Development Of A Hemagglutination Inhibition Test Antigen For Detecting Antibody Of Infectious Bronchitis Virus And A IB-ND-IBD-EDS-AI Inactivated Vaccine

Infectious bronchitis (IB) is a kind of highly contagious disease of chicken which have caused substantial economic losses for poultry industry of the world. In this study, we developed the HI test antigen and established method to detect HI antibody of infectious bronchitis virus (IBV), and then we developed an IB-ND-IBD-EDS-AI inactived vaccine.1Development of a hemagglutination inhibition test antigen for detecting antibody of IBVWe compared the HI test antigens concentrated by different methods (Ultracentrifugation, PEG precipitation, PEG dialsis) and enzyme treatments (Type A culture filtrate of Clostridium perfringens, Type I lecithinase C and Neuraminidase). Finally, the best antigen preparation was determined:IBV (M41strain) was inoculated into10-day-old SPF embryos, then the SPF embryos were incubated for36hours at37℃; allantoic fluid was harvested and centrifugated at4000g for30min, supernatant was centrifugated at25000g for70min, and the virus precipitate was treated with3U/mL of PLC1, incubated for2h at37℃. The HI test antigen possessed high specificity and the established HI test could detect the antibody of IBV.2Development of an IB-ND-IBD-EDS-AI Inactivated VaccineIBV (LH strain) was passaged to10generations continuously, EID50of P1, P5and P10generations reached more than106.5/mL and that showed the IBV (LH strain) possessed high stability. The IBV (LH strain) were not polluted with bacteria, mycoplasma and other exogenous viruses.14-day-old chickens were inoculated with H120vaccine firstly,3weeks later chickens underwent blood sampling and inoculated with inactivated vaccine. After3weeks, chickens underwent blood sampling again. The geometric mean HI antibody titer of the second vaccination was three times of the first vaccination. IBV (LH strain) has good immunogenicity.NDV (LaSota strain), IBV (LH strain), IBDV (NF8strain), EDSV (AV127strain) and H9subtype AIV (F strain) were multiplicated by SPF chicken embryos or duck embryos, and then completely inactivated by0.1%formaldehyde for24h and emulsified with mineral oil to to produce the inactivated IB-ND-IBD-EDS-AI vaccine.The chickens were inoculated the inactivated IB-ND-IBD-EDS-AI vaccine. Three weeks after immunization, NDV HI antibody titer geometric mean was6.5log2, IBV HI antibody titer geometric mean was7.1log2, IBDV antibody titer geometric mean was583(positive, more than391). Four weeks after immunization, EDSV HI antibody titer geometric mean was up to8.8log2, H9subtype AIV HI antibody titer geometric mean was7.5log2. The inactivated vaccine provided adequate protection for chickens against IBV (LH strain), NDV (F48E8strain) and H9subtype AIV (F strain) challenges after immunization.The immune duration of the inactivated IB-ND-IBD-EDS-AI vaccine was at least12weeks for IB, ND, IBD, EDS and H9subtype AI.