Development of genetic crossing methods to identify genes associated with macrocyclic lactone resistance in the sheep nematode parasite, Haemonchus contortus.

Abstract

There is a pressing need to develop strategies to reduce the emergence of macrocyclic lactone anthelmintic resistance in sheep flocks. Management practices aimed at maintaining anthelmintic susceptible nematodes in refugia while achieving a satisfactory level of production may prove to be useful. However, sensitive molecular tests are required to monitor the subtle effects of these practices on the frequency of resistance alleles within nematode populations. To-date, conventional studies of candidate genes coding for the known methods of action of macrocyclic lactone anthelmintics have produced a complex picture, highlighting the relevance of different approaches to the identification of resistance markers. This thesis describes the development of a single nematode parent genetic crossing method and discusses its application to identify molecular markers for anthelmintic resistance. Parasitological and molecular verification of successful inbreeding of the MHco3 strain of H. contortus derived from the progeny of a genetic cross between single nematode parents is described. The single parent genetic crossing method has enabled the production of diverse inbred lines of the MHco3 H. contortus and may prove useful for genome assembly, or for the development of a genetic map. The study has afforded insights to the biology of H. contortus and effects of host immunity on nematode parasites. New information is presented concerning the period during which adult female nematodes continue to shed fertilised eggs after removal of males, the development of unfertilised H. contortus eggs, and the population genetics of mixed infections of two different strains of H. contortus. Novel backcrossing experiments initially between a macrocyclic lactone resistant (MHco4 or MHco10) and a susceptible (MHco3) strain of H. contortus and then between ivermectin treated backcross generations and the parental susceptible strain are described. The resources provided by these experiments should enable comparative genomic analysis and conventional molecular biology to identify resistance genes derived from the parental resistant strains in fourth backcross generations that are the same as a parent ivermectin susceptible population, apart from the presence of alleles linked to anthelmintic resistance, derived from parent resistant strains.