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Research article - Peer-reviewed, 2021

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

Reslova, Nikol; Skorpikova, Lucie; Kyrianova, Iveta Angela; Vadlejch, Jaroslav; Hoglund, Johan; Skuce, Philip; Kasny, Martin;

Abstract

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

Gastrointestinal nematode; Sheep; Multiplex detection; Real-time PCR; Cell-free DNA

Published in

Parasites and Vectors

2021, volume: 14, number: 1, article number: 391
Publisher: BMC

Authors' information

Reslova, Nikol
Masaryk University Brno
Skorpikova, Lucie
Masaryk University Brno
Kyrianova, Iveta Angela
Czech University of Life Sciences Prague
Vadlejch, Jaroslav
Czech University of Life Sciences Prague
Skuce, Philip
Moredun Research Institute
Kasny, Martin
Masaryk University Brno

UKÄ Subject classification

Clinical Science

Publication Identifiers

DOI: https://doi.org/10.1186/s13071-021-04882-4

URI (permanent link to this page)

https://res.slu.se/id/publ/113421