| Objective To investigate the genotype and virulence related effectors of the Toxoplasma gondii isolates from cat in Xuzhou, Jiangsu, and to analyze the relationship between the genotype and the virulence related genes.Methods After anesthesia, the brain, tongue and heart of the 40 cats from Xuzhou, Jiangsu were collected. After digestion and filtration, the tissues were prepared into suspension. The Kunming mice were infected with intraperitoneal injection. Collect the ascites of infected mice, purified T. gondii strains, extraction of genomic DNA.10 genetic markers (SAG1 and SAG2 and SAG3 and BTUB, GRA6, c22-8, c29-2 and 1358, PK1 and apico) were amplified by PCR-RFLP, and the amplified products were observed and analyzed by restriction enzyme digestion and electrophoresis. The genotypes of the T. gondii strains were determined by comparing with the reference strains. At the same time, the virulence was analyzed via observation of the survival time and survival rate after techyzoites inoculation, and BALB/c mice and Kunming mice were infected respectively with T. gondii. The coding sequences of virulence related effectors of GRA15, ROP5, ROP16 and ROP18 were amplified by PCR, and the polymorphism was compared with type Ⅰ, Ⅱ and Ⅲ.Results A total of 6 strains of T. gondii were isolated from cats (TgCtxz9, TgCtxz34, TgCtxz37, TgCtxz38, TgCtxz39, TgCtxz40). They were the tachyzoite and not cysts. The genotyping results showed that 2 strains belong to type Chinese 1 (ToxoDB#9) and 4 strains were found to be ToxoDB#205. All of the six isolates exhibited strong virulence, leading to 100% death after 5-7 days in BALB/c mice and 9-12 days in Kunming mice post-infection by 1 x 103 tachyzoites. The analysis of virulence related GRA15, ROP5, ROP16 and ROP18 genes showed that the Chinese 1 strains possess the polymorphic GRA15Ⅱ, ROP5μ-2, ROP16Ⅰ/Ⅲ and ROP18n, whereas the ToxoDB#205 strains carry GRA15Ⅱ, ROP5μ-1, ROP 16Ⅱ and ROP18Ⅲ. It shows that the consistency of the two factors of ROP18 and GRA15 is demonstrated, and obvious differences between ROP5 and ROP 16 of the two genotypes.ConclusionThe T. gondii isolates collected from Xuzhou have limited genotypes, which showed that 2 strains were type Chinese 1 (ToxoDB#9 type), and 4 strains were ToxoDB#205. It confirmed that two genotypes were virulent and the six T. gondii strains can cause all the mice died within a short period of time.the virulence associated molecular GRA15, rop5, ropl6 and rop18 gene sequence of two genotypes were amplified, and it confirmed that type Chinese 1 strains and type ToxDB#205 of T. gondii, were different from traditional type I, II and III in these 4 virulence associated molecular gene sequences. It share the polymorphic virulence effectors and more factors involved in the virulence. |
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