title: Development of molecular tools for the rapid assessment of benzimidazole resistance and investigation of possible risk factors in resistance development in Nematodirus battus populations
Creators: name: Melville, LA
Publisher: name: University of Liverpool Repository
Year: 2019

description: Nematodirus battus is an economically important gastrointestinal nematode (GIN) which threatens lamb health and sustainability of UK farming. In recent years apparent changes in the hatching and infection dynamics have occurred in conjunction with the first reports of fenbendazole resistance The aims of this project were to develop molecular tools to study the BZ-resistant allele frequency in UK N. battus populations; examine the hatching preferences (with or without a chill stimulus) of UK N. battus populations; assess farmers’ perceptions of N. battus infection and to gather farm management data to assess current control methods. Finally, to develop generalised linear mixed effect models (GLMM) to investigate possible associations between management decisions and the development of anthelmintic resistance (AR) and the ability of eggs to hatch without chilling. Three DNA-based detection methods were evaluated within the current project; pyrosequencing, deep-amplicon sequencing by Illumina MiSeq and Loop-mediated isothermal amplification (LAMP) to detect and quantify the single nucleotide polymorphisms (SNPs) within the β-tubulin isotype 1 gene associated with BZ-resistance; namely F176Y, E198A and F200Y. Pyrosequencing and deep amplicon sequencing assays were developed, evaluated and used to conduct a genotyping survey of N. battus populations from UK commercial farms. Results were comparable between the two platforms, indicating that either method would be suitable as a laboratory diagnostic tool. LAMP was also evaluated within the project with a view to on-farm diagnosis. The prototype LAMP assay successfully identified the resistant allele when tested using plasmid DNA and was capable of producing semi-quantitative results. However, further evaluation of the prototype assay using field samples produced inconsistent results. A total of 282 N. battus populations were analysed in the genotyping survey. The F200Y resistant allele was identified in 26% of the populations tested, albeit at low overall allele frequency (~2%) with a focal region of high resistant allele frequency identified in North West England. The F167Y mutation was identified for the first time in N. battus, at low prevalence and frequency and no mutations were identified at codon 198. Generalised linear mixed effect model analysis investigating the drivers of BZ-resistance in N. battus highlighted set stocked grazing, reseeding of heavily contaminated pasture and the observation of symptoms predominantly in spring as potential risk factors with the administration of quarantine practice as a protective factor. The farm management questionnaire highlighted significant regional variation in the observation of N. battus. The perception of increasing severity of symptoms were reported from respondents in the North whilst the time at which N. battus is becoming more apparent to respondents has changed in the South. Diagnostic methods also varied regionally, with greater uptake of faecal egg counting and online risk maps to determine anthelmintic treatments in the South. The requirement for chilling of N. battus eggs prior to hatching was tested in 90 UK N. battus populations in vitro. Up to 87% of eggs were able to hatch without chilling, with higher non-chill hatching observed in Scottish populations. GLMM analysis, identified associations between spring temperature, lambing date, N. battus faecal egg count and grazing management strategies with non-chill hatching. The results indicated that the requirement for chilling may vary throughout the year, possibly in response to host immune development. This project represents the largest survey of UK N. battus populations ever conducted and has provided a valuable insight into how this species has changed in recent years. Epidemiological differences observed North to South questions the suitability of the current ‘one fits all’ approach to advice. Validation of the risk factors identified, could provide the basis for novel control strategies to minimise production losses and the associated economic cost of N. battus infection.