Investigations into the biology and genetics of Cylicocyclus nassatus as relating to the mechanism of action and selection for resistance to avermectin-milbemycin anthelmintics
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The objectives of this dissertation were to study the feasibility of using Drenchrite® bioassay for the diagnosis of anthelmintic resistance in horse parasitic nematodes, determine the effects of moxidectin (an anthelmintic drug) selection on the population genetic diversity of Cylicocyclus nassatus, a common cyathostomin species and also to clone, sequence and characterize the orthologs of nematode glutamate-gated chloride channel (GluCl) genes from C. nassatus. Cyathostomin are the principal parasitic pathogen and most important intestinal nematodes of horses. Resistance against two of the three available classes of anthelmintics has been reported in cyathostomins. Although no clinical case report of resistance against avermectin-milbemycin (AM) class exists at present, it is highly probable that cyathostomin will develop resistance to this group of drugs in near future. First objective: Studies on validation of a larval development assay (LDA) for detection of anthelmintic resistance in horses in the current situation of anthelmintic resistance determined that a LDA cannot be recommended for use in the field in the present scenario of anthelmintic resistance in horses. Second objective: Selection using a subtherapeutic dose of moxidectin for a period of 29 months (21 doses) leads to a statistical decrease in population genetic diversity within C. nassatus as determined by Amplified Fragment Length Polymorphism (AFLP) for two of the three primer combinations tested. A high level of inherent genetic polymorphism in C. nassatus individuals was discovered in this study. Third objective: A homology based PCR approach was used to clone and sequence full-length cDNAs for two genes from C. nassatus which are orthologous to invertebrate glutamate-gated chloride channel genes. Phylogenetic analysis determined the close relationship of these genes to GluCl genes in other species. Radioligand studies using GluCl subunits expressed in mammalian cells demonstrated the presence of a high affinity ivermectin-binding site on the C. nassatus GluCl- subunit but not on the GluCl- subunit. Together these findings provide insights into some potential genetic aspects of AM resistance in cyathostomin nematodes and may serve as a basis for future studies aimed towards understanding the process of development of AM resistance in cyathostomins, as well as for the development of molecular diagnostic tools for detecting resistance.