Evaluation of Benzimidazole Resistance in Equine Cyathostomins in the Kermanshah Province of Iran Using Coprological Analysis and Allele-Specific PCR
-
Rezvan Jamshidpour
,
Reza Nabavi
,
Hossein Moadab
,
Farid Rezaei
,
Abdolali Chale Chale
,
Neil Sargison
Abstract
Background: Resistance to benzimidazole (BZ) by cyathostomin nematodes has become a major threat to equine health around the world. We aimed to evaluate the efficacy of BZ drugs against small strongyle nematodes in horses of western Iran using coprological and molecular examination.
Methods: Faecal egg count reduction tests were performed on 398 horses kept in 16 stables in western Iran (Kermanshah Province), to detect benzimidazole resistance in small strongyle nematodes. Allele-specific PCR was used to identify the F200Y (TAC/TTC) SNP in the beta-tubulin gene codon in cyathostomin larvae.
Results: There was a 96.1%-98.3% (90% CI) reduction in faecal egg counts following mebendazole treatment and a 96.6% - 98.7% (90% CI) reduction in faecal egg counts following fenbendazole treatment. The allele-specific PCR showed BZ-susceptible homozygote genotypes in all examined samples (Two pools of 10 to 50 L3 of cyathostomin nematodes from each of the 18 selected horses).
Conclusion: Benzimidazole resistance in equine small strongyles has been reported globally, and the results of the present study were unexpected. The probable reasons for the slow development of BZ-resistance are climatic conditions in the country that have significant negative effects on the ability of free stages of the resistant strongyle nematode to survive and develop.
1. Corning S. Equine cyathostomins: a review of biology, clinical significance and therapy. Parasit Vectors. 2009; 2 Suppl 2(Suppl 2):S1.
2. Lyons ET, Tolliver SC, Drudge JH. Historical perspective of cyathostomes: prevalence, treatment and control programs. Vet Parasitol. 1999;85(2-3):97-111.
3. Reinemeyer CR, Nielsen MK. Parasitism and colic. Vet Clin North Am Equine Pract. 2009;25(2):233-245.
4. Drudge JH, Elam G. Preliminary observations on the resistance of horse strongyles to phenothiazine. J Parasitol. 1961;47(4):38-39.
5. Traversa D, Castagna G, Von Samson-Himmelstjerna G, et al. Efficacy of major anthelmintics against horse cyathostomins in France. Vet Parasitol. 2012;188(3-4):294-300.
6. Kaplan RM, Nielsen MK. An evidence-based approach to equine parasite control: it ain’t the 60s anymore. Equine Vet Educ. 2010;22(6):306-316.
7. Relf VE, Lester HE, Morgan ER, Hodgkinson JE, Matthews JB. Anthelmintic efficacy on UK Thoroughbred stud farms. Int J Parasitol. 2014;44(8):507-514.
8. Bellaw JL, Krebs K, Reinemeyer CR, et al. Anthelmintic therapy of equine cyathostomin nematodes-larvicidal efficacy, egg reappearance period, and drug resistance. Int J Parasitol. 2018;48(2):97-105.
9. Raza A, Qamar AG, Hayat K, et al. Anthelmintic resistance and novel control options in equine gastrointestinal nematodes. Parasitology. 2019;146(4):425-437.
10. Pook JF, Power ML, Sangster NC, et al. Evaluation of tests for anthelmintic resistance in cyathostomes. Vet Parasitol. 2002;106(4):331-343.
11. Kaplan RM, Denwood MJ, Nielsen MK, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine. Vet Parasitol. 2023; 318:109936.
12. Vidyashankar AN, Hanlon BM, Kaplan RM. Statistical and biological considerations in evaluating drug efficacy in equine strongyle parasites using fecal egg count data. Vet Parasitol. 2012;185(1):45-56.
13. Ihler CE. A field survey on anthelmintic resistance in equine small strongyles in Norway. Acta Vet Scand. 1995; 36:135-143.
14. Von Samson-Himmelstjerna G, Pape M, Von Witzendorff C, Schnieder T. Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages. J Parasitol. 2002;88(2):254-257.
15. Pape M, Posedi J, Failing K, Schnieder T, Von Samson-Himmelstjerna G. Analysis of the beta-tubulin codon 200 genotype distribution in a benzimidazole-susceptible and-resistant cyathostome population. Parasitology. 2003; 127(Pt 1):53-9.
16. Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB. The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol. 2008;38(10):1149-1160.
