Background: Genetic resistance against benzimidazole (BZ) anthelmintics is widespread in cyathostomins, the commonest group of intestinal parasitic nematodes of horses. Studies of BZ-resistant nematodes of sheep, particularly Haemonchus contortus, have indicated that an anthelmintic resistance-conferring T/A polymorphism, encoding an F (phenylalanine) to Y (tyrosine) substitution, in beta-tubulin isotype 1 is present at two loci, codons 167 and 200 (F167Y, F200Y). Recent studies using complementary (c) DNA derived from BZ-susceptible and -resistant cyathostomins identified statistical differences in the frequency of the BZ-resistant A allele at these loci. However, the lack of high-throughput genomic DNA-based detection of polymorphisms limits the study of eggs or larvae from field isolates. In the present study, we report genomic DNA sequences for beta-tubulin isotype 1 from multiple cyathostomin species, thus facilitating the development of pyrosequencing assays to genetically characterize third-stage larvae (L3s) of cyathostomins from mixed-species field isolates.
Results: Sequence analysis of the beta-tubulin isotype 1 gene in a common species, Cylicocyclus nassatus, indicates a revised genomic structure to published data, revealing that codons 167 and 200 are located on separate exons. A consensus sequence was generated from 91 and 76 individual cyathostomins for the regions spanning codons 167 and 200, respectively. A multi-species genomic DNA-based assay was established to directly pyrosequence individual L3 from field samples of unknown species and BZ sensitivity in a 96-well plate. In this format, the assay to detect F167Y gave a 50-90% success rate. The optimisation of the assay at codon 200 is currently underway. Subsequently, the genotype at F167Y was determined for 241 L3s, collected prior to and after BZ treatment. These results demonstrated a reduction in the heterozygous genotype, TTC/TAC, and an increase in the homozygous resistant genotype TAC/TAC in post-treatment samples. However, the differences in allele frequencies determined before and after BZ treatment were not statistically significant.
Conclusion: Extensive genomic DNA sequence, spanning codons 167 and 200 of the beta-tubulin isotype 1 gene, was generated from multiple cyathostomin species. The data facilitated the development of a pyrosequencing assay, capable of detecting the genotype of individual cyathostomin L3s derived from mixed-species field samples. Differences in codon 167 allele frequencies were observed in L3s isolated pre- and post-BZ treatment.