Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus

Objectives We have found that in a laboratory strain of the nematode parasite of ruminants, Haemonchus contortus, repeated ivermectin treatment, in vivo in sheep, selected against TTC, coding for Phe, and for TAC, coding for Tyr, at codon 200 in beta-tubulin isotype 1. This 200Tyr single nucleotide polymorphism (SNP) has been associated with benzimidazole anthelmintic resistance. In this study, we investigated the alpha-tubulin and beta-tubulin sequences of 17 different field and laboratory strains/isolates of H. contortus with known treatment history and status for susceptibility or resistance to macrocyclic lactone NO or benzimidazole anthelmintics. Basic methods DNA (genomic or cDNA) from 10 to 32 male parasites was sequenced for alpha-tubulin or beta-tubulin for each strain/isolate and the frequency of coding SNPs was compared between the different strains/isolates with known treatment history and drug resistance status. Main results We have analyzed alpha-tubulin and beta-tubulin isotypes 1 and 2 genes from the different H. contortus strains/isolates and found that repeated ivermectin or moxidectin (both ML anthelmintics) use changes the frequency of beta-tubulin isotype 1 alleles and selects for the following SNPs: either from TTC (Phe) to TAC (Tyr) at codon 200 or codon 167, or from GCA (Ala) to GAA (Glu) at codon 198. 200Tyr and 167Tyr were associated with GTT (Val) or CTC/CTT (Leu) at codon 368, whereas 200Phe or 167Phe were associated with either ATT (IIe) or GTT (Val) at codon 368 in beta-tubulin isotype 1. The SNPs 200Tyr, 167Tyr in beta-tubulin isotype 1 have been described to confer benzimidazole resistance in nematodes and recently 198AIa has also been associated with benzimidazole resistance. Conclusions Although MLs exert their antiparasitic effect through binding to ligand-gated chloride channels, benzimidazoles interact with beta-tubulin. These genetic analyses indicate that there is a correlation between exposure and/or resistance to MLs and an increase in the frequency of the beta-tubulin alleles containing codons, which are determinant for benzimidazole resistance. ML use may predispose parasitic nematodes to benzimidazole resistance. This has major implications for parasite control programs dependent on MLs/ benzimidazole rotations and on the use of ML/ benzimidazole combination therapy, such as for human lymphatic filariasis and nematode control in ruminant livestock.