von Samson-Himmelstjerna G, Walsh T K, Donnan A A, Carrière S, Jackson F, Skuce P J, Rohn K, Wolstenholme A J
Institute for Parasitology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.
Parasitology. 2009 Mar;136(3):349-58. doi: 10.1017/S003118200800543X. Epub 2009 Jan 21.
Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in beta-tubulin genes have been associated with BZ-resistance. In the present study, we investigated beta-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000-10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the 'egg hatch test', and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.
苯并咪唑（BZ）被广泛用于治疗人和动物的寄生线虫感染，但在兽医寄生虫中耐药性普遍存在。β-微管蛋白基因中的几种多态性与BZ耐药性有关。在本研究中，我们调查了18株对噻苯达唑具有不同耐药水平的捻转血矛线虫分离株的β-微管蛋白同型1序列。在耐药分离株中频率显著增加的唯一多态性是密码子200处的TTC突变为TAC。使用实时PCR（使用来自100个三期幼虫，即L3的DNA）和焦磷酸测序（来自1000 - 10000个L3的DNA）来测量这些分离株密码子200处的等位基因频率，结果相似；药物敏感性随着TAC频率的增加而降低。焦磷酸测序还用于测量第167和198位的等位基因频率。我们表明，这种测量足以评估大多数捻转血矛线虫分离株的BZ耐药状态。在不同实验室进行的实时PCR和焦磷酸测序结果之间的一致性表明，这些工具适用于捻转血矛线虫BZ耐药性的常规诊断。分子方法比“虫卵孵化试验”更敏感，并且比当前的体内或体外抗蠕虫药耐药性检测方法耗时更少。因此，它们为农场常规分子耐药性检测提供了一个切实可行的选择。
