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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 32 times.
            1. Osterman-Lind E, Holmberg M, Grandi G. Selective Anthelmintic Treatment in Horses in Sweden Based on Coprological Analyses: Ten-Year Results. Animals (Basel) 2023 Aug 28;13(17).
              doi: 10.3390/ani13172741pubmed: 37685005google scholar: lookup
            2. Stummer M, Frisch V, Glitz F, Hinney B, Spergser J, Krücken J, Diekmann I, Dimmel K, Riedel C, Cavalleri JV, Rümenapf T, Joachim A, Lyrakis M, Auer A. Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic. Pathogens 2023 Aug 15;12(8).
              doi: 10.3390/pathogens12081043pubmed: 37624003google scholar: lookup
            3. Martins AV, Coelho AL, Corrêa LL, Ribeiro MS, Lobão LF, Palmer JPS, Moura LC, Molento MB, Barbosa ADS. First microscopic and molecular parasitological survey of Strongylus vulgaris in Brazilian ponies. Rev Bras Parasitol Vet 2023;32(3):e006323.
              doi: 10.1590/S1984-29612023036pubmed: 37377279google scholar: lookup
            4. El-Gameel SM, Al-Mokaddem AK, Salaeh NMK, Attia MM. Morphomolecular characterization of Strongylus vulgaris isolated from donkeys with special references to histopathological study on the affected organs. J Parasit Dis 2022 Sep;46(3):795-803.
              doi: 10.1007/s12639-022-01498-ypubmed: 36091280google scholar: lookup
            5. Jürgenschellert L, Krücken J, Bousquet E, Bartz J, Heyer N, Nielsen MK, von Samson-Himmelstjerna G. Occurrence of Strongylid Nematode Parasites on Horse Farms in Berlin and Brandenburg, Germany, With High Seroprevalence of Strongylus vulgaris Infection. Front Vet Sci 2022;9:892920.
              doi: 10.3389/fvets.2022.892920pubmed: 35754549google scholar: lookup
            6. Hedberg-Alm Y, Tydén E, Tamminen LM, Lindström L, Anlén K, Svensson M, Riihimäki M. Clinical features and treatment response to differentiate idiopathic peritonitis from non-strangulating intestinal infarction of the pelvic flexure associated with Strongylus vulgaris infection in the horse. BMC Vet Res 2022 Apr 23;18(1):149.
              doi: 10.1186/s12917-022-03248-xpubmed: 35461295google scholar: lookup
            7. Roeber F, Webster M. Protecting dogs and cats against the Australian paralysis tick, Ixodes holocyclus (Acari: Ixodidae): A review of the Australian acaricide registration process. Curr Res Parasitol Vector Borne Dis 2021;1:100054.
              doi: 10.1016/j.crpvbd.2021.100054pubmed: 35284866google scholar: lookup
            8. Steuer AE, Anderson HP, Shepherd T, Clark M, Scare JA, Gravatte HS, Nielsen MK. Parasite dynamics in untreated horses through one calendar year. Parasit Vectors 2022 Feb 8;15(1):50.
              doi: 10.1186/s13071-022-05168-zpubmed: 35135605google scholar: lookup
            9. Gehlen H, Wulke N, Ertelt A, Nielsen MK, Morelli S, Traversa D, Merle R, Wilson D, Samson-Himmelstjerna GV. Comparative Analysis of Intestinal Helminth Infections in Colic and Non-Colic Control Equine Patients. Animals (Basel) 2020 Oct 19;10(10).
              doi: 10.3390/ani10101916pubmed: 33086590google scholar: lookup
            10. Tydén E, Enemark HL, Franko MA, Höglund J, Osterman-Lind E. Prevalence of Strongylus vulgaris in horses after ten years of prescription usage of anthelmintics in Sweden. Vet Parasitol X 2019 Nov;2:100013.
