The Study Of Newcastle Disease Virus?avian Influenza Virus H9 Subtype And Infectious Bursal Disease Virus Combined Immunization

Newcastle Disease,Avian Influenza and Infectious Bursal Disease are highly contagious viral diseases of birds and considered three of the most important poultry infectious diseases worldwild.These diseases are the focus for concern throughout much of the world's agricultural community because of severe economic loses that have occurred from illness,death,and reduced egg production following infection with pathogenic or disease causing strains.Therefore,the co-immunization with IBDV,NDV and AIV not only plays a role in preventing these three kinds of infection,but also improves the efficiency of immunization and reduces the stress response caused by multiple vaccination.This has become an important means of immunization in large-scale livestock farming.In this study,a strain of vv IBDV with excellent biological characteristics was isolated from a chicken farm in a prevalent area of IBD in China and adapted to attenuated strain C1 on DF-1 cells.And it was combined with NDV vaccine strain La Sota and AIV(H9)vaccine strain XZ13,then selected suitable vaccine adjuvants and prepared as inactivated combined immunological products.Their immune effects and the growth and decline law of the body's antibody levels were studied.It laid the foundation for the development of commercial triple-inactivated vaccine against NDV,AIV(H9 subtype),and IBDV.This study mainly includes the following aspects:1.The VP2 genes of six isolated strains were cloned and sequenced,and a vvIBDV strain X1 was selected for its adaptation to immortalized cells DF-1,named as C1.Comparing the VP2 hypervariable regions and heptapeptide regions of strain X1 and strain C1.At the same time,the prokaryotic expression of the VP2 protein of strain C1 was performed.It revealed that the IBDV strain X1 was a very virulent strain and the strain C1 was an attenuated strain from sequencing analysis.In the VP2 hypervariable region and the heptapeptide region,indicating that some of the sites which determine the virulence of the virus have changed.Meanwhile,this study successfully expressed 70 ku VP2 protein in E.coli.2.To select suitable adjuvants,triple adjuvant immunoprecipitations prepared from different adjuvants were used to inoculate SPF chickens with different antigen levels.Through comparison of antibody titers and challenge protection,it was compared which adjuvants had the potential to reduce the use of antigens.The results showed that ISA 71 had a significant reduction in immunization dose of IBDV antigen compared to Marcol 52;Within a range of antigenic concentrations,ISA 71 was significantly better than Marcol 52 when immunized with a lower dose of H9 antigen;The titers against NDV HI of the ISA 71 1/8 antigen dose group were significantly higher than those of the Marcol 52 group against NDV.It also showed that the immunogenicity of strain C1 was good.3.In order to understand the effect of the triple-inactivated immunization preparation after immunization on SPF chickens,the chickens were immunized respectively and the antibody level of the chickens were measured and challenged after 28 days of inoculation.The results showed that the ND HI antibody levels of the immunized chickens in each group were ? 4 log2,the AIV H9 HI antibody levels were ? 9.2 log2,and the geometric mean titer of IBD neutralizing antibody was ?1:5000,with the highest value 1:14117.After each group attacked NDV Beijing strain F48E9,AIV strain XZ13 and IBDV strain BC6/85,the three groups of immunized chickens were 100% protected and the control group was not protected.The results showed that the triple-inactivated immune preparation was safe,reliable,and stable.It had a good immune effect on SPF chickens.4.In order to investigate the law of antibody lengthening of the three antigenic components of the triple immunosuppressive agent,SPF chickens were vaccinated and their serum antibody titer was periodically tested.At 28 days of age,each SPF chicken was vaccinated with 0.3 m L of the vaccine.The three antigens produced antibodies in the second week after immunization,and reached an ideal high-level antibody in the third to fourth weeks.Anti-NDV HI antibody values were ? 4 log2 in 33 weeks,anti-AIV HI antibody values were ? 6 log 2 in 32 weeks,anti-IBDV neutralizing antibody values were still relatively high at 53 weeks,5.05,4.53,5.12(log10).The results showed that the level of antibodies induced by co-immunization was relatively stable and lasted for a relatively long period of time,which is at least 32 weeks,as seven and a half months.