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Home / Equine Research Bank / Int J Parasitol / Detection and semi-quantification of Strongylus vulgaris DNA...
International journal for parasitology2007; 38(3-4); 443-453; doi: 10.1016/j.ijpara.2007.07.014

Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR.

Abstract: Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.
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Publication Date: 2007-08-14 PubMed ID: 17889881DOI: 10.1016/j.ijpara.2007.07.014Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The article discusses the development of a new diagnostic tool using a real-time quantitative PCR assay for identifying Strongylus vulgaris, a harmful horse-specific nematode, in equine faeces. The study showed that the new testing methodology is more accurate compared to traditional larval culture methods.

Study Objective

  • One of the main goals of this study was to create a more efficient diagnostic tool to detect S. vulgaris in horses. The existing method involves identifying L3 larvae after a two-week coproculture procedure, making it time-consuming and less accurate.
  • The researchers developed a real-time quantitative PCR assay capable of identifying S. vulgaris eggs in equine faecal samples.

Procedure

  • Researchers designed species-specific primers and a TaqMan probe by aligning known ribosomal DNA sequences of cyathostomin and Strongylus spp. nematodes.
  • The assay was tested and optimized using genomic DNA obtained from identified male worms of Strongylus and cyathostomin species.
  • To affirm the quantitative potential of the assay, eggs collected from adult female worms were investigated.
  • A statistical link was noted between egg numbers and cycle of threshold (Ct) values.
  • The PCR results remained unchanged in the presence of cyathostomin DNA, indicating no influence on results due to its presence.
  • A field evaluation was conducted on faecal samples from four Danish horse farms to substantiate the results.

Results

  • PCR assay performance was found to be significantly better than the traditional method, as represented by a high kappa-value of 0.78.
  • A strong association was found between Ct values and S. vulgaris larval counts, indicating a reliable detection of the presence of S. vulgaris.
  • The new assay could semi-quantitatively detect minute quantities of S. vulgaris eggs in equine faecal samples, confirming the reliability of this new diagnostic method.

Cite This Article

APA
Nielsen MK, Peterson DS, Monrad J, Thamsborg SM, Olsen SN, Kaplan RM. (2007). Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR. Int J Parasitol, 38(3-4), 443-453. https://doi.org/10.1016/j.ijpara.2007.07.014

Publication

International journal for parasitology
ISSN: 0020-7519
NlmUniqueID: 0314024
Country: England
Language: English
Volume: 38
Issue: 3-4
Pages: 443-453

Researcher Affiliations

Nielsen, Martin K
  • Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark. mkn@life.ku.dk
Peterson, David S
    Monrad, Jesper
      Thamsborg, Stig M
        Olsen, Susanne N
          Kaplan, Ray M

            MeSH Terms

            • Animals
            • DNA, Helminth / analysis
            • Feces / parasitology
            • Female
            • Horses / parasitology
            • Intestinal Diseases, Parasitic / diagnosis
            • Intestinal Diseases, Parasitic / veterinary
            • Male
            • Parasite Egg Count
            • Reverse Transcriptase Polymerase Chain Reaction
            • Strongyle Infections, Equine / diagnosis
            • Strongylus / genetics

            Citations

            This article has been cited 26 times.
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