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

Abstract

76 SummaryThe 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.

First report of the giant liver fluke (Fascioloides magna) infection in farmed fallow deer (Dama dama) in Poland – pathomorphological changes and molecular identification

“…The parasites were identified based on the sequence of the internal transcribed region 2 (ITS-2) with the use of primers designed by Wobeser and Schumann (23), Kralova-Hromadova et al (9), and Bazsalovicsova et al (2). A PCR method was used, as described by Kasny et al (7) with some modifications concerning reaction mixture and the time of amplification conditions.…”

Molecular discrimination of eggs of cervid trematodes using the Teflon (PTFE) technique for eggshell disruption

“…2). The PCR products were of corresponding size as amplicons obtained on genomic DNA isolated from adult flukes (Bazsalovicsová et al, 2010).…”

“…The efficiency of the method was checked microscopically and followed by the amplification of egg DNA using ribosomal internal transcribed spacer 2 (ITS2) species-specific primers. Intraspecific variation within the ITS2 region has recently been evaluated in worldwide geographic populations of all four flukes (Králová-Hromadová et al, 2008;Bazsalovicsová et al, 2010). The studies revealed low (F. hepatica, D. dendriticum) or no (F. magna, P. cervi) intraspecific variation and consistent interspecific differences amongst the four liver and stomach flukes.…”

“…DNA extraction of D. dendriticum and P. cervi eggs obtained from faecal samples was carried out with QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), following the manufacturers´ recommendations. PCR conditions, sequences and annealing positions of species-specific primers, employed in the current work, were described in detail in studies of Králová-Hromadová et al (2008) and Bazsalovicsová et al (2010).…”

“…The studies revealed low (F. hepatica, D. dendriticum) or no (F. magna, P. cervi) intraspecific variation and consistent interspecific differences amongst the four liver and stomach flukes. Selected regions within ITS2 spacer which exhibited no intraspecific variation and showed interspecific sequence heterogeneity, were applied for design of species-specific primers as promising markers in taxonomic and population studies of the flukes (Králová-Hromadová et al, 2008;Bazsalovicsová et al, 2010). The primers were utilized for amplification of species-specific ITS2 regions on DNA isolated from eggs of studied species.…”