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

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.