Development of high-throughput metabarcoding approaches to explore genetic diversity and anthelmintic resistance in sheep gastrointestinal nematodes

Abstract

Sheep farming in the UK, integral to its cultural and economic fabric, contends with significant challenges from gastrointestinal nematodes (GINs) which impair livestock productivity and increase mortality, leading to substantial economic losses. The emergence of anthelmintic resistance, compounded by the impacts of climate change, has necessitated integrated parasite management (IPM) strategies and the adoption of advanced DNA technologies such as next-generation sequencing (NGS) for diagnosis and targeted treatment. In this project, rDNA ITS-2 and mt-ND4 genetic markers were employed to analyse GINs across various locations, farms, and treatment groups. The ITS-2 analysis, also known as the Nemabiome, provides robust identification and relative abundance of GIN species when combined with faecal egg count, while mt-ND4 markers offer insights into the origins, gene flow, and infection patterns of individual species. By leveraging these technologies, this thesis aims to enhance the management and understanding of GINs, contributing to the sustainability and productivity of UK sheep farming. A significant portion of the work focused on refining these techniques. Despite the effectiveness of Nemabiome sequencing in detecting all clade V nematodes, identifying lesser-known GIN species remains challenging due to reference sequence gaps, particularly in environments with cross-infections from GINs infecting diverse grazing animals. To address this, the reference library was expanded using various GIN sample sets, listing anticipated GIN species, extracting their ITS-2 rDNA sequences from NCBI GenBank, and enhancing the library through iterative BLAST searches and phylogenetic analyses. Concurrently, mt-ND-4 markers were developed for T. circumcincta and H. contortus, the two most prevalent GIN species in the UK. These mitochondrial markers, typically species-specific, were adapted into a primer multiplex to process both species simultaneously, significantly increasing efficiency and reducing costs. Reference libraries were developed for both species, along with the post-sequencing analysis pipelines, and the multiplex was tested on various laboratory strains, verifying its accuracy and reliability. To implement both these techniques in the field, a DNA biobank was established, collecting over 250 field samples from across Scotland and England to enable comprehensive comparisons across various locations, treatments, and farms. This biobank was further enhanced by the inclusion of nine characterised laboratory strains of H. contortus and seven of T. circumcincta to assist in the comparison of resistance mechanisms and genetic diversity. Nemabiome analysis identified seven GIN species within the UK, with T. circumcincta being the most prevalent, followed by H. contortus and Trichostrongylus vitrinus. Together, these three species accounted for approximately 88% of infections. Significant spatial differences in GIN distribution were observed between areas, with T. circumcincta being prevalent in Scotland and H. contortus in England, likely influenced by climatic conditions. Despite its generally lower presence, H. contortus was consistently found on some Scottish farms, suggesting an influence of local environmental or management factors. It also showed a higher prevalence and diversity in alpacas compared to sheep or other hosts, raising questions about the role of alpacas as a maintenance host. The effectiveness of levamisole, ivermectin, and their combined anthelmintic treatments was assessed through faecal egg count reduction tests (FECRT) across 18 farms in England. Treatments were more effective against some species than others: Resistance was indicated in T. circumcincta, while Trichostrongylus axei and Trichostrongylus colubriformis displayed mixed results. Notably, the combination treatment proved less effective than individual drugs on some farms, which could suggest antagonistic interactions between these drugs. In the mt-ND4 analysis, 60 H. contortus and 35 T. circumcincta amplicon sequence variants (ASVs) were identified, with a single ASV representing over one-third and over 62% of the total reads, respectively. Phylogenetic analysis suggested a common origin for H. contortus, but potentially two distinct origins for T. circumcincta. H. contortus showed a general trend towards balancing selection or population contraction across both areas, while T. circumcincta showed population expansion or purifying selection in Scotland and balancing selection or contraction in England. There was significant gene flow with minimal genetic differentiation between Scottish and English populations across both species. Post-treatment analysis revealed a reduction in genetic diversity for H. contortus but the opposite for T. circumcincta. Ivermectin-treated groups displayed higher H. contortus diversity compared to those treated with levamisole, with the combination yielding moderate diversity levels, supporting the idea of possible antagonistic interactions. No difference was observed between treatment groups for T. circumcincta. Overall, the study underscores regional and inter-species variations, alongside the challenges of emerging anthelmintic resistance, especially in T. circumcincta. It highlights the critical need for continuous monitoring and targeted management strategies to effectively control GIN prevalence and manage resistance within UK sheep farming. Additionally, the findings lay a foundation for utilising more diverse and specific genetic markers to further investigate genetic diversity and resistance mechanisms