17. Sazmand A, Bahari A, Papi S, Otranto D. Parasitic diseases of equids in Iran (1931–2020): a literature review. Parasit Vectors. 2020;13:586.
18. Bello TR, Allen TM. Comparison of two faecal egg recovery techniques and larval culture for cyathostomins in horses. Am J Vet Res. 2009;70(5):571-573.
19. Wagner EL, Tyler PJ. A comparison of weight estimation methods in adult horses. J Equine Vet Sci. 2011;31(12):706-710.
20. Nielsen M, Mittel L, Grice A, et al. AAEP internal parasite control guidelines, American Association of Equine Practitioners. 2019.
21. Coles GC, Bauer C, Borgsteede FH, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol. 1992;44(1-2):35-44.
22. Kaplan RM, Nielsen MK, Levecke B, et al. Online tools for planning FECRT studies and analysing FECRT data. http://www.fecrt.com. 2023; Accessed 12 May 2024.
23. Denwood MJ, Kaplan RM, McKendrick LJ, et al. A statistical framework for calculating prospective sample sizes and classifying efficacy results for faecal egg count reduction tests in ruminants, horses and swine. Vet Parasitol. 2023;314:109867.
24. Varady M, Königová A, Čorba J. Benzimidazole resistance in equine cyathostomes in Slovakia. Vet Parasitol. 2000;94(1-2):67-74.
25. Lichtenfels JR. Helminths of domestic equids, illustrated keys to genera and species with emphasis on North American forms. Proceedings of the Helminthological Society of Washington. 1975; 42:92.
26. Cernea M, Carvalho LMM, Cozma V, et al. Atlas of diagnosis of equine strongylidosis. 1st ed. Edutura Academic Press, Cluj-Napoca; 2008.
27. Santos DW, De Castro LLD, Giese EG, Molento MB. Morphometric study of infective larvae of cyathostomins of horses and their distribution. J Equine Vet Sci. 2016; 44:49-53.
28. Kaplan RM, Vidyashankar AN. An inconvenient truth: global worming and anthelmintic resistance. Vet Parasitol. 2012;186(1-2):70-78.
29. Denwood MJ, Love S, Innocent GT, et al. Quantifying the sources of variability in equine faecal egg counts: Implications for improving the utility of the method. Vet Parasitol. 2012;188(1-2):120-126.
30. Van Wyk JA, Mayhew E. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide. Onderstepoort J Vet Res. 2013;80(1):539.
31. Craven J, Bjørn H, Henriksen S, et al. Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine Vet J. 1998;30(4):289-293.
32. Cirak VY, Güleğen E, Bauer C. Benzimidazole resistance in cyathostomin populations on horse farms in western Anatolia, Turkey. Parasitol Res. 2004; 93:392-395.
33. Slocombe JOD, Cote JF, De Gannes RV. The persistence of benzimidazole-resistant cyathostomes on horse farms in Ontario over 10 years and the effectiveness of ivermectin and moxidectin against these resistant strains. Can Vet J. 2008;49(1):56-60.
34. Elard L, Humbert JF. Importance of the mutation of amino acid 200 of the isotype 1 β-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta. Parasitol Res. 1999; 85:452-456.
35. Gholamian A, Galehdari H, Eslami A, Nabavi L. Study of β-tubulin gene polymorphisms in Haemonchus contortus isolated from sheep populations in Khouzestan, southwestern Iran. Iran J Vet Res. 2007;8(3):239-243.
36. Mohseni H, Taghavi RA, Borji H, Rashtibaf M. Study on the efficacy of albendazole and levamisole against gastrointestinal helminthes in sheep flocks in Mashhad. Iran J Vet Clin Sci. 2019;13(1):23-29.
37. Ebrahimi R, Yakhchali M, Malekinejad H. Anthelmintic resistance to albendazole and fenbendazole in gastrointestinal n`ematodes of sheep in Saghez Municipality, Iran. J Vet Res. 2020;75(1):1-7.
38. Nabavi R, Shayan P, Shokrani HR, Eslami A, Bokaie S. Evaluation of benzimidazole resistance in Haemonchus contortus using comparative PCR-RFLP methods. Iran J Parasitol. 2011;6(2):45-53.
39. Shokrani HR, Shayan P, Eslami A, Nabavi R. Benzimidazole-Resistance in Haemonchus contortus: New PCR-RFLP method for the detection of point mutation at codon 167 of isotype 1 β-tubulin gene. Iran J Parasitol. 2012;7(4):41-48.