              doi: 10.1016/j.vpoa.2019.100013pubmed: 32904767google scholar: lookup
            11. Hedberg-Alm Y, Penell J, Riihimäki M, Osterman-Lind E, Nielsen MK, Tydén E. Parasite Occurrence and Parasite Management in Swedish Horses Presenting with Gastrointestinal Disease-A Case-Control Study. Animals (Basel) 2020 Apr 7;10(4).
              doi: 10.3390/ani10040638pubmed: 32272754google scholar: lookup
            12. Santos LL, Salgado JA, Drummond MG, Bastianetto E, Santos CP, Brasil BSAF, Taconeli CA, Oliveira DAA. Molecular method for the semiquantitative identification of gastrointestinal nematodes in domestic ruminants. Parasitol Res 2020 Feb;119(2):529-543.
              doi: 10.1007/s00436-019-06569-3pubmed: 31834492google scholar: lookup
            13. Harvey AM, Meggiolaro MN, Hall E, Watts ET, Ramp D, Šlapeta J. Wild horse populations in south-east Australia have a high prevalence of Strongylus vulgaris and may act as a reservoir of infection for domestic horses. Int J Parasitol Parasites Wildl 2019 Apr;8:156-163.
              doi: 10.1016/j.ijppaw.2019.01.008pubmed: 30815358google scholar: lookup
            14. Yang S, Gao X, Meng J, Zhang A, Zhou Y, Long M, Li B, Deng W, Jin L, Zhao S, Wu D, He Y, Li C, Liu S, Huang Y, Zhang H, Zou L. Metagenomic Analysis of Bacteria, Fungi, Bacteriophages, and Helminths in the Gut of Giant Pandas. Front Microbiol 2018;9:1717.
              doi: 10.3389/fmicb.2018.01717pubmed: 30108570google scholar: lookup
            15. Kaspar A, Pfister K, Nielsen MK, Silaghi C, Fink H, Scheuerle MC. Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment. BMC Vet Res 2017 Jan 11;13(1):19.
              doi: 10.1186/s12917-016-0918-ypubmed: 28077153google scholar: lookup
            16. Nielsen MK, Scare J, Gravatte HS, Bellaw JL, Prado JC, Reinemeyer CR. Changes in Serum Strongylus Vulgaris-Specific Antibody Concentrations in Response to Anthelmintic Treatment of Experimentally Infected Foals. Front Vet Sci 2015;2:17.
              doi: 10.3389/fvets.2015.00017pubmed: 26664946google scholar: lookup
            17. Nielsen MK, Vidyashankar AN, Bellaw J, Gravatte HS, Cao X, Rubinson EF, Reinemeyer CR. Serum Strongylus vulgaris-specific antibody responses to anthelmintic treatment in naturally infected horses. Parasitol Res 2015 Feb;114(2):445-51.
              doi: 10.1007/s00436-014-4201-5pubmed: 25358238google scholar: lookup
            18. Clough D, Råberg L. Contrasting patterns of structural host specificity of two species of Heligmosomoides nematodes in sympatric rodents. Parasitol Res 2014 Dec;113(12):4633-9.
              doi: 10.1007/s00436-014-4154-8pubmed: 25273630google scholar: lookup
            19. Roeber F, Jex AR, Gasser RB. Next-generation molecular-diagnostic tools for gastrointestinal nematodes of livestock, with an emphasis on small ruminants: a turning point?. Adv Parasitol 2013;83:267-333.
              doi: 10.1016/B978-0-12-407705-8.00004-5pubmed: 23876874google scholar: lookup
            20. Andersen UV, Howe DK, Dangoudoubiyam S, Toft N, Reinemeyer CR, Lyons ET, Olsen SN, Monrad J, Nejsum P, Nielsen MK. SvSXP: a Strongylus vulgaris antigen with potential for prepatent diagnosis. Parasit Vectors 2013 Apr 4;6:84.
              doi: 10.1186/1756-3305-6-84pubmed: 23557195google scholar: lookup
            21. Roeber F, Jex AR, Gasser RB. Advances in the diagnosis of key gastrointestinal nematode infections of livestock, with an emphasis on small ruminants. Biotechnol Adv 2013 Dec;31(8):1135-52.
