关键词: 副猪嗜血杆菌, ABC转运体周质底物结合蛋白, 间接ELISA, 免疫评价

Abstract:

【Background】Haemophilus parasuis (HPS) is the pathogen of upper respiratory tract of pigs, causing Glaser’s disease, mainly in pigs before and after weaning and in the nursery stage, which is usually seen in young pigs aged 5-8 weeks, and the incidence rate is generally 10%-15%. There are 15 serotypes of the bacteria and the serotypes currently prevalent in major pig-raising countries are 4, 5, 12 and 13, and it is one of the main bacterial pathogens affecting the development of pig industry. At present, there is no commercial kit for detecting antibody of the bacteria in China.【Objective】The development of a rapid, sensitive and specific antibody detection kit could provide the technical support for the effective prevention and control of the disease.【Method】Three different periplasmic substrate binding proteins of OppA, DppA and HbpA were expressed and purified. The positive and negative serum was used to screen one of the above three proteins with sound immunoreactivity and specificity. Using the screened protein as the coating antigen, an indirect ELISA method for detecting HPS antibody was established, the reaction conditions of indirect ELISA were optimized, and the kit was assembled. On this basis, the sensitivity and specificity of the kit were evaluated; the practicability of the kit was evaluated by testing 2 000 clinical pig serum samples collected at different times and from different pig farms; based on the above clinical serum samples, 200 samples were selected randomly and tested with this kit, indirect hemagglutination test, and imported commercial kit, respectively, and the test results were compared to verify the compliance rate of the kit; finally, the kit developed in this study was used to detect immune and challenge pigs. The collected serum was used to evaluate the growth and decline of antibodies against the bacteria after immunization.【Result】Three proteins of OppA, DppA and HbpA were successfully expressed and purified, and it was found that OppA had the best immunoreactivity and specificity. After optimizing the reaction conditions, the coating concentration of OppA was determined to be 1 μg·mL^-1, the blocking solution was 0.5% BSA-PBS solution, the sample dilution was 1% BSA-PBST solution, the sample incubation time was 30 min, the sample dilution was 1:50, the enzyme labeled secondary antibody incubation time was 30min, the substrate action time was 15min, and the cut-off value was 0.18; the sensitivity and specificity of the kit were 96.67%, and the kit could detect the HPS positive sera against HPS with common serotypes prevalent in China and no cross-reaction with positive sera of other common pathogens in pigs; the positive rate of 2 000 clinical serum samples was 34.65%; 200 serum samples were randomly selected, the coincidence rate with indirect hemagglutination test was 92.50%, and the coincidence rate with imported commercial kit was 87.00%; using the kit to detect the swine serum collected at different times after immunization and challenge, the HPS antibody fluctuation rule was in line with expectations.【Conclusion】The ELISA antibody detection kit for Haemophilus parasuis developed in this study had high specificity and sensitivity, and hads a high coincidence rate with the commercial kit and indirect hemagglutination test, so it could be used for clinical HPS antibody detection and vaccine immunity evaluation.

Key words: Haemophilus parasuis, ABC transporter periplasmic substrate binding proteins, indirect ELISA, immunization evaluation