Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants

The species-specific ribosomal internal transcribed spacer 2 (ITS2) markers were designed for PCR-based molecular differentiation of Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, liver and stomach flukes of domestic and free living ruminants. Complete ITS2 sequences were obtained for D. dendriticum and P. cervi, for the later species, ITS2 structure was determined for the first time. Intraspecific variation within geographically distant populations was found to be either very low (F. hepatica; D. dendriticum) or even absent (F. magna; P. cervi). ITS2 regions with the absence of intraspecific polymorphisms but with interspecific sequence heterogeneity were applied for design of speciesspecific primers. The specificity of developed primers was tested on genomic DNA isolated from adult individuals of studied fluke species. Application of the primers is of particular value for molecular differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs after coprological examinations.