Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice

Abstract

Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in the USA. However, still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, 7 of 49 deer, and one of one goose. DNA isolated from culture-derived tachyzoites of these isolates was characterized initially using multilocus PCR-RFLP markers. Nine genotypes were revealed, including ToxoDB PCR-RFLP #1 (4 isolates), #2 (2 isolates), #3 (4 isolates), #4 (6 isolates), #5 (4 isolates), #54 (1 isolate), #141 (1 isolate), #143 (1 isolate), and #216 (6 isolates), indicating high genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of six T. gondii isolates (5 of #216 and #141) was determined in outbred Swiss Webster mice. Three of #216 and the #141 isolates were acute virulent to mice, and the other 2 #216 isolates were intermediate virulent. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results indicated that the #216 isolates are likely derived from recombination of the clonal type I and III lineages. To determine if T. gondii virulence can be predicted by typing, we genotyped a collection of strains using PCR-RFLP markers for polymorphic genes ROP5, ROP16, ROP18 and GRA15, which are known to interact with host immune response. The results showed that there is an association of genotypes of ROP5 and ROP18 with mouse-virulence, however, additional gene(s) may also contribute to virulence in distinct T. gondii genotypes.

Keywords: Genotyping; Isolation; Pennsylvania; Seroprevalence; Toxoplasma gondii; USA; Wildlife.

Fig. 1

Neighbor-net analysis using concatenated 4 intron sequence markers comprising 1775 bp and 10 RFLP markers. The, wildlife isolates (TgFoxPa7, 8, 9) of T. gondii from Pennsylvania are closely related to haplogroup 3 strains. T. gondii haplogroups are shown in the circle numbers as designated previously (Su et al., 2012). Mouse-virulent strains are in red boxes, intermediate virulent in, green, and non-virulent strains in blue, the unknowns are in black. Samples TgFoxPa7, TgFoxPa8 and TgFoxPa9 are from this study. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

Genetic relationship of 23 T. gondii strains based on ROP5, ROP16, ROP18 and GRA15 PCR-RFLP polymorphisms. Ten, genotypes were identified among these strains. Mouse-virulent strains are in red boxes, intermediate virulent in green, and non-virulent, strains in blue. The number in the parenthesis is ToxoDB PCR-RFLP genotypes. T. gondii haplogroups are shown in the circle numbers, as designated previously (Su et al., 2012). Isolates TgWtdPa4, TgWtdPa5, TgFoxPa7, TgFoxPa8 and TgFoxPa9 are from current study. The result shows potential association of genotypes with virulence phenotype. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)