| H9N2 subtype avian influenza,Newcastle disease and chicken hepatitis-hydropericardium syndrome were all widespread and serious in Chinese poultry.Newcastle disease,one of the three major infectious diseases of chickens,was an infectious disease that chickens must guard against.Due to the constant variation of the antigenicity of the H9N2 subtype avian influenza virus,the immune effect of the existing vaccine decreased year by year,and the immune failure often occurred.Chicken hepatitis-hydropericardium syndrome was a new infectious disease which was seriously harmful to Chinese poultry industry in recent years.In order to reduce the harm of the above three epidemics to Chinese poultry industry and reduce the immune stress,we carried out the development of H9N2 subtype avian influenza,Newcastle disease,chicken hepatitis-hydropericardium syndrome tritivalent inacativated vaccine.In this study,phylogenetic analysis and cross-challenge after vaccination were conducted to select H9N2 subtype avian influenza vaccine strains that had good cross-immunoprotection and were closely related to the epidemic strains.This strain was then used for vaccine production with Newcastle disease virus and fowl adenovirus serotype-4,and the physical properties and safety of the vaccine were evaluated.Antibody titer determination and challenge protection test were conducted to evaluate the immune protection of tritivalent vaccine and determine the minimum immunization dose,in order to provide a certain theoretical basis for clinical application of tritivalent vaccine.The specific contents were as follows:Experiment I Screening of vaccine strains for H9N2 avian influenzaFive strains of H9N2 subtype avian influenza virus kept in the laboratory,were isolated from several infected chicken farms and live poultry markets in Jiangsu province,and their HA and NA gene sequences were analyzed.The results showed that the HA gene of strain 1609JR2#D and 1704CZ3-2 belonged to the lineage h9.4.2.1,the NA gene belonged to the lineage F/98-like,and the nucleotide and amino acid sequences of HA and NA genes were less homologous with the recent epidemic strains.The HA genes of strains 18092902,18092904 and 18092909 all belonged to the main predominant epidemic lineage h9.4.2.5 in China,and the NA gene belonged to the Y280-like lineage,whose nucleotide and amino acid sequences of HA and NA genes were highly homologous with the recent epidemic strains,suggesting that these three strains had close relationship with the recent epidemic strains.After inactivation of viruses 18092902,18092904 and 18092909,the corresponding oil emulsion inactivated vaccine was made.Through cross-hemagglutination inhibition test and cross-challenge protection test,it was found that the antibody induced by vaccine 18092902 can produce good cross reaction with homologous virus and heterologous virus,and the protection rate of immunized chickens against the homologous virus and heterologous virus can reach 100%,indicating that the virus 18092902 can be used as the H9N2 subtype avian influenza vaccine strain.Experiment II Preparation and quality evaluation of avian influenza(H9N2 subtype),Newcastle disease,chicken hepatitis-hydropericardium syndrome tritivalent inactivated vaccineStrain 18092902 of H9N2 avian influenza virus,strain La Sota of Newcastle disease virus(CVCC AV1615)and strain HN of fowl adenovirus serotype-4 were obtained through the chicken embryo inoculation and cell culture,with virus contents of 108.5 EID50/0.1 mL,109-17 EID50/0.1 mL,105.5 TCID50/0.1 mL,respectively.Each virus solution was inactivated with a final concentration of 0.2%formaldehyde solution.The H9N2 subtype avian influenza virus,Newcastle disease virus and fowl adenovirus serotype-4 solution that passed the inactivation test were prepared into tritivalent oil emulsion inactivated vaccines at a ratio of 1:1:1.The quality evaluation of the tritivalent vaccine was carried out,and the physical property test and safety test results were all qualified.Experiment Ⅲ Evaluation of immune efficacy of avian influenza(H9N2 subtype),Newcastle disease,chicken hepatitis-hydropericardium syndrome tritivalent inactivated vaccineCombined with the vaccine-induced antibody level and immune protection against virus attack,the immune effect of the avian influenza(H9N2 subtype),Newcastle disease,chicken hepatitis-hydropericardium syndrome tritivalent inactivated vaccine was evaluated.It was found that the antibody titers of H9N2 subtype avian influenza virus and Newcastle disease virus showed a significant and continuous increase within 0 to 14 days after immunization(P<0.05),and antibody production peaked at 21 to 28 days after immunization.The immune efficacy of the vaccine was evaluated,and the results showed that the chickens immuned with tritivalent vaccine could produce higher levels of antibodies to H9N2 subtype avian influenza virus,Newcastle disease virus and fowl adenovirus serotype-4,and could effectively defend against H9N2 subtype avian influenza virus and fowl adenovirus serotype-4 attacks,and the immune protection rate was up to 100%,which indicated that tritivalent vaccine had good immune efficacy.In order to determine the minimum immunization dose of the tritivalent vaccine,chickens were immunized subcutaneously with vaccines of 0.1 mL,0.3 mL,and 0.9 mL.By measuring antibody levels and immune protection rate,it was found that the average antibody titer of H9N2 avian influenza induced by the three immune doses all reached 10.00±0 log2,and the protection rate against H9N2 avian influenza virus and fowl adenovirus serotype-4 all reached 100%.However,there were differences in antibody induction of Newcastle disease virus and fowl adenovirus serotype-4 at different doses.When the immune dose was 0.1 mL and 0.3 mL,the average antibody titer of Newcastle disease virus and the average neutralizing antibody titer of fowl adenovirus serotype-4 increased significantly with the increase of dose(P<0.01).When the immune dose was 0.9 mL,there was no significant change in the average antibody titer of Newcastle disease virus and the average neutralizing antibody titer of fowl adenovirus serotype-4(P>0.05).The results showed that the minimum immunization dose.of avian influenza(H9N2 subtype),Newcastle disease,chicken hepatitis-hydropericardium syndrome tritivalent inactivated vaccine was 0.1 mL. |
