Departamento de Patobiología, Unidad de Enfermedades Parasitarias, Facultad de Veterinaria, UDELAR. Av. Lasplaces 1620, CP 11600 Montevideo, Uruguay.
Departamento de Patobiología, Unidad de Enfermedades Parasitarias, Facultad de Veterinaria, UDELAR. Av. Lasplaces 1620, CP 11600 Montevideo, Uruguay.
Ticks Tick Borne Dis. 2021 Sep;12(5):101747. doi: 10.1016/j.ttbdis.2021.101747. Epub 2021 May 19.
The resistance of Rhipicephalus microplus to acaricides is a serious control problem, so its early diagnosis by a molecular technique is important. This study aims to develop a multiplex allele-specific polymerase chain reaction (PCR) for single-nucleotide polymorphisms (SNPs) in the para-sodium channel gene and in the GABA-Cl gene, associated with pyrethroids (cypermethrin and flumethrin) and fipronil resistance, respectively. We used 22 tick field isolates from farms with tick control problems (sampling convenience). These farms are located in departments of northern Uruguay. Three mutations in the sodium channel gene (Domain II S4-5: C190A and G215T; domain III S6: T2134A) and one in the GABA-Cl gene (A286S/L: CG856CC/TG) were studied. Mutations G215T and T213A were not detected. In all field isolates, the resistant allele (R) for C190A mutation (knockdown resistance, kdr) was detected, mainly in heterozygous individuals (SR) (11.1% to 86.7%). The highest incidence of the kdr mutant allele occurred in the Tacuarembó tick field isolates, where on 7 out of 10 farms >30% of individuals were SR and on one farm > 30% of individuals were RR. The next highest was Artigas (half of farms had>30% SR individuals and a quarter had >30% RR individuals). The resistance to dieldrin locus (rdl) mutation (CG856CC/TG) was absent in five field isolates. The highest incidenceof the mutant allele was observed in ticks from farms in Rivera (all farms had SR in >30% of individuals and two farms had RR in >12.5 and >16.7% of individuals) followed by farms in Tacuarembó (3 of 10 farms had >30% SR and 2 with >30% RR). Less than half of the farms had rdl in homozygous individuals. No significant association was observed between phenotypic bioassays and the rdl resistance allele. Several field isolates were phenotypically susceptible to the presence of the rdl allele. Several causes are possible (bioassay sensitivity, discriminating concentration). Individuals with simultaneous kdr and rdl mutations were present in 17 field isolates, and their frequency varied between 0.06% and 60%. Genotypic analysis shows that tick resistance to both acaricides, especially pyrethroids, is a serious problem. It is important to monitor the resistance using molecular techniques to plan efficient control measures. This is the first report describing kdr and rdl detection in R. microplus in Uruguay.
牛皮蝇蛆对杀螨剂的抗性是一个严重的控制问题，因此早期通过分子技术进行诊断非常重要。本研究旨在开发一种用于检测钠离子通道基因和 GABA-Cl 基因单核苷酸多态性（SNP）的多重等位基因特异性聚合酶链反应（PCR），这些 SNP 与拟除虫菊酯（氯菊酯和氟氯苯菊酯）和氟虫腈抗性有关。我们使用了 22 个来自有蜱虫控制问题的农场的蜱虫田间分离株（采样方便）。这些农场位于乌拉圭北部的各个部门。研究了钠离子通道基因（域 II S4-5：C190A 和 G215T；域 III S6：T2134A）中的三个突变和 GABA-Cl 基因中的一个突变（A286S/L：CG856CC/TG）。未检测到突变 G215T 和 T2134A。在所有的田间分离株中，均检测到钠离子通道基因 C190A 突变（击倒抗性，kdr）的抗性等位基因（R），主要存在于杂合个体（SR）中（11.1%至 86.7%）。在 Tacuarembó 蜱田间分离株中，kdr 突变体等位基因的发生率最高，在 10 个农场中的 7 个农场中，超过 30%的个体为 SR，在一个农场中，超过 30%的个体为 RR。其次是 Artigas（一半的农场中，超过 30%的个体为 SR，四分之一的农场中，超过 30%的个体为 RR）。五个田间分离株中未检测到狄氏剂抗性基因（rdl）突变（CG856CC/TG）。在 Rivera 的农场蜱虫中观察到突变等位基因的最高发生率（所有农场中，超过 30%的个体为 SR，两个农场中，超过 12.5%和超过 16.7%的个体为 RR），其次是 Tacuarembó 的农场（10 个农场中有 3 个农场的 SR 超过 30%，2 个农场的 RR 超过 30%）。不到一半的农场中，rdl 等位基因在纯合个体中存在。表型生物测定法与 rdl 抗性等位基因之间未观察到显著相关性。一些田间分离株对 rdl 等位基因的存在表现出表型敏感性。可能存在多种原因（生物测定法的敏感性、区分浓度）。在 17 个田间分离株中存在同时具有 kdr 和 rdl 突变的个体，其频率在 0.06%至 60%之间变化。基因型分析表明，蜱对这两种杀螨剂，尤其是拟除虫菊酯的抗性是一个严重的问题。使用分子技术监测抗性以制定有效的控制措施非常重要。这是首次在乌拉圭描述牛皮蝇蛆中的 kdr 和 rdl 检测。
