In vitro selection and differentiation of ivermectin resistant cyathostomin larvae

Cyathostomins are considered to be the primary helminth pathogen of horses and macrocyclic lactones (ML) are the most frequently used anthelmintics. Therefore, ML resistance is a serious threat for the control of these parasites. In the present study ivermectin resistant cyathostomin L3 were in vitro selected, using a reiterative larval migration inhibition assay (rLMIA) and differentiated by reverse line blot (RLB). Larvae were obtained from two populations, one from a never treated, free-roaming horse population in the nature reserve Oostvaardersplassen (OVP) and the other from regularly ivermectin-treated ponies of Utrecht University (UU). In the rLMIA the proportion of larvae that migrated increased with each passage, demonstrating that the applied procedure indeed selects for larvae the least susceptible for ivermectin. This was further supported by the fact that glutamate addition to this procedure reversed the selection effect, which also suggests that glutamate-gated chloride channels (GluCls) play a role in the ivermectin resistance of the selected L3. In both populations the predominant species were Cyathostomum catinatum, Cylicostephanus longibursatus and Cylicocyclus nassatus. After in vitro selection in the rLMIA in the presence of ivermectin the predominant species became C. catinatum in both larval populations, while C. nassatus disappeared in the never treated OVP larval population but not in the regularly ivermectin-exposed UU population. It is concluded that the rLMIA and RLB can be used to study anthelmintic resistance in cyathostomin populations and to study differences and changes in species composition between populations with different anthelmintic exposure histories.