Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM-resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. Over the last few years, studies have reported genetic selection or reduced responses to IVM in some O. volvulus populations. The risk of a recrudescence of the disease was recently reported with the emergence of resistant adult parasite population in Ghana. It is important to understand the effects of IVM on O. volvulus populations to be able to identify genetic markers to follow IVM selection in the field. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following three years (1994-1997) of IVM treatments. There were four treatment groups: 150mug/kg (1xp.a. or 4xp.a.) and 800mug/kg (1xp.a. or 4xp.a.). DNA from macrofilariae was genotyped for beta-tubulin and P-glycoprotein-like protein (PLP) gene, as well as two control genes and other loci. Reproductive organs of female worms were analyzed by microscopy. A correlation was established between the reproductive status of the female worms and beta-tubulin genotype with the beta-tubulin heterozygous female worms being less fertile than the homozygous female worms. This disadvantage in fertility seemed to disappear after repeated exposure with IVM. We have found evidence that repeated IVM treatment selects for specific alleles of beta-tubulin and PLP. We observed that IVM selection pressure was higher in the female worms than in the male worms. Additionally, loss of polymorphism and selection pressure were higher following thirteen three-monthly doses of IVM compared to annual doses of IVM. Moreover, we found evidence of excess of homozygosity in O. volvulus population, that may be caused by non-random mating and/or subdivision population, which may have implication for the control program. PLP and beta-tubulin genes appear to be promising DNA markers for field use to follow IVM selection. In this perspective, alternative control measures could be considered locally in regions where gene selection is apparent, reducing the likelihood that IVM resistance would develop further and spread.

Onchocerciasis, commonly known as "River blindness" is a disease affecting over 37 million people, worldwide. It is caused by the parasitic nematode Onchocerca volvulus and transmitted by the blackfly vector of the genus Simulium. The drug ivermectin (IVM) is the principal means of controlling the disease. As a result of recent reports on sub-optimal response to ivermectin and genetic selection in O. volvulus, we carried out a 21 month epidemiological study to investigate the response of O. volvulus to repeated rounds of IVM treatments in 2501 subjects from 19 Ghanaian communities that have received between 6 and 18 annual treatments and one IVM naive community. Skin microfilaria (mt) assessments were done before IVM treatment and at days 30, 90, 180 and 364 post-IVM treatment. At day 90 after the second IVM treatment, nodulectomies were carried out on 140 patients and embryogrammes constructed on female worms. We found IVM is still an effective microfilaricide, with efficacy of 98-100%. However, its effect on adult worm fertility has been reduced. Day 90 and 180 post-treatment showed significantly higher (p<0.05) skin mf repopulation of 7.1% to 53.9%, and >100% of pretreatment counts at day 364 post-treatment in four communities compared with the other six communities, which had <80% of pretreatment mf counts on day 364. From these results we classified the 10 communities into good IVM response (four communities), intermediate IVM response (two communities), poor IVM response (three communities), and the previously IVM naive community. Nodule and worm viability and worm densities were significantly higher (p<0.05) in the poor response communities compared with the good response communities, with the intermediate falling between the two. Embryogramme analysis showed significantly higher reproductive activity and output in worms from poor response communities with up to 41% of females having live stretched mf in utero compared with good response communities which had no intra-uterine stretched mf. These results show evidence of lack of sustained response of adult O. volvulus to IVM in the poor response communities, manifested as a rapid return to fertility after IVM treatment. We conclude that IVM resistance is emerging in onchocerciasis and is manifested as a loss of effect of IVM on suppression of parasite reproduction. / Beta tubulin isotype 1 gene has been shown to be linked to IVM treatment and selection in O. volvulus and veterinary nematodes. Genetic analysis of the full length genomic DNA sequence of beta-tubulin from worms obtained in the three IVM response categories and IVM naive community showed single nucleotide polymorphisms (SNPs) at 24 sites on the entire 3696 bp. The frequencies of eight SNPs were significantly different (p< 0.05) between the poor response communities and the good response/naive communities. Four SNPs, 183 T/G, 1188 T/C, 1309 CIT and 1545 A/G resulting in a genotype configuration GG/CC/TT/GG (183/1188/1309/1545) was significantly higher in the poor IVM response communities than the other communities. The phenotypic and genotypic analyses are consistent with a conclusion that IVM resistance has been selected. These four SNPs could be used to develop a genetic marker for early detection of IVM resistance. This study has shown for the first time that IVM resistance is emerging in Onchocerca volvulus and that there are genetic changes associated with IVM resistance which could be used for epidemiological monitoring for emerging resistance.

Onchocerca volvulus is a parasitic filarial nematode responsible for human onchocerciasis, a disease commonly known as "River Blindness". Although there are no well documented cases of ivermectin resistance in O. volvulus, reports of suboptimal responses to ivermectin have appeared. The purpose of this thesis was to examine genetic polymorphisms in O. volvulus and to determine whether there was genetic evidence of ivermectin selection on O. volvulus genes. Analysis of 17 genes from O. volvulus was undertaken in two populations of worms, either from ivermectin-naive patients or from patients who had been repeatedly treated with ivermectin annually. In 14 of the genes no differences in genetic polymorphism were found (although polymorphisms were identified). However, chi square analysis (chi2=0.05) indicated significant differences in allele frequencies for a P-glycoprotein, a beta-tubulin and a putative dyf=8 gene. Analysis of the O. volvulusbeta-tubulin alleles identified three amino acid substitutions in the H3 region with ivermectin selection. Microtubules play a key structural role in the formation of neurons, and in ivermectin-resistant Haemonchus contortus, amphidial neurons show distorted microtubule bundles. Polymerization and depolymerization assays of the recombinant O. volvulus beta-tubulin alleles showed interesting differences between the polymerized tubulin using the two different alleles. It is speculated that similar differences could cause the disorganization of the microtubules identified in the amphidial neurons in ivermectin resistant H. contortus. In addition to the coding mutations, a 24 bp deletion in the adjacent intron to the H3 was detected. A PCR diagnostic assay was developed to genotype individual macro- and microfilariae. Further analyses were conducted to investigate the possibility of a direct relationship between ivermectin and beta-tubulin. Data obtained from equilibrium dialysis experiments indicated that BODIPY FL ivermectin bound to purified O. volvulus alpha- and beta-tubulins. More interesting, non-fluorescent ivermectin and taxol competed with the BODIPY FL ivermectin. The work presented in this thesis provides evidence of genetic selection by ivermectin on O. volvulus and suggests a putative binding site for ivermectin on tubulin. These data provide novel information on ivermectin selection in O. volvulus and on the possible involvement of tubulin in ivermectin resistance.

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