Genetic characterization of GRA6 genes from Toxoplasma gondii from pigs in Okinawa, Japan

Toxoplasma gondii from pigs in Okinawa Prefecture was characterized by nested PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analysis of the dense granule antigen GRA6 gene. By nested PCR, parasite DNA was detected in 33 out of 91 lymph node samples with lesions similar to those found in toxoplasmosis samples that had been collected from,pigs at an abattoir. RFLP analysis with MseI was successfully conducted in 29 of 33 PCR-positive samples to group the isolates into one of the three genotypes of T. gondii. Genotyping of the 29 studied samples rendered the following results: 13 of type 1 (44.8%), 14 of type 11 (48.3%), and 2 of type 111 (6.9%). The GRA6 genes of 12 Okinawa isolates were cloned and sequenced. Nine new nucleotide sequences were found, and nucleotide substitutions specific for the Okinawa isolates were found at 13 positions. Phylogenetic analysis indicated that all GRA6 sequences were divided into one of the 3 main groups, and Okinawa isolates of GRA6 genotypes II and III seemed to be closely related to the Beverley strain and the NED strain, respectively. The results from this study may provide basic and useful information for the analysis of the molecular epidemiology of T. gondii infection within Japan.