Development of molecular markers for the typing and genetic analysis of Toxoplasma gondii
To develop robust and reproducible methods for molecular typing of Toxoplasma strains, the DNA regions of 5S rDNA, 28S-18S rDNA IGS SAG2, and GRA6 loci were examined. The 5S sequences were identical among 24 different strains; sequencing of the IGS region showed a few polymorphisms (0.66%) distinguishing virulence types. The IGS PCR-RFLP methods were developed and used to examine 29 strains of different virulence types. Sequence analysis of the IGS 5'-end showed great diversity between Neospora caninum and T. gondii. The IGS-RFLPs also clearly distinguished between those two closely related species. Nucleotide sequencing of the SAG2 locus (a surface antigen coding gene) showed 1.37% polymorphisms among 24 strains. Apart from a single nucleotide change at the 5'-flanking region, the type III and type I strains were identical. However, three new alleles of this locus were identified in minor variants of the strains. Analysis of the coding region of the GRA6 locus (a dense granule antigen coding gene) revealed a great degree of polymorphisms (3.24%) among 33 strains. Nine different alleles, representing the three current types and the minor variants of strains were characterised at this locus. A PCR-RFLP based on GRA6 polymorphisms was developed which could distinguish the three major types of T. gondii. This marker proved to be a suitable tool for a population study of the Toxoplasma parasite. The predominance of non-synonymous nucleotide substitutions in SAG2 and GRA6 genes confirmed positive selection in these loci, suggesting they play an important role in the parasite virulence. Phylogenetic analysis based on the multi-locus sequence alignment showed the existence of more than three lineages in Toxoplasma populations.