Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 β-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 β-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

中文翻译：

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开发用于胃肠道线虫田间种群苯并咪唑抗性等位基因频率分析的扩增子测序技术。

抗蠕虫病的胃肠蠕虫已成为绵羊和山羊健康状况不佳的主要原因。需要灵敏的和特定的分子标记来监测田间寄生虫种群中抗性的基因型频率。对苯并咪唑的胃肠道线虫耐药性是由同型1β-微管蛋白基因中三个位置之一的突变引起的。在没有抗其他广谱驱虫剂标记的情况下，这些提供了相关的研究实例。使用常规分子方法检查单个寄生虫可能无法确定野外线虫种群中这些单核苷酸多态性的普遍性。在确定寄生虫种群中的低水平抗药性时，这可能很费力并且缺乏敏感性。这里，我们报告了一种基于Illumina MiSeq深度扩增子测序平台的新型方法的开发，该方法可对小反刍类胃肠道线虫Teladorsagia circumcincta的同型1β-微管蛋白基因座进行测序，并确定苯并咪唑抗性突变的频率。我们通过评估序列表示偏倚，比较Illumina MiSeq和焦磷酸测序的结果，并将该方法应用于包含已知比例的抗性和易感幼虫的种群，验证了该方法。我们将该方法应用于从三个农场的一年多的母羊和羔羊采集的田间样品，每个样品都强调了绵羊管理和寄生虫控制方法的不同方面。结果表明，有机会根据选择压力建立假设，这些选择压力会导致采样日期，农场和母羊或羔羊之间的抗性等位基因频率差异，并考虑其遗传固定或其他方面的影响。这项研究提供了一种实用，准确，灵敏和可扩展的方法的概念证明，该方法可以在田野研究中确定胃肠道线虫中驱虫药抗药性突变的频率，并作为牲畜养殖者的管理工具。