBDV indirect ELISA antibody detection method.The anti-FAd V-4 antibody level was detected by FAd V-4indirect ELISA antibody detection method constructed previously.The results showed that high levels of anti-IBDV and anti-FAd V-4 specific antibodies were induced 21 days after immunization.The results of the viral challenge test showed that the r FAd V4-IBDV/VP2 oil-emulsion inactivated vaccine could provide full protection against the hypervirulent IBDV variant strains and the pathogenic FAd V-4 strain.The results showed that r FAd V4-IBDV/VP2 can be used for preparation of a cost-effective and highly efficient bi-valent vaccine for the prevention and control of HHS and IBD.