SRA

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs, which in the case of livestock parasites, are severely compromised by the widespread development of drug resistance. More recently, there are concerns regarding the emergence of anthelmintic resistance in human parasitic nematodes in response to the selection pressure resulting from mass drug administration (MDA) programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance.. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. We describe the development and validation of deep-amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance. We have used parasite communities in sheep, to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each with varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated SNPs at three different codons of the ß-tubulin gene, in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.