Benzimidazole-resistant β-tubulin alleles in a population of parasitic nematodes ( Cooperia oncophora) of cattle

Three anthelmintic classes with distinct mechanisms of action are commercially available. Selection of nematode populations resistant to all these drugs has occurred, particularly in trichostrongyloid parasites of sheep. Anthelmintic resistance in cattle parasites has only recently been recognized and appears to be less pronounced, even though very similar species infect both hosts. To understand the bases for differences in the rate of resistance development in sheep versus cattle parasites, it is important to first demonstrate that the same kinds of resistance alleles exist in both. The benzimidazoles (BZ), which have been used for more than 40 years, were chosen as an example. BZ-sensitive (BZ S) and BZ-resistant (BZ R) nematodes that parasitize sheep have been distinguished at the molecular level by a single nucleotide change in the codon for amino acid 200 of a β-tubulin gene, a switch from T T ̄ C (phenylalanine) to T A ̄ C (tyrosine). PCR primers were designed to completely conserved regions of trichostrongyloid β-tubulin genes and were used to amplify DNA fragments from Haemonchus contortus (cDNA from a BZ S and a BZ R library) as positive controls. The technique was then extended to the cattle parasites, Cooperia oncophora and Ostertagia ostertagi (from genomic DNA). Sequence analysis proved the presence of amplified BZ S alleles in all three species and BZ R alleles in the BZ R population of H. contortus. Based on these data, nested PCR primers using the diagnostic T ̄ or A ̄ as the most 3′ nucleotide were designed for each species. Conditions for selective PCR were determined. To demonstrate feasibility, genomic DNA was recovered from individual H. contortus L 3 larvae from both BZ S and BZ R populations. Genomic DNA was also isolated from >70 individual adult male C. oncophora collected from a cattle farm in New Zealand with reported BZ resistance. Allele-specific PCR discriminated among heterozygotes and homozygotes in both species. This method could find utility in studying the molecular epidemiology of BZ resistance in cattle parasites and for defining the variables that limit the development and spread of anthelmintic resistance in this host.