K. Oberhauserová, E. Bazsalovicsová, I. Králová-Hromadová, P. Major and M. Reblánová
Molecular comparative analysis of eggs of four liver and stomach flukes of cervids and domestic ruminants, Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, was performed using a new methodological approach for eggshell disintegration. Eggs of all species were crushed mechanically by the Teflon method (PTFE) without use of chemical reagents and an efficient disruption of eggshell was checked microscopically. The egg suspension was then subjected to DNA isolation and PCR amplification using species-specific primers that annealed to the internal transcribed spacer 2 (ITS2) region of ribosomal DNA. The size of PCR products of individual species corresponded well to the size of amplicons obtained from adult flukes. The results provided evidence that the Teflon method does not destroy the structure of egg DNA, thus making the procedure broadly applicable during coprological examinations. Molecular markers introduced here are particularly important for blanket screening and differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs.
E. Bazsalovicsová, I. Králová-Hromadová, M. Špakulová, M. Reblánová and K. Oberhauserová
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.