To study the prevalence of the polymorphism in position 200 of the beta-tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naïve horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42·3% of the adults (n=104) were homozygous TTC/TTC, 55·8% were heterozygous TTC/TAC and only 1·9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.

Three nucleotide data sets, one nuclear (ITS-2) and two mitochondrial (COI and l-rRNA), have been investigated in order to determine relationships among species of Strongylinae and Cyathostominae, intestinal parasites of the horse.Nucleotide sequence data reported in this paper are available in the NCBI, GenBank, accession numbers: AF263472 to AF263472 and AF268479. The data exhibited a strong mutational bias towards A and T and in the COI gene, silent sites appeared to saturate rapidly partly due to this substitution bias. Thus, the COI gene was found to be less phylogenetically informative than the l-rRNA and ITS-2 genes. Combined analysis of the l-rRNA and ITS-2 genes supported a monophyletic clade of the cyathostomes with Tridentoinfundibulum gobi, which had previously been classified as a nematode of ‘uncertain origin'. The Strongylinae grouped consistently outside the clade containing the cyathostomes and T. gobi. Molecular analysis failed to provide strong evidence for the separation of cyathostomes into classical genera, as previously defined by morphological classification.

The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstrating the potential of the approach for the diagnosis of equine strongyloidosis. The establishment of this PCR assay also has implications for studying the biology and epidemiology of equine strongyles and anthelmintic resistance using faecal egg count reduction tests.