Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
ISP, INRA, Université Tours, UMR1282, 37380, Nouzilly, France.
Int J Parasitol Drugs Drug Resist. 2021 Apr;15:168-176. doi: 10.1016/j.ijpddr.2021.03.002. Epub 2021 Mar 26.
The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing "resistant" allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous "resistant" genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the "resistant" allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.
旋毛虫是小反刍动物中最普遍和最具致病性的寄生虫之一。尽管通常使用驱虫药进行控制，但寄生虫对药物的耐药性已成为畜牧业生产中的一个主要问题。虽然旋毛虫中苯并咪唑类药物耐药性的分子检测已经很成熟，但检测莱克多巴胺耐药性的分子工具和方案远不够先进。hco-acr-8 基因编码一个关键的乙酰胆碱敏感亚基，使受体对莱克多巴胺敏感。在这里，我们报告了一种数字 PCR 检测方法的开发，作为一种分子工具来检测先前与莱克多巴胺耐药性相关的 hco-acr-8 中的 63 bp 缺失。对单个成年旋毛虫的 Sanger 测序产生了 56 个高质量的共识序列，围绕着包含缺失的区域。根据测序数据，设计了新的引物和探针，并使用一种新的数字 PCR 检测方法对基因组 DNA 样本中缺失的“耐药”等位基因进行了定量验证。对六个表型描述的分离株（n=60）和两个瑞典绵羊养殖场（n=30）的单个成虫进行了测试，在这些养殖场中，莱克多巴胺是有效的。尽管在耐药和敏感参考分离株之间发现了基因型频率的显著差异（p=0.01），但在敏感分离株以及一些瑞典旋毛虫样本中，均观察到纯合“耐药”基因型大量存在。此外，在七个瑞典绵羊养殖场中，在莱克多巴胺根据粪便卵囊计数减少试验结果完全有效的情况下，在进行莱克多巴胺治疗之前（n=7）和之后（n=6）收集的幼虫培养场样品进行了检测，以评估每个样品中“耐药”等位基因的频率。在治疗前的样本中，缺失的频率范围为 35%至 80%，而在治疗后的样本中则没有检测到可扩增的旋毛虫基因组 DNA。这些数据表明，在单个旋毛虫和田间幼虫培养物中，hco-acr-8 中的 63 bp 缺失频率相对较高，并对在绵羊养殖场中使用 hco-acr-8 缺失作为莱克多巴胺耐药性检测的分子标记的实用性提出了质疑。
