Improvement Of Indirect ELISA For Detecting Antibody Of Haemophilus Parasuis And Serological Survey Of Haemophilus Parasuis In Some Areas In Sichuan Province

Haemophilus parasuis (HPS) is a commensal organism of the upper respiratory tract of conventional pigs, but under appropriate conditions can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. HPS spreads worldwide, which is an economically important respiratory pathogen that affects swine industry. This study had improved an indirect ELISA for detecting antibody of Haemophilus parasuis and serological survey of Haemophilus parasuis in some areas in Sichuan province.1.The improvement of an indirect ELISA for detection of antibody against HPSThis study was based on the established indirect ELISA by author's laboratory with some improvements so as to satisfy the demand in serological survey of Haemophilus parasuis in practice. First, we prepared the positive control sera of Haemophilus parasuis serovar 5 derived from pigs. The bacteria was cultured in TSB medum and inactivated after 18h as the antigen, the pig was immunized intramuscularly at the dose of 2mL(1×109CFU/ml) at 14-day intervals, One week after the second booster immunization the pig was immunized ear-vein intravenously with the same dose. Blood was obtained from the pig 14 days after the last immunization and the antibody titers against HPS were detected by IHA test, and the results showed that antibody titer had reached 1:1024. In this study, we had successfully prepared the positive control sera of Haemophilusparasuis serovar 5 which can be applied to the establishment of indirect ELISA.Secondly, Indirect ELISA for detection of porcine serum antibodies against HPS has been improved and optimized. This assay was optimized for an antigen coating concentration of 1:100 (30μg/ml) and a serum dilution of 1:160 by chessboard titration tests.The optimal dilution of HRP-Rabbit Anti-Swine IgG was 1:5000. Finally, this method has improved sensitivity, specificity and compared with the indirect haemagglutination assay (IHA).The sensitivity of improved indirect ELISA method is much higher than IHA kit, the former is 2.5 to 10 times of the latter. It is determined as negative when detecting positive-serum of APP, E.coli, SS, PM, JEV by indirect ELISA. The comparison of indirect ELISA method and IHA kit, the 86 serum samples of pig were tested the antibody against HPS by indirect ELISA and IHA were 11.6%(10/86) and 9.3%(8/86) respectively. It indicated that indirect ELISA was the same as IHA to detect antibody against HPS in swine sera and the positive detection rate of HPS were higher than IHA. 2. The serological survey of Haemophilus parasuis in some areas in Sichuan provinceThe improved indirect ELISA described in this study was used to test 632 serum collected from 15 regions in Sichuan. The results showed that 103 of the 632 were positive and the total positive rate is 16.3% with positive rate from 0% to 66.6%.Testing pigs in different growth stages, results showed that the positive rate in nursery pigs, fattening pigs and adult pigs are 28.2%(29/103),19.4% (57/294) and 6.8%(16/235) respectively. The results of serological survey indicated that the pig herds in most parts of Sichuan are in different levels of HPS infection. Leshan, Emeishan, Liangshan, Dazhou and Ziyang are the regions of higher infection rate. The positive ratio of samples from Liangshan, Dazhou, Leshan, Emeishan, Ziyang were 66.6%,34.6%,30.2%,17.1%,16.3%, respectively. The positive rate in nursery pigs is obviously higher than that in other growth stages.