A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage is crucial for minimising production losses by determining which drugs remain most effective. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. In this paper, we described a mixed deep amplicon sequencing approach to determine the frequency of LEV resistant S168T in Haemonchus spp., coupled with BZ resistance (beta-tubulin isotype-1) and ITS-2 nemabiome. At this time, this constitutes the first multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing of LEV resistant H. contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp., confirming that LEV is still an important anthelmintic class for small ruminants in New South Wales. It is hoped that the mixed amplicon metabarcoding approach described herein, paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into concrete recommendations for the livestock sector.