Isolation and RFLP genotyping of Toxoplasma gondii from the gray wolf (Canis lupus)

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study feral gray wolves (Canis lupus) from Minnesota were examined for T. gondii infection. Antibodies to T. gondii were detected in 130 (52.4%) of 248 wolves tested by the modified agglutination test (cut-off titer of 25). Tissues (hearts, brains or both) of 109 wolves were bioassayed in mice for protozoal isolation. Viable T. gondii was isolated from 25 and the isolates were further propagated in cell culture. T. gondii DNA from these isolates was characterized using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Four genotypes were detected. Twenty-one isolates were Type 12 (ToxoDB PCR-RFLP genotype #5), 2 were Type II clonal (ToxoDB #1), 1 was Type II variant (ToxoDB #3), and 1 was a new genotype designated as ToxoDB genotype #219.