Genotyping And Virulence Of Toxoplasma Gondii Isolates From Cats In Different Regions Of China

Objective This study is aimed to reveal whether T. gondii genotypes in cats changes with the location variation and also to inspect the virulence of the isolated T. gondii.Method Totally 89 cats were bought from Anhui (22), Hubei (23), Guangdong (20) and Shanxi province (24) respectively.All the cats were anesthetized before sacrificed. Brain, tongue and heart were removed from each cat for T. gondii examination and isolation. In order to isolate the parasite from the cats, protocol described by Dubey was used. Briefly,20 g of tongue, brain, and heart tissue of each animal were pooled and were homogenized in ten volumes (w/v) of saline solution, mixed with ten volumes of acidic pepsin and the mixture was incubated in a shaker for lh at 37℃. The digest was centrifuged, neutralized, and added with antibiotics. Total of 0.5 ml of the homogenate was inoculated subcutaneously into five mice for each cat. Then,0.5 ml Dexamethasone was injected into each mouse in the first three days after inoculation of tissue homogenate. The mice were sacrificed under anesthesia and examined for viable T. gondii tachyzoites as soon as obvious clinical manifestations were observed. Totally 14 T. gondii isolated were obaitained. To detect the virulence of these isolates, each of ten mice for each isolate was inoculated with 1000 tachyzoites. Mortality was recorded based on the death of date after inoculationDNA lysates were extracted from the infected mice peritoneal lavage,inorder to get pure tacyzoite of T. gondii density gradient centrifugation was used. We use fast DNA purification kit to extract DNA samples.9 unlinked three-way markers were selected including 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1b, C22-8, C29-2 and an apicoplast locus Apico. For each candidate marker, the target DNA sequence was amplified by PCR using Extac DNA polymerase (TaKaRa). The reaction was carried out in 25ul containing 2ul PCR buffer,2.5ul MgC12,2ul dNTPs,0.5 mM each of the forward and reverse primers,0.3 units of EXtac DNA polymerase, lul of DNA lysate and 18.2ul H2O. Reference types wereⅠ(RH) andⅡ(PRU) andⅢ(CTG). In order to reveal the pattern of each strain,10 ul of PCR products digest with restriction enzymes. The reaction was carried out by incubating at the proper temperature for each restriction enzyme by the manufacturer's instruction (Fermentas). The digested PCR products were resolved in a 2.5% agarose gel by electrophoresis with 0.3 mg/ml ethidium bromide and visualised under UV light. Compare with the three reference strains the new T.gondii strains genotype can be detected.Results 14 T. gondii was isolated from tissues of 89 cats and these isolates were designated Tgctysl-2, Tgctwhl-8 and CopI-IV.The T. gondii isolates positive rate changes with different locations, Hubei province with highest 34.8% much higher than the other three provinces. Genotyping of the 14 isolates revealed only two genotypes. Neither of the two belong to the three clonal lineage types (Ⅰ,Ⅱ,Ⅲ).12 of the 14 strains had TypeⅡpatterns at SAG2, GRA6, L358, Pk1,C22-8, and C29-2 loci but displayed a TypeⅢpattern at the SAG3,BTUB loci and type I in Apico locus.we designated this type #1.It is highly pathogenic in mice,all the mice died 4-7 days after inoculated subcutaneously with 1000 tachyzoites. Interestingly, in isolate Tgctys1, Tgctwh1 and Tgctwh4 group one or two mice can survive for more than one and half month and cysts can be found in this mouse brain after dead. The other two isolates(Tgctwh6, Tgctwh8) revealed unique genotype in SAG3,BTUB and GRA6 loci and the rest loci type are the same with #1,we named it #2. It's avirulence to mice and many cysts can be found in mice brain too. This two isolates were indistinguishable from each other by PCR-RFLP method. As far as we know #2 is a unique combination of archetypal and has never been found in China before. No typical TypeⅠ,ⅡandⅢlineages that predominate in north America and Europe was found in this study.Conclusions 1. In our study, we found that geographicical changes may do a little infection to the T.gondii genotyping. The T.gondii genotypes we have isolated from cats are all atypical and #1 is up to 85.7% may be it is the major genotype spread in China and it is also high virulent to mice.#2 as the best as we know it has never been found in China before. It means that some other genotypes may spread in cats in China too.