Molecular method for the semiquantitative identification of gastrointestinal nematodes in domestic ruminants

Santos, Lucíola Licínio de Castro Paixão; Salgado, Jordana Andrioli; Drummond, Marcela Gonçalves; Bastianetto, Eduardo; Santos, Cláudia Portes; Brasil, Bruno S. A. F.; Taconeli, César Augusto; Oliveira, Denise Aparecida Andrade de

doi:10.1007/s00436-019-06569-3

Cited by 19 publications

(19 citation statements)

References 49 publications

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“…A next step would be to investigate which helminth species are shared between roe deer and the neighbouring domestic animals. Advanced molecular tools are now available to analyse parasite DNA in faecal samples or coproculture-derived larvae (Avramenko et al ., 2017; Santos et al ., 2020). This could be used to compare the nemabiome composition of wild and domestic ungulates sharing the same area.…”

Section: Discussionmentioning

confidence: 99%

Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density

et al. 2020

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Because of their continuing expansion, wildlife ruminant species that prosper in rural landscapes may be increasingly affected by and/or contribute to the circulation of certain generalist pathogens also infecting domestic ruminants, when they share common spaces or resources. In this study, we aimed to test the hypothesis that parasitism with gastrointestinal nematodes (GINs) of wild roe deer inhabiting different rural landscapes is correlated with livestock density. We used faecal egg counts of GINs and spatial data of 74 GPS-collared roe deer, inhabiting various landscapes from closed forests to open fields, together with weekly records of livestock abundances on pasture. We tested whether the excretion of GIN eggs in roe deer was influenced by the density of livestock in their home range over the grazing season. Our results showed that all of the roe deer home ranges, except four, contained pastures occupied by livestock. Excretion of GIN eggs occurred in 77% of the roe deer. The excretion of GIN eggs in roe deer tended to increase with livestock density in their home range. This result suggests, but does not prove, a higher risk of ingesting GIN larvae originating from livestock dung. In the context of increasing overlap between roe deer and livestock ranges, the exchange of pathogens between both hosts is plausible, although species identity of the parasites present was not determined. Assessing which GIN species are shared between wild and domestic ruminants, and how this may affect the health of both hosts, is a central question for future research in the context of interspecific pathogen circulation.

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Section: Discussionmentioning

confidence: 99%

Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density

et al. 2020

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“…The whole process of species differentiation using morphological characteristics is time-consuming and laborious, requires an experienced helminthologist, and is not always reliable. Therefore, molecular confirmation [28][29][30] . Until now, multiplex PCR techniques for the detection of A. sidemi and H. contortus tended to target the nuclear ribosomal region and mitochondrial NADH dehydrogenase subunit 4 (ND4) 40,41 .…”

Section: Discussionmentioning

confidence: 98%

“…Therefore, molecular confirmation is appropriate if not downright necessary. For this reason, GIN diagnostics based on molecular methods is fast becoming a preferred method, because it allows for a rapid , safe , and sensitive species identification 28 – 30 .…”

Section: Discussionmentioning

confidence: 99%

“…Taxonomic identification based on morphological/morphometric features is possible, especially in adult male specimens, but in adult females and immature nematodes, these methods are unreliable and in the larval stages and eggs they are not applicable at all. Moreover, because morphological examination is time-consuming and requires a helminthologist of considerable experience, we witness a growing use of molecular tools for species identification and demand for novel approaches 28 – 30 .…”

Section: Introductionmentioning

confidence: 99%

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

Škorpíková

Reslová

Magdálek

et al. 2020

Sci Rep

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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.

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“…The advantages are manifold and besides simultaneous detection of multiple parasites present in a sample (use of three different dyes for the six targets means that only one third of the number of reactions must be performed to diagnose all six GINs) include high-throughput capacity, wide dynamic range and avoidance of inaccuracies related to end-point analysis. High-throughput sequencing devices are being increasingly applied to the challenge of GIN identi cation [34] and complex nematode community studies [35,36,37]. This 'nemabiome'-type technology is still relatively expensive and requires expertise and complicated bioinformatic analysis, so is still the preserve of specialist laboratories.…”

Section: Discussionmentioning

confidence: 99%

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

Reslová

Škorpíková

Kyriánová

et al. 2021

Preprint

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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.

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Molecular method for the semiquantitative identification of gastrointestinal nematodes in domestic ruminants

Cited by 19 publications

References 49 publications

Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density

Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density

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

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

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