Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance.
The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, K alpha, and maximum binding, Bmax, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The K alpha was of the same order of magnitude (x 10(7) M-1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of Bmax by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larvae to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.