Characterisation of anthelmintic resistance in a multiple drug resistant Teladorsagia circumcinta isolate
The purpose of this study was to undertake detailed molecular and phenotypic characterisation of a MDR isolate of T. circumcincta (MTci5) with particular focus upon the mechanisms underlying benzimidazole (BZ) resistance. MTci5 was isolated from a farm in Central Scotland, which employed a suppressive anthelmintic dosing regime and was closed in 2002 when control of the parasite population became unsustainable. Underpinning all of the experiments in this study was an anthelmintic selection process whereby the MTci5 isolate was pressured individually with three broad-spectrum anthelmintics (benzimidazole, ivermectin and levamisole). There are three main areas of investigation in this study, the first being an investigation of the population genetic structure or a MDR isolate. A central question was whether the MDR phenotype of MTci5 is conferred by the inheritance of genes present in a single interbreeding population or whether there is genetic sub-structuring, whereby discrete sub-populations of the isolate each show resistance to different anthelmintics. Microsatellite analysis was employed to investigate the population genetic structure of the MTci5 isolate. The results suggest that the MTci5 isolate is a single, freely interbreeding population with triple resistance, showing no evidence of genetic sub-structuring. The second area of investigation was the role of the F200Y isotype I ß-tubulin mutation in the determination of BZ resistance and the potential involvement of this mutation in resistance to ivermectin (IVM) and levamisole (LEV). There was no evidence of an effect of IVM or LEV selection upon the F200Y isotype I ß-tubulin mutation. The third area of investigation was the origin and diversity of BZ resistance alleles in the MTci5 isolate. Single strand conformation polymorphism (SSCP) analysis of small region extending through exons 1 and 2 and intron 1 of the isotype I ß-tubulin gene was used to assess the genetic diversity of this locus in the MTci5 isolate and of five other UK T. circumcincta populations. Results are consistent with the theory of multiple independent, spontaneous mutations at the P200 locus of the isotype I ß-tubulin gene.