              doi: 10.1016/j.biotechadv.2013.01.008pubmed: 23376340google scholar: lookup
            22. Studzińska MB, Tomczuk K, Demkowska-Kutrzepa M, Szczepaniak K. The Strongylidae belonging to Strongylus genus in horses from southeastern Poland. Parasitol Res 2012 Oct;111(4):1417-21.
              doi: 10.1007/s00436-012-3087-3pubmed: 22961235google scholar: lookup
            23. Zhang W, Yie S, Yue B, Zhou J, An R, Yang J, Chen W, Wang C, Zhang L, Shen F, Yang G, Hou R, Zhang Z. Determination of Baylisascaris schroederi infection in wild giant pandas by an accurate and sensitive PCR/CE-SSCP method. PLoS One 2012;7(7):e41995.
              doi: 10.1371/journal.pone.0041995pubmed: 22911871google scholar: lookup
            24. Nielsen MK. Restrictions of anthelmintic usage: perspectives and potential consequences. Parasit Vectors 2009 Sep 25;2 Suppl 2(Suppl 2):S7.
              doi: 10.1186/1756-3305-2-S2-S7pubmed: 19778468google scholar: lookup
            25. Carlsgart J, Roepstorff A, Nejsum P. Multiplex PCR on single unembryonated Ascaris (roundworm) eggs. Parasitol Res 2009 Mar;104(4):939-43.
              doi: 10.1007/s00436-008-1307-7pubmed: 19096878google scholar: lookup
            26. Lauri A, Mariani PO. Potentials and limitations of molecular diagnostic methods in food safety. Genes Nutr 2009 Mar;4(1):1-12.
              doi: 10.1007/s12263-008-0106-1pubmed: 19067016google scholar: lookup
            27. Nielsen MK, Pyatt A, Perrett J, Tydén E, van Doorn D, Pihl TH, Schmidt JS, von Samson-Himmelstjerna G, Beasley A, Abbas G, Jabbar A. Global equine parasite control guidelines: Consensus or confusion?. Int J Parasitol Drugs Drug Resist 2025 Aug;28:100600.
              doi: 10.1016/j.ijpddr.2025.100600pubmed: 40472642google scholar: lookup
            28. Phetkarl T, Fungwithaya P, Lewchalermvong K, Sontigun N. Prevalence of gastrointestinal and blood parasites in horses of Nakhon Si Thammarat province, Thailand. Vet World 2024 Nov;17(11):2460-2468.
              doi: 10.14202/vetworld.2024.2460-2468pubmed: 39829659google scholar: lookup
            29. Baghdadi HBA, Abdelsalam M, Attia MM. Diagnostic innovations in Equine Parasitology: a Nanogold-ELISA for sensitive serodiagnosis of migratory strongylus vulgaris larvae infections. BMC Vet Res 2024 Dec 27;20(1):579.
              doi: 10.1186/s12917-024-04389-xpubmed: 39731087google scholar: lookup
            30. Hedberg Alm Y, Tydén E, Martin F, Lernå J, Halvarsson P. Farm size and biosecurity measures associated with Strongylus vulgaris infection in horses. Equine Vet J 2025 May;57(3):703-711.
              doi: 10.1111/evj.14212pubmed: 39171858google scholar: lookup
            31. Diekmann I, Blazejak K, Krücken J, Strube C, von Samson-Himmelstjerna G. Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse. Equine Vet J 2025 Mar;57(2):522-529.
              doi: 10.1111/evj.14134pubmed: 39012065google scholar: lookup
            32. Buono F, Veneziano V, Veronesi F, Molento MB. Horse and donkey parasitology: differences and analogies for a correct diagnostic and management of major helminth infections. Parasitology 2023 Oct;150(12):1119-1138.
              doi: 10.1017/S0031182023000525pubmed: 37221816google scholar: lookup
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