Haemonchus contortus: the role of two beta-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics.

The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.