Department of Veterinary Pharmacy, Clinics and Comparative Medicine, School of Veterinary Medicine and Animal Resources, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P. O. Box 7062, Kampala, Uganda; Research Center for Ticks and Tick-borne Disease Control (RTC), College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P. O. Box 7062, Kampala, Uganda; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-eCho, Obihiro 080-8555, Hokkaido, Japan.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-eCho, Obihiro 080-8555, Hokkaido, Japan; United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan.
Pestic Biochem Physiol. 2017 Nov;143:181-190. doi: 10.1016/j.pestbp.2017.07.009. Epub 2017 Aug 5.
We previously reported emergence of super synthetic pyrethroid (SP) resistant Rhipicephalus (Boophilus) decoloratus ticks in Uganda. This study investigated the genetic basis of phenotypic resistance against SP in R. (B.) decoloratus and sought to identify novel diagnostic mutations for rapid detection of SP resistance in the above tick species. Genomic DNA was extracted from pooled larvae of 20 tick populations (19 of known SP susceptibility and 1 unknown susceptibility). The voltage sensitive sodium channel (VSSC) domain II S4-5 linker (SP target) and partial carboxylesterase (SP metabolizing enzyme) genes were amplified by PCR, cloned and sequenced. The resultant sequences were analyzed to determine single nucleotide polymorphisms (SNPs) associated with phenotypic resistance in the various tick populations investigated. Novel SNPs that introduced Eco RI and Eco RII restriction sites in carboxylesterase gene were identified in silco and validated with restriction fragment length polymorphism (RFLP) against 18 tick populations of known SP susceptibility. The study identified a super knock down resistance (kdr) mutation T58C in R. (B.) decoloratus VSSC associated with stable SP resistance. We further identified multiple nonsynonymous mutations in carboxylesterase of SP resistant ticks; one of which conferred novel EcoRII (G195C) restriction site for PCR-RFLP detection of SP resistance. In conclusion, this study is the first to report super kdr mutation in sodium channel domain II and multiple mutations in carboxylesterase genes that may concurrently mediate stable resistance against synthetic pyrethroids in R. (B.) decoloratus ticks from Uganda. The Eco RII based PCR-RFLP is a useful tool for rapid detection of stable SP resistant R. (B.) decoloratus ticks.
我们之前曾报道过，在乌干达出现了对超级合成拟除虫菊酯（SP）具有抗性的 Rhipicephalus（Boophilus）decoloratus 蜱。本研究调查了 R.（B.）decoloratus 对 SP 表型抗性的遗传基础，并试图鉴定用于快速检测上述蜱种中 SP 抗性的新型诊断突变。从 20 个蜱种群（19 个已知对 SP 敏感，1 个未知敏感）的幼虫中提取基因组 DNA。通过 PCR 扩增电压敏感钠通道（VSSC）结构域 II S4-5 接头（SP 靶标）和部分羧酸酯酶（SP 代谢酶）基因，然后克隆和测序。分析所得序列以确定与所研究的各种蜱种群中表型抗性相关的单核苷酸多态性（SNP）。在羧酸酯酶基因中引入 Eco RI 和 Eco RII 限制位点的新型 SNP 是在计算机中鉴定的，并通过对 18 个已知对 SP 敏感的蜱种群的限制片段长度多态性（RFLP）进行验证。研究发现 R.（B.）decoloratus VSSC 中与稳定 SP 抗性相关的超级击倒抗性（kdr）突变 T58C。我们进一步在 SP 抗性蜱的羧酸酯酶中鉴定了多个非同义突变；其中之一赋予了新型 EcoRII（G195C）限制位点，用于 PCR-RFLP 检测 SP 抗性。总之，本研究首次报道了钠离子通道 II 结构域中的超级 kdr 突变和羧酸酯酶基因中的多个突变，这些突变可能同时介导乌干达 R.（B.）decoloratus 蜱对合成拟除虫菊酯的稳定抗性。基于 Eco RII 的 PCR-RFLP 是一种快速检测稳定 SP 抗性 R.（B.）decoloratus 蜱的有用工具。
