Eurasian beaver (Castor fiber) is a well-established faunal element in the Czech Republic, even though, historically, its populations were almost eradicated in this region. Nowadays, its distribution and population density are well monitored; nonetheless, the beaver's parasites, as potential threats to the environment, are often neglected in wildlife management. Therefore, we investigated the endoparasitic helminth diversity of 15 beaver individuals from three collection sites in the Czech Republic. Three parasite species were collected: Stichorchis subtriquetrus (Digenea), Travassosius rufus, and Calodium hepaticum (Nematoda), of which the two nematode species were reported for the first time from C. fiber in the Czech Republic. The highest prevalence and intensity of infection were observed in S. subtriquetrus (P = 93%, I = 1-138), while the two other species were collected only from one beaver individual. Subsequent analysis of the genetic diversity of the specimens using highly variable genetic markers revealed a weak population structure among the individuals collected from different beaver hosts. There was only a weak association of COI haplotypes with geography, as the haplotypes from the Berounka basin formed homogeneous groups, and individuals from the Dyje basin and Morava partially shared a haplotype. Even though common population genetic markers (i.e., microsatellites) did not reveal any structure in the hosts, our results suggest that the genetic diversity of their parasites may shed more light on population partition and the historical migration routes of Eurasian beavers.

• Keywords
• Genetic diversity, Parasite spill-over, Population genetics, Reintroduction,
• MeSH
• Genetic Variation MeSH
• Rodentia MeSH
• Humans MeSH
• Trematoda * MeSH
• Trichostrongyloidea * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

BACKGROUND: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. METHODS: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. RESULTS: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. CONCLUSIONS: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

• Keywords
• Cell-free DNA, Gastrointestinal nematode, Multiplex detection, Real-time PCR, Sheep,
• MeSH
• Feces parasitology   MeSH
• Gastrointestinal Diseases diagnosis   parasitology   veterinary   MeSH
• Gastrointestinal Tract parasitology   MeSH
• Nematoda classification   genetics   MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Nematode Infections diagnosis   parasitology   veterinary   MeSH
• Sheep Diseases diagnosis   parasitology   MeSH
• Sheep MeSH
• Parasite Egg Count MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

• MeSH
• DNA, Helminth genetics   MeSH
• Haemonchus classification   genetics   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• DNA, Ribosomal Spacer genetics   MeSH
• Polymorphism, Genetic MeSH
• Ruminants parasitology   MeSH
• Reproducibility of Results MeSH
• Trichostrongyloidea classification   genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• DNA, Helminth MeSH
• DNA, Ribosomal Spacer MeSH

Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.

• MeSH
• Yersinia Infections diagnosis   microbiology   MeSH
• Humans MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Foodborne Diseases diagnosis   microbiology   parasitology   MeSH
• Toxoplasma genetics   isolation & purification   MeSH
• Toxoplasmosis diagnosis   parasitology   MeSH
• Yersinia enterocolitica genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH