Biochemical and molecular investigation into levamisole-resistance in trichostrongylid nematodes of sheep

LEV-resistance in trichostrongylid nematode parasites of sheep is of considerable economic importance and a growing problem in Auatralia and other countries. To combat LEVresistance, a greater understanding of the mode of action of LEV and mechanisms of resistance to LEV are necessary. In this project, biochemical and molecular approaches have been used to investigate LEV-resistance and mode of action. A pharmacological study of 20 LEV-analogues confirmed that LEV is a highly specific molecule. Most modifications to the LEV-structure reduced its effectiveness against both LEV-resistant and -susceptible strains of H. contortus, as measured by in vitro egg hatch assay, ligand binding assay and adult contraction assay. The nAChR-specific ligand, a-Bgt, was used to characterise the putative cholinceptor from H. contortus. a—Bgt bound specifically to homogenates of L3 H. contortus with the pharmacological characteristics of a nAChR. A single, high—affinity binding-site(Kd 2 — 3 x 10'9 M) was detected in the two LEV-susceptible strains tested. Binding was inhibited by the LEV-analogue, MAL. This confirmed that LEV binds to a nAChR in H. contorlus. A second, lowaffinity binding-site (Kd ~10'6 M) was detected in the LEV—resistant strains tested.This indicated that there was an alteration in receptor structure or population in the LEV-resistant strains. The cloning of a nAChR a-subunit candidate gene from T. colubriformis has provided a major advancement in the knowledge of cholinoceptors from parasitic nematodes and the genomic organisation of T. colubriformis, From the proposed coding sequence of this gene, a predicted peptide structure of a T. colubriformis nAChR (it-subunit has been determined. Comparison with peptide sequences of subunits from other species showed that the nematode receptors form a distinct subtype of the nAChR family. They cannot be strictly classified as most like neuromuscular of neuronal receptor types. The genomic organisation of the T. colubriformis nAChR a-subunit candidate gene revealed some conserved features with genes from C. elegans and eukaryotes in general. Intron/exon splice sites are highly conserved and are most similar to those from C. elegans. A region of high sequence homology to mariner-like transposable elements has also been described. This is the first report of such an element from T. colubriformis. An investigation of the proposed major Ach-binding-site sequence of the T. colubriformis ocsubunit candidate gene from individual LEV-resistant and -susceptible worms did not reveal any changes in this region that would alter the encoded protein. Therefore, a change in this binding-site region does not appear to be responsible for LEV-resistance in the strain examined. Sequencing of this region identified a polymorphism in the intron sequence that would result in an RFLP for the enzyme NlaIII. The frequencies of each allel in male and female worms were determined and provided strong evidence that the polymorphism, and therefore the a—subunit gene, is on the X-chromosome in T. colubriformis. This is of particular interest as LEV-resistance is believed to be due to a sex-linked gene or linked-gene complex in this species (Martin and McKenzie, 1990). The identification of an RFLP in this sequence also provides a marker for the X-chromosome in this species