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Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing

Published online by Cambridge University Press:  21 January 2009

G. von SAMSON-HIMMELSTJERNA
Affiliation:
Institute for Parasitology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559Hannover, Germany
T. K. WALSH
Affiliation:
Department of Biology and Biochemistry, University of Bath, BathBA2 7AY, UK
A. A. DONNAN
Affiliation:
Division of Parasitology, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, MidlothianEH26 0PZ, UK
S. CARRIÈRE
Affiliation:
Department of Biology and Biochemistry, University of Bath, BathBA2 7AY, UK
F. JACKSON
Affiliation:
Division of Parasitology, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, MidlothianEH26 0PZ, UK
P. J. SKUCE
Affiliation:
Division of Parasitology, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, MidlothianEH26 0PZ, UK
K. ROHN
Affiliation:
Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine, Bünteweg 2, 30559Hannover, Germany
A. J. WOLSTENHOLME*
Affiliation:
Department of Biology and Biochemistry, University of Bath, BathBA2 7AY, UK
*
*Corresponding author: Department of Biology and Biochemistry, University of Bath, BathBA2 7AY, UK. Tel: +44 1225 386553. Fax: +44 1225 386779. E-mail: A.J.Wolstenholme@bath.ac.uk

Summary

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

Type
Research Article
Copyright
Copyright © 2009 Cambridge University Press

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References

REFERENCES

Albonico, M., Allen, H., Chitsulo, L., Engels, D., Gabrielli, A.-F. and Savioli, L. (2008). Controlling soil-transmitted helminthiasis in pre-school-age children through preventive chemotherapy. PLoS Neglected Tropical Diseases 2, e126.Google Scholar
Albonico, M., Engels, D. and Savioli, L. (2004 a). Monitoring drug efficacy and early detection of drug resistance in human soil-transmitted nematodes: a pressing public health agenda for helminth control. International Journal for Parasitology 34, 12051210.Google Scholar
Albonico, M., Wright, V. and Bickle, Q. (2004 b). Molecular analysis of the beta-tubulin gene of human hookworms as a basis for possible benzimidazole resistance on Pemba Island. Molecular and Biochemical Parasitology 134, 281284.CrossRefGoogle ScholarPubMed
Alvarez-Sanchez, M. A., Perez-Garcia, J., Cruz-Rojo, M. A. and Rojo-Vazquez, F. A. (2005). Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep. Veterinary Parasitology 129, 291298.CrossRefGoogle ScholarPubMed
Braasch, D. A. and Corey, D. R. (2001). Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chemistry & Biology 8, 17.CrossRefGoogle ScholarPubMed
Coles, G. C., Bauer, C., Borgsteede, F. H. M., Geerts, S., Klei, T. R., Taylor, M. A. and Waller, P. J. (1992). World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology 44, 3544.CrossRefGoogle ScholarPubMed
Coles, G. C., Jackson, F., Pomroy, W. E., Prichard, R. K., von Samson-Himmelstjerna, G., Silvestre, A., Taylor, M. A. and Vercruysse, J. (2006). The detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology 136, 167185.CrossRefGoogle ScholarPubMed
Drogemuller, M., Schnieder, T. and von Samson-Himmelstjerna, G. (2004). Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations. Journal of Parasitology 90, 868870.CrossRefGoogle ScholarPubMed
Elard, L., Comes, A. M. and Humbert, J. F. (1996). Sequences of beta-tubulin cDNA from benzimidazole-susceptible and -resistant strains of Telodorsagia circumcincta, a nematode parasite of small ruminants. Molecular and Biochemical Parasitology 79, 249253.CrossRefGoogle Scholar
Elard, L. and Humbert, J. F. (1999). Importance of the mutation of amino acid 200 of the isotype 1 beta-tubulin gene in the benzimidazole resistance of the small ruminant parasite Teladorsagia circumcincta. Parasitology Research 85, 452456.CrossRefGoogle ScholarPubMed
Eng, J. K. L., Blackhall, W. J., Osei-Atweneboana, M. Y., Bourguinat, C., Galazzo, D., Beech, R. N., Unnasch, T. R., Awadzi, K., Lubega, G. W. and Prichard, R. K. (2006). Ivermectin selection on β-tubulin: Evidence in Onchocerca volvulus and Haemonchus contortus. Molecular and Biochemical Parasitology 150, 229235.CrossRefGoogle ScholarPubMed
Ghisi, M., Kaminsky, R. and Maser, P. (2007). Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Veterinary Parasitology 144, 313320.CrossRefGoogle ScholarPubMed
Hodgkinson, J. E., Clark, H. J., Kaplan, R. M., Lake, S. L. and Matthews, J. B. (2008). The role of polymorphisms at β-tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. International Journal for Parasitology 38, 11491160.CrossRefGoogle ScholarPubMed
Hoffmann, C., Minkah, N., Leipzig, J., Wang, G., Arens, M. Q., Tebas, P. and Bushman, F. D. (2007). DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations. Nucleic Acids Research 35, e91.CrossRefGoogle ScholarPubMed
Kaplan, R. M. (2004). Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, 477481.CrossRefGoogle ScholarPubMed
Kaplan, R. M., Klei, T. R., Lyons, E. T., Lester, G., Courtney, C. H., French, D. D., Tolliver, S. C., Vidyashankar, A. N. and Zhao, Y. (2004). Prevalence of anthelmintic resistant cyathostomes on horse farms. Journal of the American Veterinary Medicine Association 225, 903910.CrossRefGoogle ScholarPubMed
