Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.