Multiplex real-time PCR assays for the identification and semi-quantitative assessment of strongyle nematodes in faeces
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| Year of publication | 2021 |
| Type | Conference abstract |
| MU Faculty or unit | |
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| Description | Background. The diagnosis of strongyle nematode (SN) infections in small ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture techniques. Such procedure is not only archaic but also demanding on time and labor, requiring a skilled expert. Nowadays, molecular methods are the cornerstone of reliable diagnostics for sustainable parasite control and accurate SN identification. Material and Methods. Two multiplex real-time PCR assays for specific detection of five main and one invasive SN species, including an internal amplification control to avoid false negative results, were designed. The assays were optimized for analysis of DNA extracted from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Eggs per gram of faeces value was assigned to each of the detected species based on a semi-quantitative evaluator which was established using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. The applicability of assays was tested on 44 individually collected faecal samples from three farms. Results were compared to those recorded using the Concentration McMaster technique and larval cultures. Results. Assays showed great specificity to target species of SN and further clarified species identification obtained by larval culture. Also proved higher sensitivity in strongylid-type egg detection over faecal egg counts techniques by revealing three false negative samples, while showing moderate agreement in evaluation of infection intensity. Conclusion. Multiplex assays proved to be rapid and accurate for analysis of the faecal samples with the focus on simultaneous and reliable species identification 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. Funding source: The work supported by an INTER-COST project by the Czech Republic Ministry of Education, Youth and Sports (LTC19018) and the COST Action COMBAR CA16230. |
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