title: Leva-LAMP: Sustainable use of levamisole through elucidation of genetic markers of resistance and molecular diagnostics in Haemonchus contortus
Creators: name: Antonopoulos, Alistair
Publisher: name: University of Glasgow
Year: 2022

description: Haemonchus contortus is a gastrointestinal parasitic nematode primarily infecting small ruminants. It is globally distributed, and a major cause of production losses and animal health concerns. Control largely relies on the use of broad spectrum anthelmintics, however, the effectiveness of many of these drugs is declining due to widespread anthelmintic resistance. One of the broad spectrum anthelmintics available to control H. contortus is levamisole (LEV), a cholinergic agonist which, when bound to the nematode acetylcholine receptor (AChR), causes paralysis in the worm. Resistance to LEV is, at the time of writing, less widespread worldwide than to other major drug classes, such as benzimidazoles (BZs) and macrocyclic lactones (MLs). However, unavailability of effective molecular diagnostics, due to the lack of fully resolved and validated molecular markers of LEV resistance in H. contortus and over reliance on the faecal egg count reduction test, which has inherent inaccuracies, and is poor at detecting emerging resistance, underline the urgent need to improve resistance monitoring. Understanding of genetic markers of LEV resistance is, therefore, key to improving the diagnosis of anthelmintic resistance and maintaining the sustainability of LEV. Therefore, the goal of this PhD was to elucidate and validate genetic markers of LEV resistance, and develop proof-of-concept molecular diagnostic assays. First, using whole genome sequencing data (WGS) from a controlled genetic cross (from a resistant and a susceptible parental line) pre- and post-LEV treatment, two quantitative trait loci (QTL) were identified. A single non-synonymous SNP variant, S168T, was identified in acr-8, a gene present within the major QTL, and previously shown to be essential for conferring LEV sensitivity to the H. contortus AChR. Extensive analysis of WGS data and single worm genotyping demonstrated that the presence of the S168T variant was predictive of a LEV resistant phenotype in all LEV resistant laboratory and field isolates examined, and absent in all susceptible isolates. Further SNP variants were also identified in LEV resistance associated genes in a second QTL on chromosome IV, which likely constitute minor markers of LEV resistance. Concurrently, putative markers of LEV resistance previously detailed in the literature were examined, but found not to constitute effective markers of LEV resistance in the isolates examined. A pilot study to validate a next-generation amplicon sequencing panel was also undertaken, with preliminary validation complete for S168T, and several additional minor markers of LEV resistance. Following on from these results, several molecular diagnostic assays with significant translational potential were developed and validated for the detection of the S168T variant in H. contortus. A two-step nested allele specific (AS)-PCR was optimised and demonstrated for the detection of the S168T variant in both laboratory and field isolates. Two emerging novel loop-mediated isothermal amplification (LAMP) technologies were then evaluated for detection of the S168T variant. Loop-primer enzymatic cleavage (LEC)-LAMP showed promising results discriminating between the resistant and susceptible alleles, with the added capacity for multiplexing. LEC-LAMP was also preliminarily adapted to point-of-care (POC) detection via a lateral flow (LF) platform. The results generated in this PhD have laid the foundation necessary to establish reliable and accurate laboratory based molecular diagnostics for the detection of LEV resistance in H. contortus, with potential for fast and low-cost application of these assays at the POC.