Toxoplasma gondii infection in selected species of free-living animals in Poland.

Introduction and objective. Free-living animals can play an important role as a reservoir of Toxoplasma gondi;, however, data concerning this issue in Poland are still limited.The aim of study was to assess the occurrence of T. gondii infection by using molecular methods in free-living animals in selected regions of Poland. Materials and method. Tissues samples of 396 animals (foxes, muskrats, birds, martens, badgers, polecats, raccoons, minks, raccoon dogs, otters, small rodents and insectivores, and grass snakes were collected from various regions of Poland. After samples digestion, DNA was isolated using QIAmp DNA Mini Kit (Qiagen). DNA extraction from small rodents and insectivores samples was performed without digestion. Next, nested PCR (B1 gene) and, for a part of nested PCR positive amplicons, RFLP PCR, were performed according to the method by Grigg and Boothroyd (2001). The other part of nested PCR positive DNA isolates were genotyped using 5 genetic markers: SAG1, SAG2 (5'- and 3'), SAG3, BTUB and GRA6, based on the method by Dubey et al. (2006). These PCR products were sequenced and compared with the NCBI database using Blast. Results. In total, in 50 of the 396 examined animals DNA of T. gondii was detected (12.6%). The highest percentages of positive results in PCR was obtained in martens (40.9%) and badgers (38.5%), lower in birds (27.3%) and the lowest in foxes (7.4%). The RFLP and multilocus PCR analysis showed the dominance of T. gondii clonal type II (or II/III). Conclusions. The results of this study indicate the frequent T. gondii infection among free-living animals in Poland, especially martens and badgers, which may indirectly indicate that these animals contribute to the spread of the parasite in the sylvatic environment in Poland. The genotyping analysis showed the dominance of T. gondii clonal type II (or II/III).