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Georgia Veterinary Scholar Program

Georgia Veterinary Scholar

Faculty Mentor

Irina Kim
University of Georgia
Class of 2005

Dr. Ray Kaplan

 

Mechanisms of avermectin resistance in parasitic nematodes: PCR cloning of the UNC-9 gene homologue from Cylicocyclus nassatus (Cyathostominae)

 

Intensive use of chemotherapy to combat equine strongyle infestations has inadvertently fostered the development of drug-resistant cyathostomes, which now reign as the principal parasitic pathogen of horses. The avermectin/milbemycin drug class remains the cornerstone of nematode control in horses, but as with other anthelmintics, its future efficacy is undermined by the inevitable development of drug resistance. In Caenorhabditis elegans, mutations in the innexin genes unc-7 and unc-9 confer a reduced sensitivity to avermectins, but the role of these genes in drug resistance among parasitic nematodes is virtually unknown. In this study, a PCR cloning strategy was employed to isolate an unc homologue from the cyathostome Cylicocyclus nassatus in a continuing effort to elucidate the mechanisms of avermectin-resistance in parasitic nematodes. Innexin protein sequences from C. elegans and Drosophila melanogaster were aligned to identify conserved regions of this gene family. Degenerate sense and antisense primers were designed for a touchdown RT-PCR reaction to amplify an interior unc fragment. The 5’ sequence was amplified using SL1 and gene specific primers and semi-nested RACE-PCR with gene specific primers. A partial 3’ end of the gene was obtained with semi-nested RACE-PCR and gene specific primers. The fragments were cloned and sequenced. A BLAST comparison of the spliced sequence confirmed a 933 base pair segment with a 79% protein sequence similarity to the uncharacterized C. elegans innexin protein Y8G1A.2 and a 69% similarity to the unc-9 gene of C. elegans. The functional C. elegans innexin genes are approximately 1150 base pairs long, so we estimate that we obtained about 80% of the entire gene.