Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Genetic polymorphism of the beta-tubulin gene of Onchocerca volvulus in ivermectin naïve patients from Cameroon, and its relationship with fertility of the worms.

Observations of low response of patients infected with Onchocerca volvulus to ivermectin suggest that the parasite may be under a selection process toward potential resistance. To limit the extension of this phenomenon, it is crucial to characterize the genes of O. volvulus that are involved. For this, O. volvulus adult worms collected before the introduction of ivermectin in an onchocerciasis endemic area of central Cameroon were genotyped for beta-tubulin. To derive a baseline to investigate the selective pressure of ivermectin, we analysed (1) the frequency distribution of the beta-tubulin alleles, and (2) the relationship between the different beta-tubulin related genotypes and the fertility status of the female worms. The frequency of allele b of the beta-tubulin gene was very low, as it was observed in West Africa. We observed a deficit of heterozygous female worms leading to Hardy Weinberg disequilibrium, which might be explained by a shorter life-span of these worms compared to the homozygous worms. Unexpectedly, our results also show that the heterozygous female worms were much less fertile than the homozygotes: more than two thirds of the homozygotes were fertile, whereas only 37% of the heterozygotes were fertile. These results will be further considered when analysing post-treatment data.