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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111844 |
| Date | January 2006 |
| Creators | Eng, Jeffrey K. L. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Institute of Parasitology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002589964, proquestno: AAINR32178, Theses scanned by UMI/ProQuest. |
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