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.

抗驱虫药寄生线虫（anthelmintic-resistant parasitic nematodes）对全球畜牧业的可持续生产构成严重威胁。早期检测抗驱虫药药性的出现，对于通过确定最优有效药物种类以最小化生产损失至关重要。尽管针对抗驱虫药药性的分子机制已开展多年研究，但目前尚无基于分子的检测工具可在田间实际环境中诊断刚出现的驱虫药抗性。本研究提出一种混合深度扩增子测序（mixed deep amplicon sequencing）方法，用于检测血矛线虫属（Haemonchus spp.）中左咪唑（Levamisole，LEV）抗性相关S168T突变的频率，同时结合苯并咪唑（Benzimidazole，BZ）抗性（β微管蛋白亚型1，beta-tubulin isotype-1）与ITS-2线虫宏基因组（ITS-2 nemabiome）分析。截至目前，该方法为首款针对兽医学重要性蠕虫的多药物、多物种分子诊断技术。在我们测试的澳大利亚田间分离菌株中，多数绵羊和山羊源血矛线虫属种群均可检测到S168T突变，但其突变频率极少超过16%——该临界阈值为我们基于抗左咪唑捻转血矛线虫（Haemonchus contortus，H. contortus）GWBII株的全基因组测序结果任意设定。总体而言，苯并咪唑抗性在血矛线虫属中更为普遍，这证实左咪唑类药物仍是新南威尔士州小反刍动物的重要驱虫药种类。本研究所述的混合扩增子宏条形码（mixed amplicon metabarcoding）技术，有望为大规模测序作为田间监测技术的应用铺平道路，其结果可转化为面向畜牧行业的具体实操指导建议。