40. Hosseini M, Sanjarani Z, Nabavi R, et al. Morpho-Molecular characterization of cattle Haemonchus nematodes from southeast of Iran. Iran J Vet Med. 2022;16(4):364-371.
41. Flores AG, Osmari V, Ramos F, et al. Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil. Rev Bras Parasitol Vet. 2020;29(3):e003820.
42. Saemi Soudkolaei A, Kalidari GA, Borji H. Anthelmintic efficacy of fenbendazole and levamisole in native fowl in northern Iran. Parasit Vectors. 2021;14(1):104.
43. Ashrafzadeh-Shiraz M, Tavassoli M, Dalir-Naghadeh B, Sazmand A. Impaired efficacy of fenbendazole and ivermectin against intestinal nematodes in adult horses in Iran. Res Vet Sci. 2024;166:105078.
44. Mfitilodze MW, Hutchinson GW. Development and survival of free-living stages of equine strongyles under laboratory conditions. Vet Parasitol. 1987; 23(1-2):121-133.
45. Genchi C, Carrara L, Malnati G. Epidemiological aspects of the gastrointestinal nematodes of animals in pasture [horses]. Clin Vet. 1978; 101:175-184.
46. Craig TM, Bowen JM, Ludwig KG. Transmission of equine cyathostomes (Strongylidae) in central Texas. Am J Vet Res. 1983;44(10):1867-1869.
47. Sargison N, Chambers A, Chaudhry U, et al. Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd. Int J Parasitol. 2022;52(12):763-774.
48. Scott L, Bishop RM, Pomroy WE. Anthelmintic resistance in equine helminth parasites- a growing issue for horse owners and veterinarians in New Zealand? N Z Vet J. 2015;63(4):188-198.
49. Bartram DJ, Leathwick DM, Taylor MA, et al. The role of combination anthelmintic formulations in the sustainable control of sheep nematodes. Vet Parasitol. 2012;186(3-4):151-158.
50. Kaplan RM, West EM, Norat‐Collazo LM, Vargas J. A combination treatment strategy using pyrantel pamoate and oxibendazole demonstrates additive effects for controlling equine cyathostomins. Equine Vet Educ. 2014;26(9):485-491.
51. Scare JA, Leathwick DM, Sauermann CW, Lyons ET, Steuer AE, Jones BA, Clark M, Nielsen MK. Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins. Int J Parasitol Drugs Drug Resist. 2020;12:28-34.
52. Nielsen MK, Kaplan RM, Thamsborg SM, Monrad J, Olsen SN. Climatic influences on development and survival of free-living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance. Vet J. 2007;174(1):23-32.
53. Mitchell CJ, O'Sullivan CM, Pinloche E, Wilkinson T, Morphew RM, McEwan NR. Using next-generation sequencing to determine diversity of horse intestinal worms: identifying the equine 'nemabiome'. J Equine Sci. 2019;30(1):1-5.
2. Lyons ET, Tolliver SC, Drudge JH. Historical perspective of cyathostomes: prevalence, treatment and control programs. Vet Parasitol. 1999;85(2-3):97-111.
3. Reinemeyer CR, Nielsen MK. Parasitism and colic. Vet Clin North Am Equine Pract. 2009;25(2):233-245.
4. Drudge JH, Elam G. Preliminary observations on the resistance of horse strongyles to phenothiazine. J Parasitol. 1961;47(4):38-39.
5. Traversa D, Castagna G, Von Samson-Himmelstjerna G, et al. Efficacy of major anthelmintics against horse cyathostomins in France. Vet Parasitol. 2012;188(3-4):294-300.
6. Kaplan RM, Nielsen MK. An evidence-based approach to equine parasite control: it ain’t the 60s anymore. Equine Vet Educ. 2010;22(6):306-316.
7. Relf VE, Lester HE, Morgan ER, Hodgkinson JE, Matthews JB. Anthelmintic efficacy on UK Thoroughbred stud farms. Int J Parasitol. 2014;44(8):507-514.
8. Bellaw JL, Krebs K, Reinemeyer CR, et al. Anthelmintic therapy of equine cyathostomin nematodes-larvicidal efficacy, egg reappearance period, and drug resistance. Int J Parasitol. 2018;48(2):97-105.
9. Raza A, Qamar AG, Hayat K, et al. Anthelmintic resistance and novel control options in equine gastrointestinal nematodes. Parasitology. 2019;146(4):425-437.
