Study On Molecular Diagnosis Methods For Toxoplasmasis And Drug Resistance Of Toxoplasma Gondii Isolates

Toxoplasmosis causes reproductive failure in pregnant women and pregnant animals, manifested as puny,and abnormal embryo,abortion and stillbirths.The Toxoplasma gondii is ubiquitous among people and animals throughout the world and is high infection in most herds.It is the enemy of human prepotency and one of the four factors leading to abnormal embryo in pregnant women.Because of above reasons,establishing quick diagnostic methods and isolating wild strains are very important to prevent and control toxoplasmosis.In this research,MIC3 of Toxoplasrna gondii was highly expressed in E.coli;rMIC3-SPA-ELISA and rMIC3-AG-ELISA diagnostic methods for swine toxoplasmosis were established.This research will be useful for vaccination,diagnosis, prevention and control of swine toxoplasmosis.The results of the research are as following:1.Expression of MIC3 in E.coliMIC3 of Toxoplasma gondii are considered as an important candidate antigen of diagnostic methods and DNA vaccine.The MIC3 are expressed during tachyzoite, bradyzoite and sporozoite period.The recombinant plasmid pGEX-KG-MIC3 was respectively introduced into E.coli BL21-CodonPlus.After induction by IPTG,high expressions were found.An average molecular weight of 66 KD was determined by SDS-PAGE.The expressed products of MIC3 could specifically bind to the antibody against Toxoplasma gondii by Western blot.2.Development of SPA-ELISA and AG-ELISAThe quick diagnostic methods of SPA-ELISA and AG-ELISA for swine toxoplasmosis were established by rMIC3.Both methods did not react with positive sera of other pathogen and could detect positive serum at a maximum dilution of 1:6400. OD₆₃₀ values of positive and negative sera remained constant under different storage time from 1 month to 8 months in 4℃.All the results indicated that SPA-ELISA and AG-ELISA had high specificity,good sensitivity and longer shelf life of products.A total of 304 serum samples from four species of animals(pigs,goats,dogs and cats) were detected by AG-ELISA and MAT methods.AG-ELISA showed a higher prevalence than MAT in pig sera and goat sera,and obtained the same prevalence in dog sera and cat sera.Kappa results were estimated and a good agreement was observed in AG-ELISA×MAT(κ=0.88,P＜0.001).In order to validate the effect of AG-ELISA,sera from experimentally infected pigs were detected by AG-ELISA,ELISA kit and MAT,and the time course of anti-T.gondii antibody responses for each of these tests was determined.AG-ELISA was able to detect all animals as positive at day 10,and MAT detected them starting at day 14.The peak of antibody level was detected at day 35 in AG-ELISA and MAT.The antibody level has remained high at the 57th day after inoculation in all the methods.Kappa results were estimated and a good agreement was observed both in AG-ELISAxMAT(κ=0.86, P＜0.001) and AG-ELISA×ELISA kit(κ=0.83,P＜0.001).3.Isolating for T.gondii wild strains and studying on its drug resistanceAnticoagulating blood samples collected from serum positive farms were used to isolate T.gondii parasites.The optimizing test indicated that the best culture medium was DMEM medium with high glucose,1%FBS and 10%SRC.19 blood samples collected from the pigs of T.gondii antibody positive farms were cultured with PK-15 cell line.Ten T.gondii strains were isolated,and the validity of the strains was confirmed by specific PCR and ELISA methods.These results demonstrated that the method of cell culture is an effective way to isolate T.gondii strain from animals.One of the ten isolates was proved to be resistant for sulfonamides.In conclusion,the rapid,accurate and convenient diagnostic methods of SPA-ELISA and AG-ELISA of animal toxoplasmosis have been established.They could be used widely for serological detection of T.gondii antibody in many species of animals.The isolation of pig-derived wild strains and the confirmation for sulfonamides resistance have laid the foundation for control of toxoplasmasis.