Isolation And Identification Of Infectious Bursal Disease Virus Variant And Efficacy Evaluation Of Inactivated Vaccine

Infectious Bursal disease(IBD)which is caused by infectious Bursal disease virus(IBDV)is one of the poultry diseases with high morbidity and mortality in China.As a highly contagious virus,IBDV mainly has a negative impact on chicks and young chickens,such as causing atrophy of Fabricius bursa and even necrosis,as well as leading to immunosuppression.IBDV which contains two pieces of double-stranded RNA is easy to mutate.Classical strains,super strains,antigenic mutant strains and Chinese new mutant strains have appeared successively.In order to rapidly identify and type IBDV,the Taqman-MGB probe and primer were designed by the sequence of highly variable region of IBDV VP2gene,and a fluorescence quantitative PCR method that took into account both IBDV identification and sequencing typing was successfully established.The method had a good linear relationship in the range of 10~1 to 10~8 copies/μL(the correlation coefficient was 0.9963)and the amplification efficiency was fine(the amplification efficiency was109%).There was no amplification for other common viruses and the detection limit was12.5 copies/μL.To understand the epidemic status of IBDV in some areas of our country,the pathogen,antibody titer and bursa index of IBDV were studied and investigated sentry chickens and large flocks of hens in five large parent breeding chicken farms at different ages in Handan,Jiangxi Yichang and Chongqing.The results showed that the IBDV antibody titer of Sentry chickens in field A turned negative at 28 days of age and remained until the transfer group.The antibody titer of Sentry chickens in field B-E decreased at28 days of age,but increased to more than 10000 after 42 days of age.IBDV was not detected in A field,and IBDV was detected in B-E field at different time points,and the sequencing results showed that it belonged to a new variant of Chinese IBDV.In summary,the new mutant IBDV is common in many poultry nursery of our country and can break through the protection of the existing IBDV vaccine.We isolated IBDV from SPF chicken embryo to further understand the characteristics of IBDV mutant strains in infected fields.IBDV’s virulence to SPF chicken embryo was measured,and then it was domesticated in DF-1 cells.We used IBDV A fragment to sequence IBD and analyze its homology.The results showed that three IBDV strains,named J2021,T2021 and T2022,were isolated from the self-positive chicken embryo samples.All the isolates could cause severe bleeding of chicken embryos,but the strains adapted to DF-1 cells were not successfully domesticated.The sequencing of the A segment of the isolates showed that all the three IBDV isolates were new variants in China.Based on the nucleotide sequence homology of VP2 gene,the results showed that the homology of the three isolates with the new IBDV variant was 96.2%-99.1%,and the amino acid homology with the new domestic variant SHG19 was 100%.T2021,T2022 and J2021 belonged to two evolutionary branches.Based on the analysis of amino acid sequences in the highly variable region of VP2 gene,the results showed that the three isolates had the characteristic amino acid sites 222T,249K,286I,318D of the mutant and the characteristic amino acid sites 221K,252I,299S of the new variant.The results comfirmed that all the IBDV isolates in the investigation field belonged to the new variant strains in China.In order to explore the effective prevention and control methods of the new IBDV variant strains,the new IBDV variant strains were added to the original new flow method of inactivated vaccine,and immunized according to the original immunization procedures.The results showed that the atrophy symptom of Fabricius bursa was releaseed in some chickens immunized with inactivated vaccine,but could not reach the normal level,while the bursa atrophy was always serious in sentry chickens which were not immunized.The index of Fabricius bursa showed that the index of Fabricius bursa of Sentry chickens at different days of age was less than 1,and the index of Fabricius bursa was close to the normal value after immunization.The results showed that the addition of inactivated vaccine of IBDV variant could alleviate the symptoms of atrophy of Fabricius bursa in chickens to a certain extent.