Establishment Of Diagnostic Methods For PPV And PRV And Immune Efficacy Of PPV-PRV Combined Inactivated Vaccine

Porcine Parvovirus infection and porcine Pseudorabies are the major diseases that caused reproductive disorders. Porcine parvovirus infection is caused by porcine parvovirus(PPV) and Pseudorabies is caused by pseudorabies virus( PRV). Both diseases are widely distributed in the world,causing huge economic losses to the swine industry. In order to effectively prevent these diseases,TaqMan real-time PCR methods were established to detect PPV and PRV, and ELISA method to detect PPV antibody was developed, and Porcine parvovirus BQ-C strain and PRV-ΔgE strains preserved in laboratory were used to developed PPV-PRV combined inactivated vaccine and evaluated its immune effect.In this study, two sensitive, specific and reproducible TaqMan real-time PCR methods were established to detect PPV and PRV according to PPV NSI gene and PRV gB gene sequence. The results showed that the two methods have no nonspecific amplification, coefficient of variation of intra-and inter-assay are less than 3%, and PPV-NS1 TaqMan real-time PCR can detect 13 copies initial template,PRV-gB TaqMan real-time PCR can detect 167 copies original template. Sensitivity of the two real-time PCR methods is 100 times than that of conventional PCR methods.In this study, PPV-VP2 protein as antigens were expressed by baculovirus expression vector system,and PPV antibodies detection indirect ELISA method was developed. After Optimization, its optimal antigen coating concentration is 10μg/mL, best HRP secondary antibody dilution is 10000 times, the best coating solution is 0.01 mol /mL carbonate buffer(PH = 9.6), the best blocking solution is 5% skim milk-PBS, the optimal blocking time is 90 min, the optimal serum dilution is 200 times, the positive Cut-off value of 0.3. The established method and commercialization ELISA kits were used to detect 360 clinical serum samples, the results show that the positive rate of established ELISA method to detect PPV antibody was 75.00%, the positive rate of commercial ELISA kit was 75.83%, Coincidence rate of the two ELISA methods is 94.17%.In this study, PPV-BQ-C strain and rPRV-ΔgE preserved in the laboratory were used as the antigens of the combined vaccine, and used β- propiolactone as inactivator. Mixed the above two inactivated virus solution with the ratio of PPV-BQ-C: rPRV-ΔgE = 2: 1. Emulsified to develop vaccine with the ratio of antigen : adjuvant = 8.5 : 1.5. Immunized replacement gilts with the developed PPV-PRV combined inactivated vaccine after preliminary examination, and PPV HI antibody levels reached a peak after immunization 4w, PPV HI antibody level of PPV-PRV combined inactivated vaccine group is 1: 776, PPV HI antibody level of PPV commercial vaccine group 1: 445.PRV neutralizing antibodies level of PPV-PRV combined inactivated vaccine group is 1: 59.46, PRV neutralizing antibodies level of PRV commercial vaccine group is 1: 32.70. In order to study the immune efficacy of PPV-PRV combined inactivated vaccine, gilts were attacks by PPV and PRV virulent, and then evaluated the protective effect of the vaccine by detecting viral load of organs. The results showed that four weeks after challenge, virus nucleic acid can not be detected in PPV-PRVcombined inactivated vaccine group after PPV virulent challenge. Virus nucleic acid can be detected in liver in PPV-PRV combined inactivated vaccine group after PRV virulent challenge. So the vaccine can resist PPV virulent attack, but can not completely resist PRV virulent attack.