Skip to main content
SpringerLink
Log in

A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Resistance to fenbendazole, ivermectin, and moxidectin was explored by a fecal egg count reduction test in four meat sheep flocks in southwestern France where anthelmintic resistance was suspected. The FECR test results of the present study confirmed the presence of benzimidazole resistance in three out of the four farms and the presence of ivermectin resistance in one flock. In addition, a suspicion of moxidectin resistance was shown in this latter farm. Both conventional morphological and molecular identifications were performed on larval cultures before and after the treatment in the studied farms. A high positive correlation was found between the number of larvae counted under binocular microscope and the number of larvae estimated by the qPCR analysis (R 2 = 0.88) and a high Cohen’s Kappa value (0.91) in the detection of strongylid larvae in larval cultures. According to qPCR results, Trichostrongylus species demonstrated high levels of BZ resistance and Teladorsagia circumcincta was involved in the IVM resistance in one farm. The molecular procedures used in this study have the potential to be beneficial for anthelmintic resistance surveillance in sheep industry.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Almeida FA, Garcia KC, Torgenson PR, Amarante AFT (2010) Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil. Parasit Int 59:622–625

    Article  CAS  Google Scholar 

  • Areskog M, Sollenberg S, Engstrom A, von Samson-Himmelstjerna G, Hoglund J (2013) A controlled study on gastrointestinal nematodes from two Sweddish cattle farms showing field evidence of ivermectin resistance. Parasit Vectors 7:13

    Article  Google Scholar 

  • Bartley DJ, Donnan AA, Jackson E, Sargison N, Mitchell GBB, Jackson F (2006) A small scale survey of ivermectin resistance in sheep nematodes using the faecal egg count reduction test on samples collected from Scottish sheep. Vet Parasitol 137:112–118

    Article  CAS  PubMed  Google Scholar 

  • Beugnet F (1992) Présence de souches des strongles gastro-intestinaux des ovins et des caprins résistants aux benzimidazoles dans l’ouest Lyonnais. Rev Med Vet 143(6):529–533

    Google Scholar 

  • Bott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, Gasser RB (2009) A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep. Int J Parasitol 39:1277–1287

    Article  CAS  PubMed  Google Scholar 

  • Cabaret J, Mage C, Bouilhol M (2002) Helminth intensity and diversity in organic meat sheep farms in Centre of France. Vet Parasitol 105:33–47

    Article  CAS  PubMed  Google Scholar 

  • Chartier C, Pors I, Hubert J, Rocheteau D, Benoit C, Bernard N (1998) Prevalence of anthelmintic resistant nematodes in sheep and goats in Western France. Small Rum Res 29:33–41

    Article  Google Scholar 

  • Coles GC, Bauer C, Borgsteede FHM, Geerts S, Klei TR, Taylor MA, Waller PJ (1992) World Association for the Advancement of the Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 44:35–44

    Article  CAS  PubMed  Google Scholar 

  • Falzon LC, O’Neill TJ, Menzies PI, Peregrine AS, Jones-Bitton A, van Leeuwen J, Mederos A (2014) A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep. Prev Vet Med 117:388–402

    Article  CAS  PubMed  Google Scholar 

  • Gasser RB, Chilton NB, Hoste H, Beveridge I (1993) Rapid sequencing of rDNA from single worms and eggs of parasite helminths. Nucleic Acid Res 21:2525–2526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gasser RB, Newton SE (2000) Genomic and genetic research on bursate nematodes: significance, implications and prospects. Int J Parasitol 30:509–534

    Article  CAS  PubMed  Google Scholar 

  • Geurden T, Hoste H, Jacquiet P, Traversa D, Sotiraki S, Frangipane di Regalbono A, Tzanidakis N, Kostopoulou D, Gaillac C, Privat S, Giangaspero A, Zanardello C, Noé L, Vanimisetti B, Bartram D (2014) Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy. Vet Parasitol 201:59–66

    Article  CAS  PubMed  Google Scholar 

  • Hoste H, Chilton NB, Gasser RB, Beveridge I (1995) Differences in the second internal transcribed spacer (ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae). Int J Parasitol 25(1):75–80

    Article  CAS  PubMed  Google Scholar 

  • Hubert J, Kerboeuf D, Nicolas JA, Dubost G, Gayaud C (1991) Résistance des strongles gastro-intestinaux aux benzimidazoles chez les petits ruminants en Limousin. Rec Med Vet 167(2):135–140

    Google Scholar 

  • Jacquiet P, Alzieu JP, Cabaret J, Vial-Novella C, Garrain C, Minéry S, Arranz JM, Prévot F, Bergeaud JP, Grisez C, Cortet J, Sauvé C, Dorchies P, Gruner L (2004) Epidémiologie comparée en Ariège et dans les Pyrénées Atlantiques des infestations des brebis à l’herbe infestées par les helminthes et par Oestrus ovis. Bulletin des GTV, Hors-série Parasitologie des ruminants laitiers, 303–309

  • Keane O, Keegan JD, Good B, de Waal T, Fanning J, Gottstein M, Casey M, Hurley C, Sheehan M (2014) High levels of treatment failure with commonly used anthelmintics on Irish sheep farms. Irish Vet J 67:16

    Article  Google Scholar 

  • Lancaster MB, Hong C (1987) Differentiation of third stage larvae of “ovine Ostertagia” type and Trichostrongylus species. Vet. Rec. 120:503

    Article  CAS  PubMed  Google Scholar 

  • MAFF (1986) Ministry of Agriculture, Fisheries and Food Manual of Veterinary Parasitological Laboratory Techniques, Reference book 418 HMSO

