Development Of An Antibody-ELISA For Trypanosoma Evansi And Preliminary Investigation Of Trypanosoma Evansi Infection Of Buffaloes In Guangxi

The disease caused by Trypanosoma evansi (T. evansi) is a broad host disease. All mammals are susceptible with T. evansi. In southern China, T. evansi usually infects cattle, particularly Buffalo and causes the cattle acute death or subclinical infection. The maim symptom are anemia, progressing emaciation, stillbirth, miscarriage, reduced lactation et al. T. evansi has become the major hindrance of Chinese animal husbandry, especially the cattle industry. As antigen of T. evansi is changing frequency, no successful vaccine is available for controlling this disease. Drug is still the maim way for controlling the disease of T.evansi. However, because the infection of T. evansi in cattle is chronic and the symptom of worm-emia is fluctuating, it is very difficult to make early detection. Therefore, developing appropriate method for early diagnosis, and monitoring the antibody level of T. evansi in herd serology is a very important guide for control of the disease by drugs.In this experiment New Zealand rabbits were inoculated with 10~7 T. evansi stock HB. Then ten clone-derived populations were isolated from each of 4 New Zealand rabbits at 3-day intervals. At the same time, 40 rabbit anti-T, evansi sera were prepared. Nighteen Variant Antigen Type (VAT) were identified from the 40 populations by the Indirecet fluorescent antibody staining tecnique(IFA). Two VATS, HbTat1.18 and HbTat1.15 which reacted with sera of different populations strongly, derived from 40 populations by the IFA.VSG of HbTat1. 18 was subtract and purified by ultrahigh speed centrifugation. During the lyse of T. evansi populations, different protein inhibitions were used, and then twice of ultrahigh speed centrifugation were applied. Throgh SDS-PAGE analysis, the molecular weight of the purified VSG was about 63.5KDa.The soluble antigen derived from the VAT HbTat1.18 was used for developing an indirect ELISA. The optimal condition of the ELISA reaction was determined. It is shown that the optimal of coating concentration of the protein was 2.59μg/ml, the optimal dilution of serum sample was 1:400, the optimal reaction time of serum to antigen was 1.5 hours, the optimal reaction time of enzyme-labeled goat anti-bovine IgG HPR was 1 hour and the optimal coloration time of substrate was 20 minutes. 26 negative serum samples was detected under the definite condition. According to the form (critical value＝mean value of negative serum + 3SD), the critical value is determined as 0.227. The recombinant protein shown no reaction with anti-fasciolias, it is indicated that indirect ELISA was specific for detecting antibody to T. evansi in serum. 16 positive serum samples which is selected from an exprimental buffalo infected with the T. evansi and 8 negative serum samples were examined by developed ELISA, 100%(16/16) gave positive tests while 100%(8/8) gave negative tests.The indirect ELISA was used for detecting the antibody variation from exprimental buffalo and prevalence of T.evansi in Guangxi. 79 buffaloes serum samples from Guangxi were examined, 23(29.1%) gave the positive tests.In this study, two VATS, HbTat1.18 and HbTat1.15 which have some specific antigenition from 40 T. evansi populations were selected by IFA. An indirect antiboy ELISA was developed successfully by using the soluble protein from the VAT HbTat1.18. For detection of the serum samples from Guangxi by the ELISA, it's indicated that T. evansi is still prevalent in Guangxi and shows a high infection rate in Guangxi buffaloes. The department must take more inttentions on detection and monitoring of T. evansi infection, and make sure the buffalo industry of guangxi develop successfully.