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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 ,
T. K. WALSH ,
A. A. DONNAN ,
S. CARRIÈRE ,
F. JACKSON ,
P. J. SKUCE ,
K. ROHN  and
A. J. WOLSTENHOLME
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: [email protected]
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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.

Keywords

nematodethiabendazoleanthelmintic resistancesingle nucleotide polymorphismtrichostrongyle
Type
Research Article
Information
Parasitology , Volume 136 , Issue 3 , March 2009 , pp. 349 - 358
Copyright
Copyright © 2009 Cambridge University Press

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