Isolation And Genotyping Of Toxoplasma Gondii Isolates From Cats In Southwest China

Objective To investigate the genotype and genotype-associated virulence ofToxoplasma gondii isolates from stray cats in Southwest China(Guiyang province)using multilocus polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technology.Methods Brain tissues of12stray cats collected from Guiyang were bioassayed inmice and viable T. gondii were isolated from the brains of two cats. The samples weregenotyped at10genetic markers, including9nuclear loci, SAG1, SAG2, SAG3, BTUB,GRA6, c22-8, c29-2, L358, PK1and an apicoplast locus Apico. the target DNAsequences were amplified by PCR using PCR Master Mix for all markers. Each PCRreaction was conducted on1.5μl of each DNA extraction sample with25μl PCRMaster Mix,1μl of each primer with the total reaction volume reaching50μl.To disclose the pattern of each strain,10μl of PCR products with restriction enzyme.thereaction was carried out by incubating at the appropriate temperature for each restrictionenzyme by the instruction book. the PCR products were disposed in a2.5%agarose gelby electrophoresis and visualised under UV light.Compare with the reference strainsthe new T.gondii strains genotype can be detected. To determine the susceptibility ofmice to China1lineage of the parasites，four strains with10mice for each (KM,BALB/c, C57BL, and ICR) were individually inoculated with1000tachyzoites of T.gondii CT-1isolate. And then mortality and survival rates were recorded daily for45days.Results Genotyping of2T. gondii isolates using10markers for PCR-RFLP analysesrevealed one genotype of China1(ToxoDB#9). SAG2、GRA6、L358、PKl、c22-8locusof the China1genotype were the same as type II, SAG3、BTUB、c29-2locus were thesame as type III, Apico locus was same as type I. Four strains ofBALB/c,C57BL,KM,ICR female mice each with ten.The mortality of C57BL, BALB/c,KM, and ICR mouse strains were100%,100%,30%, and20%,respectively, which hada significant difference (P <0.01); comparision of C57BL with BALB/c mice, andKM with ICR mice showed no significant difference (P>0.01).Conclusion A limited genetic diversity was found among isolates of T. gondii inChina and genotype China1(ToxoDB#9) is dominantly prevalent even in the SoutwestChina where a high biodiversity is presented.