10. Pook JF, Power ML, Sangster NC, et al. Evaluation of tests for anthelmintic resistance in cyathostomes. Vet Parasitol. 2002;106(4):331-343.
11. Kaplan RM, Denwood MJ, Nielsen MK, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine. Vet Parasitol. 2023; 318:109936.
12. Vidyashankar AN, Hanlon BM, Kaplan RM. Statistical and biological considerations in evaluating drug efficacy in equine strongyle parasites using fecal egg count data. Vet Parasitol. 2012;185(1):45-56.
13. Ihler CE. A field survey on anthelmintic resistance in equine small strongyles in Norway. Acta Vet Scand. 1995; 36:135-143.
14. Von Samson-Himmelstjerna G, Pape M, Von Witzendorff C, Schnieder T. Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages. J Parasitol. 2002;88(2):254-257.
15. Pape M, Posedi J, Failing K, Schnieder T, Von Samson-Himmelstjerna G. Analysis of the beta-tubulin codon 200 genotype distribution in a benzimidazole-susceptible and-resistant cyathostome population. Parasitology. 2003; 127(Pt 1):53-9.
16. Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB. The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol. 2008;38(10):1149-1160.
17. Sazmand A, Bahari A, Papi S, Otranto D. Parasitic diseases of equids in Iran (1931–2020): a literature review. Parasit Vectors. 2020;13:586.
18. Bello TR, Allen TM. Comparison of two faecal egg recovery techniques and larval culture for cyathostomins in horses. Am J Vet Res. 2009;70(5):571-573.
19. Wagner EL, Tyler PJ. A comparison of weight estimation methods in adult horses. J Equine Vet Sci. 2011;31(12):706-710.
20. Nielsen M, Mittel L, Grice A, et al. AAEP internal parasite control guidelines, American Association of Equine Practitioners. 2019.
21. Coles GC, Bauer C, Borgsteede FH, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol. 1992;44(1-2):35-44.
22. Kaplan RM, Nielsen MK, Levecke B, et al. Online tools for planning FECRT studies and analysing FECRT data. http://www.fecrt.com. 2023; Accessed 12 May 2024.
23. Denwood MJ, Kaplan RM, McKendrick LJ, et al. A statistical framework for calculating prospective sample sizes and classifying efficacy results for faecal egg count reduction tests in ruminants, horses and swine. Vet Parasitol. 2023;314:109867.
24. Varady M, Königová A, Čorba J. Benzimidazole resistance in equine cyathostomes in Slovakia. Vet Parasitol. 2000;94(1-2):67-74.
25. Lichtenfels JR. Helminths of domestic equids, illustrated keys to genera and species with emphasis on North American forms. Proceedings of the Helminthological Society of Washington. 1975; 42:92.
26. Cernea M, Carvalho LMM, Cozma V, et al. Atlas of diagnosis of equine strongylidosis. 1st ed. Edutura Academic Press, Cluj-Napoca; 2008.
27. Santos DW, De Castro LLD, Giese EG, Molento MB. Morphometric study of infective larvae of cyathostomins of horses and their distribution. J Equine Vet Sci. 2016; 44:49-53.
28. Kaplan RM, Vidyashankar AN. An inconvenient truth: global worming and anthelmintic resistance. Vet Parasitol. 2012;186(1-2):70-78.
29. Denwood MJ, Love S, Innocent GT, et al. Quantifying the sources of variability in equine faecal egg counts: Implications for improving the utility of the method. Vet Parasitol. 2012;188(1-2):120-126.
30. Van Wyk JA, Mayhew E. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide. Onderstepoort J Vet Res. 2013;80(1):539.
31. Craven J, Bjørn H, Henriksen S, et al. Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine Vet J. 1998;30(4):289-293.
32. Cirak VY, Güleğen E, Bauer C. Benzimidazole resistance in cyathostomin populations on horse farms in western Anatolia, Turkey. Parasitol Res. 2004; 93:392-395.
33. Slocombe JOD, Cote JF, De Gannes RV. The persistence of benzimidazole-resistant cyathostomes on horse farms in Ontario over 10 years and the effectiveness of ivermectin and moxidectin against these resistant strains. Can Vet J. 2008;49(1):56-60.
34. Elard L, Humbert JF. Importance of the mutation of amino acid 200 of the isotype 1 β-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta. Parasitol Res. 1999; 85:452-456.
35. Gholamian A, Galehdari H, Eslami A, Nabavi L. Study of β-tubulin gene polymorphisms in Haemonchus contortus isolated from sheep populations in Khouzestan, southwestern Iran. Iran J Vet Res. 2007;8(3):239-243.
