The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

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. Graphical Abstract

The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture 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 PCR assays for specific detection of six main 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 larval cultures (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 3 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.

Nematode Ashworthius sidemi Schulz, 1933 (Trichostrongylidae: Haemonchinae) in mountain ecosystems – potential risk for the Tatra chamois Rupipacra rupicapra tatrica (Blahout, 1971/1972)

Background Native to Asia highly pathogenic nematode Ashworthius sidemi is now in Europe, and several dozen years after its introduction is a widespread parasite of all wild cervids. For bovids, the nematode is a significant threat to the European bison (Bison bonasus) population and has also been found in mouflon (Ovis aries musimon). This study aimed to assess the risk of infection for the endemic subspecies of northern (Alpine) chamois (Rupicapra rupicapra) – Tatra chamois (R. r. tatrica), having a critically endangered status. Methods The study was conducted in mountainous areas of Slovakia and Poland occupied by Tatra chamois (R. r. tatrica), Alpine chamois (R. r. rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus). Animals (n = 93) shot during licensed hunting and killed in road accidents (roe deer, red deer), or died a natural death (chamois) were post-mortem examined on the presence of Haemonchinae. Results A. sidemi affected all of the roe deer, and 90.0% of red deer examined. As regards chamois, it was found in one R. rupicapra originating from the Low Tatras, but not in any pure R. r. tatrica individual living in High and Western Tatras. The present work is the first confirmation of northern chamois infection with this alien, blood-sucking nematode. Conclusions Due to an important health hazard related to A. sidemi infection for the Tatra chamois (R. r. tatrica), appropriate measures should be taken to reduce the possibility of parasite transmission between various cervid species living in the Tatra area, as well as the affected population of chamois, and the pure Tatra chamois population existing in the higher parts of the mountains, constituting their natural habitat.

The use of high resolution melting analysis of ITS-1 for rapid differentiation of parasitic nematodes Haemonchus contortus and Ashworthius sidemi

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.

Parasitofauna of European bison Bison bonasus (L.) in Borecka Forest

The aim of the study was to determine the parasite species occurring currently in European bison from the Borecka Forest and to estimate prevalence and intensity of their infections. Anatomopathological and parasitological necropsy of four bison from the Borecka Forest has been carried out. 24 bison faecal samples were also analyzed using flotation, decantation and the Baermann method. As a result of performed necropsies and coproscopical analysis, 13 nematode species, 2 species of flukes and 7 species of coccidia were found. In comparison with previous studies, parasitofauna of European bison from the Borecka Forest has been augmented with 6 nematodes species (Ashworthius sidemi, Haemonchus placei, Aonchotheca bilobata, Ostertagia antipini, Ostertagia lyrata and Trichuris ovis), 1 species of fluke (Paramphistomum cervi) and 3 species of coccidia (Eimeria auburnensis, E. canadensis and E. alabamensis). All necropsied bisons were infected with nematodes A. sidemi. The Borecka Forest has been recognized as a new focus of ashwortiosis. Post-mortem examinations confirm the role of parasites, both nematodes and flukes, in the formation of gross lesions in infected bisons. .

Parasitofauna of European bison Bison bonasus (L.) in Borecka Forest

The aim of the study was to determine parasite species occurring currently in European bison from the Borecka Forest and to estimate prevalence and intensity of their infections. Anatomopathological and parasitological necropsy of four bison from the Borecka Forest has been carried out. 24 bison faecal samples were also analyzed using flotation, decantation and the Baermann method. As a result of performed necropsies and coproscopical analysis 13 nematode species, 2 species of flukes and 7 species of coccidia were found. In comparison with previous studies, parasitofauna of European bison from the Borecka Forest enriched with 6 nematodes species (Ashworthius sidemi, Haemonchus placei, Aonchotheca bilobata, Ostertagia antipini, Ostertagia lyrata and Trichuris ovis), 1 species of fluke (Paramphistomum cervi) and 3 species of coccidia (Eimeria auburnensis, E. canadensis and E. alabamensis). All necropsied bisons were infected with nematodes A. sidemi. The Borecka Forest has been recognized as a new focus of ashwortiosis. Post-mortem examinations confirm role of parasites, both nematodes and flukes, in formation of gross lesions in infected bisons.

Expansion of Ashworthius sidemi in red deer and roe deer from the Lower Silesian Wilderness and its impact on infection with other gastrointestinal nematodes

Nematodes