Development And Preliminary Application Of ELISA Differentiation Diagnosis For Acute And Chronic Porcine Toxoplasmosis

Toxoplasmosis is an amphixenosis caused by an obligate intracellular protozoan Toxoplasma gondii. T. gondii infection is worldwide, and it is transmitted among almost all worm-blooded animals including human, livestock and other wild animals. It has been estimated that one third of the word population has been infected. Usually the infection does not cause serious illness, but create cysts in tissues of host. Tissue cysts are long-lived and not associated with disease. However, in immunocompromised individuals, such as AIDS or malignancies, rupture of tissue cysts and the transformation of bradyzoites to tachyzoites results in disease reactivation. This reactivation can result in serious clinic sympotoms even be lethal. Infection in humans generally occurs either through ingestion of infectious oocysts in food or water, or ingestion of viable tissue cysts in meat. Pork is considered as a major source of meatborne tissue cysts. China has a big pig-raising industry and pork-consumption market. Thus, we urgently need a method to distinguish those pigs which carry T. gondii tissue cysts, so the measures to reduce the stress stimulation and the measures to reduce the cysts floating into market can be carried on.In this study, we acquired two kinds of recombinant T. gondii proteins by prokaryotic expression, the bradyzoite-special protein BAG1and the tachyzoite-special protein SAG1. Using rBAG1and rSAG1, we established two kinds of indirect ELISA assays. We can easily distinguish those bradyzoite-positive porcine serums, using rBAG1-ELISA and rSAG1-ELISA combined rMIC3-ELISA which was built at an earlier time.1. We acquired the complete CDS of BAG1gene of T. gondii Pru strain by RT-PCR. Then we constructed the prokaryotic expression plasmid pET28a(+)-BAG1and expressed BAG1gene in E.coli. SDS-PAGE confirmed molecular weight of rBAG1is about32kD, and western-blot confirmed the recombinant protein can be identified by T. gondii positive porcine serum.2. We acquired SAG1gene of T. gondii RH strain by PCR. Then we constructed the prokaryotic expression plasmid pET28a(+)-SAG1and expressed partial sequence of SAG1. SDS-PAGE confirmed molecular weight of rSAG1is about35kD, and western-blot confirmed the recombinant protein can be identified by T. gondii positive porcine serum.3. We established rBAG1-ELISA, and figured out that the best protein concentration is1:200, the best serum concentration is1:100, the best BSA concentration is1.00%, the best blocking time is30min, the best serum incubating time is30min, the best concentration of Gaot-anti-Porcine antibody is1:7000, the best incubating time of Gaot-anti-Porcin antibody is60min, the threshold value is OD630=0.38.4. We established rSAG1-ELISA, and figured out that the best protein concentration is1:200, the best serum concentration is1:100, the best BSA concentration is1.00%, the best blocking time is60min, the best serum incubating time is30min, the best concentration of Gaot-anti-Porcine antibody is1:6000, the best incubating time of Gaot-anti-Porcin antibody is60min, the threshold value is OD630=0.35.5. T. gondii epidemiological survey of parts of pig farms in Hubei province was carried out, using rBAG1-ELISA and rSAG1-ELISA combined rMIC3-ELISA which was built at an earlier time. The results revealed that the total T. gondii-positive rate of Hubei province is21%, and the rate of chronic infection is12.00%, and the rate of acute infection is4.30%. These results provided a good basis to us for preventing and controlling T. gondii infection.