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

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in beta-tubulin genes have been associated with BZ-resistance. In the present study, we investigated beta-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.