36. Mohseni H, Taghavi RA, Borji H, Rashtibaf M. Study on the efficacy of albendazole and levamisole against gastrointestinal helminthes in sheep flocks in Mashhad. Iran J Vet Clin Sci. 2019;13(1):23-29.
37. Ebrahimi R, Yakhchali M, Malekinejad H. Anthelmintic resistance to albendazole and fenbendazole in gastrointestinal n`ematodes of sheep in Saghez Municipality, Iran. J Vet Res. 2020;75(1):1-7.
38. Nabavi R, Shayan P, Shokrani HR, Eslami A, Bokaie S. Evaluation of benzimidazole resistance in Haemonchus contortus using comparative PCR-RFLP methods. Iran J Parasitol. 2011;6(2):45-53.
39. Shokrani HR, Shayan P, Eslami A, Nabavi R. Benzimidazole-Resistance in Haemonchus contortus: New PCR-RFLP method for the detection of point mutation at codon 167 of isotype 1 β-tubulin gene. Iran J Parasitol. 2012;7(4):41-48.
40. Hosseini M, Sanjarani Z, Nabavi R, et al. Morpho-Molecular characterization of cattle Haemonchus nematodes from southeast of Iran. Iran J Vet Med. 2022;16(4):364-371.
41. Flores AG, Osmari V, Ramos F, et al. Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil. Rev Bras Parasitol Vet. 2020;29(3):e003820.
42. Saemi Soudkolaei A, Kalidari GA, Borji H. Anthelmintic efficacy of fenbendazole and levamisole in native fowl in northern Iran. Parasit Vectors. 2021;14(1):104.
43. Ashrafzadeh-Shiraz M, Tavassoli M, Dalir-Naghadeh B, Sazmand A. Impaired efficacy of fenbendazole and ivermectin against intestinal nematodes in adult horses in Iran. Res Vet Sci. 2024;166:105078.
44. Mfitilodze MW, Hutchinson GW. Development and survival of free-living stages of equine strongyles under laboratory conditions. Vet Parasitol. 1987; 23(1-2):121-133.
45. Genchi C, Carrara L, Malnati G. Epidemiological aspects of the gastrointestinal nematodes of animals in pasture [horses]. Clin Vet. 1978; 101:175-184.
46. Craig TM, Bowen JM, Ludwig KG. Transmission of equine cyathostomes (Strongylidae) in central Texas. Am J Vet Res. 1983;44(10):1867-1869.
47. Sargison N, Chambers A, Chaudhry U, et al. Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd. Int J Parasitol. 2022;52(12):763-774.
48. Scott L, Bishop RM, Pomroy WE. Anthelmintic resistance in equine helminth parasites- a growing issue for horse owners and veterinarians in New Zealand? N Z Vet J. 2015;63(4):188-198.
49. Bartram DJ, Leathwick DM, Taylor MA, et al. The role of combination anthelmintic formulations in the sustainable control of sheep nematodes. Vet Parasitol. 2012;186(3-4):151-158.
50. Kaplan RM, West EM, Norat‐Collazo LM, Vargas J. A combination treatment strategy using pyrantel pamoate and oxibendazole demonstrates additive effects for controlling equine cyathostomins. Equine Vet Educ. 2014;26(9):485-491.
51. Scare JA, Leathwick DM, Sauermann CW, Lyons ET, Steuer AE, Jones BA, Clark M, Nielsen MK. Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins. Int J Parasitol Drugs Drug Resist. 2020;12:28-34.
52. Nielsen MK, Kaplan RM, Thamsborg SM, Monrad J, Olsen SN. Climatic influences on development and survival of free-living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance. Vet J. 2007;174(1):23-32.
53. Mitchell CJ, O'Sullivan CM, Pinloche E, Wilkinson T, Morphew RM, McEwan NR. Using next-generation sequencing to determine diversity of horse intestinal worms: identifying the equine 'nemabiome'. J Equine Sci. 2019;30(1):1-5.
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| Issue | Vol 20 No 3 (2025) | |
| Section | Original Article(s) | |
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How to Cite
1.
Jamshidpour R, Nabavi R, Moadab H, Rezaei F, Chale Chale A, Sargison N. Evaluation of Benzimidazole Resistance in Equine Cyathostomins in the Kermanshah Province of Iran Using Coprological Analysis and Allele-Specific PCR. Iran J Parasitol. 2025;20(3):389-399.
