Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

Benzimidazole resistance in Trichostrongylus axei in sheep: long-term monitoring of affected sheep and genotypic evaluation of the parasite.

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance.

Regulation of Teladorsagia circumcincta and Trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status.

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.

A short-term divergent selection for resistance to Teladorsagia circumcincta in Romanov sheep using natural or artificial challenge.

This experiment was conducted to assess the efficiency of selection on the basis of response to artificial challenges in order to breed sheep resistant to natural infection. A short-term divergent selection process was designed to estimate the genetic parameters of these two traits. Two flocks, including 100 Romanov ram lambs each, were challenged in 1990 when they were 6 months old. One flock received three artificial infections with 20 000 third-stage Teladorsagia circumcincta larvae, at intervals of 7 weeks. Faecal egg counts (FEC) were performed on Days 22, 25 and 28 post infection (p.i.) and the animals were drenched on Day 28 p.i. The other flock was grazed for 5 months on a pasture contaminated with the same species. Faecal samples were taken from the lambs at similar ages. About 5 rams with the lowest FEC and 5 with the highest FEC were selected in each flock and mated with unselected ewes. Their offspring (200 animals) were challenged in 1992, half in the same way as their sires, and the other half by the other method. Because of a drought in the summer of 1990, it was necessary to repeat part of the experiment, and in 1992 the 5 and 8 rams with the lowest and highest FEC, respectively, were selected from the offspring challenged on the pasture in 1992 and were mated with unselected ewes. Their progeny (about 80 animals) were challenged in 1994, half by natural infection, half by artificial infection. The mean FEC of the flock increased from the first to the third artificial infection. The natural infection was highly variable in different years, reflecting the difficulty of assessing resistance using this mode of challenge. Genetic parameters were estimated using animal models and REML solutions. The repeatabilities of the FEC following artificial and natural infection were 0.49 and 0.70 respectively within a period of one week, and 0.22 and 0.41 respectively for periods separated by intervals of 7 weeks; the heritabilities of the single egg count were 0.22 and 0.38 respectively. The genetic correlation was 0.87: the FEC recorded under natural or artificial infection appear to depend on the same genetic potential.