Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, P.O. Box 7036, Uppsala, Sweden.
Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, P.O. Box 7036, Uppsala, Sweden.
Int J Parasitol Drugs Drug Resist. 2018 Aug;8(2):278-286. doi: 10.1016/j.ijpddr.2018.04.005. Epub 2018 Apr 26.
Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT). Initially, we designed dyf-7 primers from a worldwide collection of adult Haemonchus contortus DNA. With the sequence data, we created a haplotype network. We then optimised and used the same sets of primers and probes in a droplet digital PCR (ddPCR) assay for precise quantification of dyf-7 allele frequencies in pre- and post-anthelmintic treatment faecal larval cultures. The fractional abundance (FA) of the mutant SNP was within the range 7.8 and 31%. However, the FA was generally stable in samples collected from the same farms, even though they were obtained on different occasions up to 25 months apart. There was also no indication that the level of IVM resistance as measured by the faecal egg count reduction test was higher on farms with high FA. Furthermore, by comparing FA in samples from the same farms pre- and post-IVM treatment, we found no evidence of a correlation between dyf-7 and level of IVM resistance. Based on these results, dyf-7 is not a suitable marker for field testing of IVM resistance in H. contortus.
在小反刍动物中，旋毛虫（Haemonchus contortus）对伊维菌素（IVM）的耐药性是一个日益严重的问题。分子诊断学的应用可以早期发现 IVM 耐药性，从而限制耐药株的传播。最近，一种候选基因，即染料填充蛋白（dyf-7）基因，被认为是 IVM 耐药性的标志物。在本研究中，我们对 A141G 和 G153T 单核苷酸多态性（SNP）在瑞典绵羊养殖场幼虫培养物中的适用性进行了严格评估，这些培养物涉及几个已经通过粪便卵减少试验（FECRT）确定耐药性的分离株。最初，我们从世界各地的旋毛虫成虫 DNA 中设计了 dyf-7 引物。根据序列数据，我们创建了一个单倍型网络。然后，我们在液滴数字 PCR（ddPCR）检测中优化并使用相同的引物和探针组，精确量化预驱虫和后驱虫粪便幼虫培养物中 dyf-7 等位基因频率。突变 SNP 的分数丰度（FA）在 7.8%至 31%之间。然而，即使在相隔长达 25 个月的不同时间从同一农场采集的样本中，FA 通常也是稳定的。此外，FA 水平与粪便卵减少试验中测量的 IVM 耐药性水平之间也没有相关性。进一步比较同一农场的 IVM 处理前后的 FA 值，我们没有发现 dyf-7 与 IVM 耐药性水平之间存在相关性的证据。基于这些结果，dyf-7 不适用于现场测试 H. contortus 对 IVM 的耐药性。
