Molecular approaches to trematode systematics: 'best practice' and implications for future study

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the ‘best molecular practice’ for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient ‘useful’ comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics

Results on search for the broad fish tapeworm Dibothriocephalus latus (Linnaeus, 1758), (syn. Diphyllobothrium latum) (Cestoda: Diphyllobothriidea), in the Danube River

Diphyllobothriosis is a fish-borne parasitic zoonosis caused by so-called “broad tapeworms” or “fish tapeworms” of different genera of the order Diphyllobothriidea. Dibothriocephalus l atus (Linnaeus 1758), (syn. Diphyllobothrium latum), is a medically important type species of the genus, whose occurrence in various European regions is either regular, e.g. in the Alpine lakes region, or occasional and sporadic, e. g. in the Danube River region. For the latter, data on the detection of D. latus plerocercoids in the second intermediate fish host (European perch Perca fluviatilis), as well as in definitive hosts (human and dog), in which infection was directly linked to the consumption of infected fish from the Danube, were published more than 50 years ago. In order to assess the current situation, we aimed to find out whether D. latus is present in the natural environment of the Danube River. In total, 700 perch from five sampling sites in the Slovak part of the Danube River were examined. Plerocercoids were not detected in any fish examined, which leads to the conclusion that D. latus is currently not present in the studied aquatic environment

First record of Cathaemasia hians (Trematoda: Cathaemasiidae) in a new bird host, the Eastern Imperial Eagle (Aquila heliaca)

An injured young individual of the Eastern Imperial Eagle (Aquila heliaca; Accipitridae) from the Protected Bird Area „Medzibodrožie” in the south-eastern Slovakia was subjected to the complete clinical examination at the Clinic for Birds and Exotic Animals of the University of Veterinary Medicine and Pharmacy. Adult trematodes were isolated from the pharynx of the eagle after oesophagoscopy. The morphological and molecular identification of the flukes confirmed a trematode Cathaemasia hians (Cathaemasiidae), the obligate parasite of black storks (Ciconia nigra) and white storks (Ciconia ciconia). This finding represents the first documented case of C. hians in new bird host species and indicates broader spectrum of definitive hosts of the fluke

Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic

Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions[...