Gilleard, John S.Levy, Michelde Queiroz, Camila A. A.2021-08-042021-01-14de Queiroz, C. A. A. (2020). The application of deep amplicon sequencing to the diagnostics and molecular epidemiology of anthelmintic resistance in sheep gastrointestinal nematodes (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/113701Gastrointestinal nematodes (GIN) are a major challenge for sheep producers worldwide. They cause a variety of clinical signs and large economic impact. Anthelmintics are commonly used in control, however, indiscriminate use of these drugs over many years has led to the problem of anthelmintic resistance. The lack of accurate resistance diagnostic methods increases the challenge of parasite control. This thesis investigates the problem of anthelmintic resistance in Western Canadian sheep for the first time. This region has been believed unsuitable for GIN due to suboptimal weather and low treatment selection pressure. Chapter 2 presents a large-scale survey of anthelmintic efficacy. The precision of the Fecal Egg Count Reduction Test (FECRT) diagnostic method was also investigated. Results revealed high GIN infection intensity and anthelmintic resistance against benzimidazoles and ivermectin, highlighting the urgent need for better diagnostic tools. Chapter 3 validates the recently developed nemabiome metabarcoding approach for the relative quantitation of GIN species in ovine fecal samples. Chapter 4 applies the validated approach integrated to FECRT to a large-scale study in Western Canada and revealed widespread resistance of H. contortus to benzimidazoles and ivermectin. Resistance to closantel was absent, but this drug was often ineffective due to its narrow spectrum activity.Chapter 5 applies deep amplicon sequencing to screen for benzimidazole resistance mutations in the isotype-1 b-tubulin gene of the main GIN in Western Canada and United States of America (USA). Codon F200Y(TTC>TAC) mutation was found at high frequency in H. contortus and at lower frequencies in other species. The relative frequencies of resistance mutations were similar between Western Canada and USA, although generally higher in USA, consistent with greater selection pressure. A pilot study also identified resistance mutations in the isotype-2 b-tubulin gene of H. contortus, suggesting this locus warrants further investigation regarding its role in benzimidazole resistance. Phylogenetic network analysis revealed that the most common resistance haplotypes were shared between Western Canada and USA, supporting a hypothesis of common origins of resistance. Given the more advanced resistance in the USA, a likely scenario is that anthelmintic resistant parasites were introduced to Western Canada from sheep imported from the USA.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.nemabiome metabarcodingovine nematodesdrug resistanceHaemonchus contortusparasite controlAgriculture--Animal Pathologydoctoral thesis10.11575/PRISM/39067