In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin.
Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.
Rufener
Lucien
L
Novartis Centre de Recherche Santé Animale, 1566 St. Aubin, Switzerland.
Kaminsky
Ronald
R
Mäser
Pascal
P
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D004351:Drug Resistance; D006189:Haemonchus / Q000187:drug effects*; D007559:Ivermectin / Q000494:pharmacology; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013827:Thiabendazole / Q000494:pharmacology*; D014404:Tubulin / Q000235:genetics*
D000871:Anthelmintics; D018509:DNA, Helminth; D014404:Tubulin; D007559:Ivermectin; D013827:Thiabendazole