Koenraadt, H., Somerville, S. C. and Jones, A. L. (1992). Characterization of mutations in the β-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. Phytopathology 82, 13481354.CrossRefGoogle Scholar
Kwa, M. S. G., Kooyman, F. N. J., Boersema, J. H. and Roos, M. H. (1993). Effect of selection for benzimidazole resistance in Haemonchus contortus on β-tubulin isotype-1 and isotype-2 Genes. Biochemical and Biophysical Research Communications 191, 413419.CrossRefGoogle ScholarPubMed
Kwa, M. S. G., Veenstra, J. G. and Roos, M. H. (1994). Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation in β-tubulin isotype 1. Molecular and Biochemical Parasitology 63, 299303.CrossRefGoogle ScholarPubMed
Laughton, D. L., Amar, M., Thomas, P., Towner, P., Harris, P., Lunt, G. G. and Wolstenholme, A. J. (1994). Cloning of a putative inhibitory amino acid receptor subunit from the nematode Haemonchus contortus. Receptors and Channels 2, 155163.Google ScholarPubMed
Ma, Z. H., Yoshimura, M. A. and Michailides, T. J. (2003). Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California. Applied and Environmental Microbiology 69, 71457152.CrossRefGoogle ScholarPubMed
Martin, P. J., Anderson, N. and Jarrett, R. G. (1989). Detecting benzimidazole resistance with fecal egg count reduction tests and in vitro assays. Australian Veterinary Journal 66, 236240.CrossRefGoogle ScholarPubMed
Melville, L. A., Sykes, A. M. and McCarthy, J. S. (2006). The beta-tubulin genes of two Strongyloides species. Experimental Parasitology 112, 144151.CrossRefGoogle ScholarPubMed
Mottier, M. D. and Prichard, R. K. (2008). Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus. Pharmacogenetics and Genomics 18, 129140.CrossRefGoogle Scholar
Petersen, M. and Wengel, J. (2003). LNA: a versatile tool for therapeutics and genomics. Trends in Biotechnology 21, 7481.Google Scholar
Prichard, R. (1994). Anthelmintic resistance. Veterinary Parasitology 54, 259268.CrossRefGoogle ScholarPubMed
Redman, E., Packard, E., Grillo, V., Smith, J., Jackson, F. and Gilleard, J. S. (2008). Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting. International Journal for Parasitology 38, 111122.CrossRefGoogle ScholarPubMed
Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996). Real-time DNA sequencing using detection of pyrophosphate release. Analytical Biochemistry 242, 8489.Google Scholar
Ronaghi, M., Uhlen, M. and Nyren, P. (1998). A sequencing method based on real-time pyrophosphate. Science 281, 363365.Google Scholar
Schwab, A. E., Boakye, D. A., Kyelem, D. and Prichard, R. K. (2005). Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment. American Journal of Tropical Medicine and Hygiene 73, 234238.CrossRefGoogle ScholarPubMed
Schwenkenbecher, J. M., Albonico, M., Bickle, Q. and Kaplan, R. M. (2007). Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR. Molecular and Biochemical Parasitology 156, 167174.CrossRefGoogle ScholarPubMed
Silvestre, A. and Cabaret, J. (2002). Mutation in position 167 of isotype 1 beta-tubulin gene of Trichostrongylid nematodes: role in benzimidazole resistance? Molecular and Biochemical Parasitology 120, 297300.Google Scholar
Silvestre, A. and Humbert, J. F. (2000). A molecular tool for species identification and benzimidazole resistance diagnosis in larval communities of small ruminant parasites. Experimental Parasitology 95, 271276.CrossRefGoogle ScholarPubMed
Troell, K., Mattson, J. G., Alderborn, A. and Hoglund, J. (2003). Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants. International Journal for Parasitology 33, 765771.CrossRefGoogle ScholarPubMed
Van Wyk, J. A. and Malan, F. S. (1988). Resistance of field strains of Haemonchus contortus to ivermectin, closantel, rafoxanide and the benzimidazoles in South Africa. Veterinary Record 123, 226228.Google Scholar
von Samson-Himmelstjerna, G. and Blackhall, W. (2005). Will technology provide solutions for drug resistance in veterinary helminths? Veterinary Parasitology 132, 223239.Google Scholar
von Samson-Himmelstjerna, G., Buschbaum, S., Wirtherle, N., Pape, M. and Schnieder, T. (2003). TaqMan minor groove binder real-time PCR analysis of beta-tubulin codon 200 polymorphism in small strongyles (Cyathostomin) indicates that the TAC allele is only moderately selected in benzimidazole-resistant populations. Parasitology 127, 489496.Google Scholar
Walsh, T. K., Donnan, A. A., Jackson, F., Skuce, P. and Wolstenholme, A. J. (2007). Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes. Veterinary Parasitology 144, 304312.Google Scholar
Wolstenholme, A. J., Fairweather, I., Prichard, R. K., von Samson-Himmelstjerna, G. and Sangster, N. (2004). Drug resistance in veterinary helminths. Trends in Parasitology 20, 469476.CrossRefGoogle ScholarPubMed
Zhou, Z. Y., Poe, A. C., Limor, J., Grady, K. K., Goldman, I., McCollum, A. M., Escalante, A. A., Barnwell, J. W. and Udhayakumar, V. (2006). Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum. Journal of Clinical Microbiology 44, 39003910.CrossRefGoogle ScholarPubMed
Zhu, W., Tenover, F. C., Limor, J., Lonsway, D., Prince, D., Dunne, W. M. and Patel, J. B. (2007). Use of pyrosequencing to identify point mutations in domain V of 23S rRNA genes of linezolid-resistant Staphylococcus aureus and Staphylococcus epidermidis. European Journal of Clinical Microbiology & Infectious Disease 26, 161165.CrossRefGoogle ScholarPubMed