Phenotypic characterization of two Ancylostoma caninum isolates with different susceptibilities to the anthelmintic pyrantel

Abstract

The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.

FIG. 1.

Force transducer circuitry and experimental apparatus. (a) The photosensitive cell is capable of sensing minute deflections of the needle upon contraction of the worm. A feedback circuit assesses the signal from the photosensitive cell and applies a modulating current to maintain the position of the needle against the contraction force. The correcting current applied to the needle can be continuously monitored by use of a USB interface and is proportional to the force applied to the needle (N, north; S, south). (b) Cannulated adult female worms were glued to the base of a plastic weigh boat and then attached to the transducer needle through the use of a nylon filament attached to the anterior region of the worm.

FIG. 2.

Dose-responses determined by LAMA. The graphs depict the reduction in the percentage of normally arrested larvae with increasing drug concentration. Error bars represent standard errors.

FIG. 3.

Dose-responses determined by LMA. Pyrantel responses describe fourth-order polynomial curves that were drawn by hand. The responses to the other drugs were fitted with sigmoidal curves. Error bars represent standard errors.

FIG. 4.

Graphical output from force transducer experiments. Force transducer traces for pyrantel against a PR and an NT adult worm. Pyr, pyrantel.

FIG. 5.

Force transduction dose-response plots for the four nicotinic agonists tested. Error bars represent standard errors.