Study On Methods For Detecting Toxoplasma Gondii In Pigmeat

Indirect ELISA was developed and applied for detection of Toxoplasma gondii IgG antibodies in pigs. In order to obtain purified soluble antigens of Toxoplasma gondii tachyzoities, 3 methods, which were trypsin digestion method, filitation method and phytohemagglutinin-p method for purifing Toxoplasma gondii tachzoities were compared. Results demonstrate that Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates to remove the host cells with phytohemagglutinin-p at a concentration of 0.01% which provide a 98.5% pure Toxoplasma gondii suspension. Development soluble protein-based indirect ELISA for detecting Toxoplasma gondii IgG antibodies in pigs.Polymerase chain reaction (PCR) method was developed and applied for detecting Toxoplasma gondii in pigs. A PCR method for detection of Toxoplasma gondii was established using double pairs of primers designed according to the sequence of B1 gene and 529bp gene segment. It was shown that double specific 194bp and 529bp fragment was amplified by this PCR procedure from all Toxoplasma gondii genomic DNA samples of NT strain, however, could not be demonstrated the double 194 bp and 529 bp bands from genomic DNA samples of Feline leucocyte, mice Ascitic fluid leucocyte, swine Ascitic fluid leucocyte, swine mycoplsma pneurnoniae, Salmonella, E.Coli, porcine circovirus, swine fever virus, PRRSV, Eimeria tenella, Sarcocystis cruzi, Sarcocystis suihominis and Trichinella. The sequence of the PCR products accorded with the known sequence of Blgene and 529 bp fragment. The results suggested that this PCR method was specific for Toxoplasma gondii. Sesitivity of the PCR was 20fg DNA level, the result demonstrate thai sensitivity of the PCR was higher.A total of 511 serum samples originating from animal hospital and 2 abattoirs were used in indirect hemagglutination test (IHA), immunoperioxidase staining method, indirect ELISA and PCR. Compared to DT ,the sensitivity and specificity and Youden index of IHA,immunoperioxidase staining method,indirect ELISA and PCR was 76.92%, 97.5%, 0.744; 66.8%, 100%, 0.668; 90.9%, 92.85%, 0.838; and 76.9%, 100%, 0.77 respectively. The positive rale by indirect ELISA was slightly higher than that by DT. The sensitivity and specificity of Immunoperioxidase staining method and PCR was lower. The data in this study demonstrated that the soluble protein-based indirect ELISA was not only reliable with high sensitivity and specificity, but also was economical and convenient in terms of antigen production. ELISA would be a very useful tool for routine diagnosiics, epidemiological surveys, and outbreak investigations.Based on all the results, an jpitimized rapid protocol for the detection of Toxoplasma gondii could be as follow ring: use indirect ELISA and IHA to screen the large number of samples firstly, and t len use immunoperioxidase staining method PCR to confirm the IHA and ELISA positive and suspected samples.