Majidi-Shad, Bijan; (1998) Development of PCR based strategies for determining starin variation in Toxoplasma gondii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Toxoplasma gondii isolates vary in pathogenic potential. Although the contribution of the host genotype to the extent of disease is documented (Suzuki et al, 1991; 1996), there is strong evidence that indicates T. gondii strain variation is also influential in outcome of infection Suzuki et al, (1989). To address this, a system was established to differentiate clinical isolates and examine them directly from patient samples. This research aimed to identify phenotypic and genotypic markers to differentiate virulent and avirulent T. gondii strains. In vitro HEL and THPl cell culture revealed that the virulent T. gondii RH and RHL strains grew faster and destroyed the monolayer earlier than the avirulent strain NED. The NED strain demonstrated a prolonged survival and produced significantly more tachyzoites than RH and RHL. These observations are comparable with the events observed in vivo. The THPl cell culture was the preferred system for study of biological differences of T. gondii strains. In the search for a molecular marker of variation, three T. gondii genes, SAG1, B1, 16S-like rDNA, were analysed. Using a polymerase chain reaction (PCR) strategy, these genes were amplified and analysed by RFLP, SSCP and sequencing. Ddel PCR RFLP of SAG1 after Southern hybridization and probing with SAG1 PCR product distinguished the virulent and avirulent genotypes of T. gondii clinical isolates, Tanzanian blood specimens and a bone marrow transplant patient sample. However, co-amplification occurred with SAG1 nested PCR of Mycobacterium tuberculosis and Leishmania brasiliensis DNA. PCR-RFLP and PCR-SSCP of the B1 gene demonstrated similarity within the sequences of the isolates studied. Alul RFLP of genomic T. gondii DNA, after Southern hybridization and probing with B1 PCR 194 bp PCR product, revealed polymorphism. PCR-RFLP and PCR-SSCP of the 16S-like rDNA gene revealed identity between isolates. The difference observed, between T. gondii and Cryptosporidium muris 16S-like rDNA PCR-RFLP pattern demonstrated the importance of this gene for differentiation of coccidian species. Sequencing of 1.3 kb of 16S-like rDNA demonstrated 1 - 2.2% sequence differences between T. gondii strains studied. The findings of this research suggest that SAG1 Ddel PCR-RFLP and B1 AluI RFLP of T. gondii genomic DNA can be used as molecular tools for differentiation of T. gondii strains at an early stage of clinical investigation with or without a limited in vitro culture.

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