The Establishment Of Serological And Pathogenic Detection Method Of Rhodococcus Equi And Its Epidemiological Investigation

Rhodococcus equi is a Gram positive facultative intracellular pathogen, which mainly causes subacute or chronic abscessating bronchopneumonia of foals in foals up to 6 months. The mortality rate of Rhodococcus equi disease is up to 80%, it can cause severe economic losses to the stud-farm and horse industry. So far, the study of R. equi in China is rarely seen, lacking of the standard pathogenic and serological survey methods. Recently research suggested, virulence-associated lipoprotein A(Vap A) is the only membrane lipoprotein associated with bacterial virulence of R. equi, which can be used as an antigen marker of virulence R. equi. Therefore, we utilized the prokaryotic expression system to express a recombinant Vap A protein(r Vap A) for developing an indirect ELISA test to detect R. equi serum antibody. Meanwhile, a colony immunoblot test was established to detect R. equi and to distinguish the virulence and non virulence R. equi.First, based on the p MAL-c5 x prokaryotic expression system, a soluble r Vap A protein was obtained and it was identified to be a good antigen reactivity by western blot.Secondly, an indirect ELISA test was developed to detect R. equi serum antibody using the purified r Vap A protein as a coating antigen and the cut-off value was 0.370. Through the specificity, repeatability and sensitivity test, the results showed that the ELISA test had no any cross reactions with other positive sera, such as the positive sera of H3N8 equine influenza, equine rhinopneumonia or equine infectious anemia. In addition, the coefficient of variations in both inter- and intra-assay were less than 15%. In some province of China, it was the first time to investigate the R. equi seroepidemiology by using the ELISA test. The results indicated that the total positive rate of R. equi serum antibody was up to 53.93%. There existed significant differences in the regions, sexes, breeds and seasons in the level of antibody, but not with ages.Finally, a colony immunoblot test was established to detect R. equi by using the Rabbit anti virulence R. equi Vap A polyclonal antibody and Rabbit anti non virulence R. equi polyclonal antibody. After optimizing the expression conditions, the colony immunoblot test can differentiate the virulence and non virulence R.equi. Compared with bacteria isolation and PCR method, the colony immunoblot test had the advantages of high sensitivity, good specificity and fast detection. By using the method to investigate the R. equi epidemiology in soil in some provinces of China, suggested that the R. equi distribution in farm soil was related with horse activity and the average positive rate of R. equi in soil was 43.21%, without virulence R. equi.The purified r Vap A protein can offer material basis for pathogenic mechanism research, diagnosis and treatment method establishment of R. equi. In this study, the indirect ELISA test and colony immunoblot test were established to provide technical support for the epidemiological investigation of R. equi. And the horses R. equi epidemiological information was obtained, it would provide basic data for the prevention and control of the Rhodococcus equi disease in our country.