  • Mahieu M, Ferré B, Madassamy M, Mandonnet N (2014) Fifteen years later, anthelmintic resistances have dramatically spread over goat farms in Guadeloupe. Vet Parasitol 205:379–384

    Article  PubMed  Google Scholar 

  • Mavrot F, Hertzberg H, Torgerson P (2015) Effect of gastro-intestinal nematode infection on sheep performance: a systematic review and meta-analysis. Parasit Vectors 8:557

    Article  PubMed  PubMed Central  Google Scholar 

  • McNally J, Callan D, Andronicos N, Bott N, Hunt PW (2013) DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces. Vet Parasitol 198:325–335

    Article  CAS  PubMed  Google Scholar 

  • O’Connor LJ, Walkden-Brown SW, Kahn LP (2006) Ecology of the free-living stages of major trichostrongylid parasites of sheep. Vet Parasitol 142:1–15

    Article  PubMed  Google Scholar 

  • Papadopoulos E, Gallidis E, Ptochos S (2012) Anthelmintic resistance in sheep in Europe: a selected review. Vet Parasitol 198:85–88

    Article  Google Scholar 

  • Paraud C, Marcotty T, Lespine A, Sutra JF, Pors I, Devos J (2016) Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France. Vet Parasitol 226:88–92

    Article  CAS  PubMed  Google Scholar 

  • Raynaud JP (1970) Etude de l’efficacité d’une technique de coproscopie quantitative pour le diagnostic de routine et le contrôle des infestations parasitaires des bovins, ovins, équins et porcins. Ann Parasitol 45:321–342

    CAS  Google Scholar 

  • Rinaldi L, Morgan ER, Bosco A, Coles GC, Cringoli G (2014) The maintenance of anthelmintic efficacy in sheep in a Mediterranean climate. Vet Parasitol 203:139–143

    Article  CAS  PubMed  Google Scholar 

  • Roeber F, Larsen JWA, Anderson N, Campbell AJD, Anderson GA, Gasser RB, Jex AR (2012) A molecular diagnostic tool to replace larval culture in conventional fecal egg count reduction testing in sheep. PLoS One 7:e37327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roeber F, Jex AR, Gasser RB (2013) Comparative evaluation of two DNA isolation techniques for PCR-based diagnosis of gastro-intestinal nematode infections in sheep. Mol Cel Probes 27:153–157

    Article  CAS  Google Scholar 

  • Roeber F, Kahn L (2014) The specific diagnosis of gastrointestinal nematode infections in livestock: larval culture technique, its limitations and alternative DNA-based approaches. Vet Parasitol 205:619–628

    Article  CAS  PubMed  Google Scholar 

  • Rose H, Rinaldi L, Bosco A, Mavrot F, de Waal T, Skuce P, Charlier J, Torgenson PR, Hertzberg H, Hendrickx G, Vercruysse J, Morgan ER (2015) Widespread anthelmintic resistance in European farmed ruminants: a systematic review. Vet Rec 176(21):546–547

    Article  CAS  PubMed  Google Scholar 

  • Stevenson LA, Chilton NB, Gasser RB (1995) Differentiation of Haemonchus placei from H. contortus (Nematoda: Trichostrongylidae) by the ribosomal DNA second internal transcribed spacer. Int J Parasitol 25(4):483–488

    Article  CAS  PubMed  Google Scholar 

  • Stevenson LA, Gasser RB, Chilton NB (1996) The ITS-2 rDNA of Teladorsagia circumcincta, T. trifurcata and T. davtiani (Nematoda: Trichostrongylidae) indicates that these taxa are one species. Int J Parasitol 26(10):1123–1126

    Article  CAS  PubMed  Google Scholar 

  • Sweeny JPA, Ryan UM, Robertson ID, Niemeyer D, Hunt PW (2012) Development of a modified molecular diagnostic procedure for the identification and quantification of naturally occurring strongylid larvae on pastures. Vet Parasitol 190:467–481

    Article  CAS  PubMed  Google Scholar 

  • Tolstrup N, Nielsen PS, Kolberg JG, Frankel AM, Vissing H, Kauppinen S (2003) OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling. Nucleic Acids Res 31:3758–3762

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Wyk JA, Cabaret J, Michael LM (2004) Morphological identification of nematode larvae of small ruminants and cattle simplified. Vet Parasitol 119:277–306

    Article  CAS  PubMed  Google Scholar 

  • Van Wyk JA, Mayhew E (2013) Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: a practical lab guide. Onders J Vet Res 80(1). doi:10.4102/ojvr.v80i1.539

  • You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res 34(8):e60

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. UMR INRA/DGER 1225 Interactions Hôtes Agents Pathogènes, UMT Santé des Petits Ruminants, Ecole Nationale Vétérinaire de Toulouse, BP 87614, 31076, Toulouse Cedex 03, France

    M. Milhes, M. Guillerm, M. Robin, M. Eichstadt, C. Grisez, F. Prévot, E. Liénard, E. Bouhsira, M. Franc & P. Jacquiet

  2. Groupement Corrézien de Défense Sanitaire (GCDS), 19001, Tulle Cedex, France

    C. Roy

Authors
  1. M. Milhes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Guillerm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Robin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Eichstadt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Grisez
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. F. Prévot
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. E. Liénard
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. E. Bouhsira
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. Franc
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. P. Jacquiet
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. Jacquiet.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Milhes, M., Guillerm, M., Robin, M. et al. A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms. Parasitol Res 116, 909–920 (2017). https://doi.org/10.1007/s00436-016-5364-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-016-5364-z

Keywords

Search

Navigation