Genotypes And Virulence Of Toxoplasma Gondii Isolates From Animals And Humans In China

Objective: This study is aimed to reveal the genotypes and virulence of Toxoplasmagondii isolates from animals and humans in China, as well as determined the kinetics ofinfection and cyst formation in KM mice following oral infection with cyst formingChinese isolate of T. gondii TgCtwh6(genotype Chinese1, ToxoDB#9).Methodology/Principal Findings: In the present study, totally of105stray cats werecollected from Hubei (31), Shandong (24) and Jiangsu (50) Provinces, respectively. Allcats were anesthetized before being sacrificed and isolated nineteen strains. Onehundred and eighteen sera samples from human patients were collected in Anhui andGuangdong Province from August2010to September2011. During the period of seracollection, eleven samples were positive for specific anti-Toxoplasma IgM antibodies,CAg and/or IgG antibodies, and four isolates were isolated from humans. We analyzed23T. gondii isolates from different host in this study (four from human and nineteenfrom cats). Reference strains of T. gondii were used as controls for genotyping,including Type I (GT1), Type II (PTG), Type III (CTG) and other strains (TgCgCa1(a.k.a. Cougar), MAS, TgCatBr5, TgCatBr64, TgRsCr1). Briefly, the target DNAsequences were amplified by PCR using PCR Master Mix (Promega, USA) for allmarkers. Each PCR reaction was conducted on1.5μl of each DNA extraction sample with25μl of PCR Master Mix,50pmol of each primer with the total reaction volumereaching50μl. Genotyping of the23T. gondii isolates using10PCR-restrictionfragment length polymorphism (PCR-RFLP) markers (SAG1, SAG2, SAG3, BTUB,GRA6, c22-8, c29-2, L358, PK1and Apico) from DNA obtained from tachyzoitesand/or cysts revealed five genotypes; among them three genotypes were atypical andtwo were archetypal. These atypical genotypes were designated as ToxoDB#9(Chinese1), ToxoDB#204, and ToxoDB#205, respectively. The genotype Chinese1showed typeII patterns at SAG2, GRA6, L358, PK1, c22-8, but c29-2, SAG3, BTUB loci displayeda type III pattern and type I at the Apico locus. Fifteen strains belong to the Chinese1lineage which was previously reported as a widespread lineage from swine, cats, andhuman in China. Two human isolates were grouped in the type I and II lineages and noclonal type III isolates were observed. The remaining isolates belonged to two newatypical genotypes which were analyzed for the first time in China. The ToxoDB#204genotype showed a unique pattern at the SAG1locus and the remaining9loci typeswere the same as type II, suggesting they are phylogenetically related. We also foundfour strains (TgCtxz3,5,7,8) that showed a mixture of clonal types I and II patterns atsome loci, while only the clonal type I pattern could be observed at the loci SAG3andL358, clonal type II alleles at SAG2, BTUB, GRA6, c22-8, c29-2, PK1, and Apico, aswell as a mixture of types II and III patterns at the SAG1locus, named ToxoDB#205.To determine the association between multilocus genotypes and virulencephenotypes in mice, ten KM mice (SPF) were intraperitoneally inoculated with1000tachyzoites of each Chinese T. gondii strain. The virulence was defined based on theresponse variables of presence or absence of peritoneal fluid, cumulative mortality, andthe number of days of survival of animals infected. We also determined the kinetics ofthe TgCtwh6isolate (Chinese1lineage) dissemination in blood and tissues of KM micewere also investigated by fluorescence quantitative PCR (qPCR) and subinoculation into fresh mice. Peripheral blood, lymph nodes, heart, liver and brain of SW mice werecollected on days2,4,7,10,14,21,35,50, and72post infection and5mice weresacrificed by cervical dislocation under anesthesia at each time of collection. The bloodwas collected from the retroorbital sinus with sodium citrate. Each tissue was collectedfrom the same organ at the same day post infection and gently wiped on sterile gauze,weighted and then homogenized with antibiotics. The parasites were detected inrecipient mice via:(i) microscopical examination of impression smears of ascitic fluidthat died during acute period for tachyzoites;(ii) brain tissue squash examination forcysts that survive45days post infection; and (iii) DNA extraction from brain or asceticfluid and detected by PCR with the same volume as described above. Our results showthat these genotypes of T. gondii isolates are highly virulent in mice except for the strainTgCtwh6, which caused parasitemia of35days post infection in mice although it hasbeen designated as Chinese1.Additionally, detection of expression of virulence-associated factors demonstratedthat GRA3was dramatically elevated only in this cyst forming strain TgCtwh6. Toensure that equal quantities of each mRNA were being compared, a housekeeping geneTgGAPDH was used as control. The rest of VFs were indistinguishable in both virulentand avirulent isolates with the same genotype.Conclusion/Significance: A limited genetic diversity was found, and Chinese1lineageis dominant circulating in mainland China. The ROP16and GRA3can be used asrepresentative virulent markers for differentiation between the virulent strain and thecyst forming strain in genotype Chinese1, a major type in China. Further studies on T.gondii from domestic and wild mammals, birds, and humans are necessary for a betterunderstanding of the potential relations between the population structure diversity andits pathogenesis in Chinese T. gondii isolates, and thus will provide insights into the development of novel drug targets and host defense mechanisms.