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

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. Graphical Abstract

The performance of field sampling for parasite detection in a wild passerine

“…High Isospora fecundity associated with most coccidia species (Burrell et al, 2020 ) may be responsible for a lower percentage of false negatives in captive samples, as oocysts will be shed in frequent and regular intervals in spite of unique shedding peaks. In studies where precise detection of a specific parasite is crucial, molecular‐based techniques may be more sensitive at detecting and quantifying parasites, especially with low infection loads (Dacal et al, 2018 ; Reslova et al, 2021 ). For either approach—molecular or microscopic—at least some individuals should be sampled repeatedly to provide an estimate of the error rate (which should then be reported).…”

Faecal metabarcoding provides improved detection and taxonomic resolution for non-invasive monitoring of gastrointestinal nematode parasites in wild moose populations

“…Given that no GIN taxa were detected solely by parasitological methods and not concurrently by metabarcoding, we instead argue that metabarcoding of GIN DNA isolated from frozen faecal samples has increased sensitivity when compared with egg and larval counts from the same samples when they are fresh, most likely in cases with low egg and larval abundance. Other PCR-based methods have been demonstrated to have increased sensitivity over traditional microscopy-based methods for GIN detection [ 50 – 53 ], but to our knowledge, this has not been previously demonstrated for DNA metabarcoding. We hypothesize that this increased sensitivity can be attributed to the metabarcoding method also detecting extracellular DNA derived from adult worms [ 54 ] in the gastrointestinal tract that may be shedding few or no eggs at the time of sampling.…”

Exploring winter diet, gut microbiota and parasitism in caribou using multi-marker metabarcoding of fecal DNA

“…For example, the Parelaphostrongylus genus includes at least three species identi ed as caribou parasites (P. odocoilei, P. tenuis, and P. andersoni) but their frequency of occurrence and impact on caribou health is quite different: P. andersoni, a muscle worm infecting caribou and other Cervidae in Canada, is common in boreal caribou, whereas P. tenuis, the meningeal worm, is rare but lethal 25 . Other complementary approaches, such as the sequencing of the ITS2 region using primers speci cally designed to target nematodes 33 or the use of qPCR detection assays allowing to differentiate species, as proposed for other nematodes (see Reslova 74 et al as an example), could be considered in this case.…”