Hodgkinson J E, Clark H J, Kaplan R M, Lake S L, Matthews J B
Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.
Int J Parasitol. 2008 Aug;38(10):1149-60. doi: 10.1016/j.ijpara.2008.02.001. Epub 2008 Feb 20.
Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of benzimidazole (BZ) anthelmintics in horses for more than 40 years and widespread drug resistance in the field, the mechanisms of resistance to this drug class in cyathostomins are not fully understood. The results presented here constitute a detailed comparison of beta tubulin gene mutations and mRNA transcript levels in populations of BZ-susceptible (BZ-S) and -resistant (BZ-R) cyathostomins. Full-length cDNA sequences were generated from individual parasites of four (n=24) and two (n=19) cyathostomin species for isotypes 1 and 2, respectively. Levels of intra- and inter-specific nucleotide sequence variation were comparable with previous findings and single amino acid substitutions were observed at several locations. On comparison of BZ-S and BZ-R parasites, differences were consistently observed at only two sites, codons 167 and 200 of the beta tubulin isotype 1 gene. Four populations of parasites were genotyped at these two loci by pyrosequencing; one that was fenbendazole (FBZ)-sensitive (FBZ-rS), two that were FBZ-resistant (FBZ-R1 and -R2) and one that was oxibendazole-resistant (OBZ-R), as previously assessed by faecal egg count reduction tests. This analysis revealed statistically significant differences between FBZ-rS and FBZ-R populations at both loci and this was highly significant for codon 167. For the OBZ-R population, the only significant difference compared with the FBZ-rS population was observed at codon 200. These observations suggest that mutations at codons 167 and 200 are important in BZ resistance and raise the possibility that selection at different loci may occur in FBZ- and OBZ-resistant parasites. Multiple parasites (n=158) were genotyped for both codons 167 and 200, the majority of which showed homozygous 'resistant' mutations at one locus only and none showed homozygous 'resistant' genotypes at both loci. No significant differences in mRNA levels of beta tubulin isotypes 1 and 2 were observed between the FBZ-rS and FBZ-R1 populations.
圆线虫被认为是马的主要寄生性病原体。尽管苯并咪唑（BZ）类驱虫药已在马身上使用了40多年，且在野外普遍存在耐药性，但圆线虫对这类药物的耐药机制尚未完全了解。本文给出的结果是对BZ敏感（BZ-S）和耐药（BZ-R）圆线虫群体中β微管蛋白基因突变和mRNA转录水平的详细比较。分别从4种（n = 24）和2种（n = 19）圆线虫物种的单个寄生虫中生成了1型和2型的全长cDNA序列。种内和种间核苷酸序列变异水平与先前的研究结果相当，并且在几个位置观察到了单个氨基酸替换。在比较BZ-S和BZ-R寄生虫时，仅在β微管蛋白1型基因的167和200密码子这两个位点始终观察到差异。通过焦磷酸测序对四个寄生虫群体在这两个位点进行基因分型；一个群体对芬苯达唑（FBZ）敏感（FBZ-rS），两个群体对FBZ耐药（FBZ-R1和-R2），还有一个群体对奥苯达唑耐药（OBZ-R），这些群体先前已通过粪便虫卵计数减少试验进行了评估。该分析揭示了FBZ-rS和FBZ-R群体在两个位点上均存在统计学上的显著差异，对于167密码子而言差异极为显著。对于OBZ-R群体，与FBZ-rS群体相比，仅在200密码子处观察到显著差异。这些观察结果表明，167和200密码子处的突变在BZ耐药中起重要作用，并增加了在FBZ和OBZ耐药寄生虫中可能在不同位点发生选择的可能性。对167和200密码子对多个寄生虫（n = 158）进行了基因分型，其中大多数仅在一个位点显示纯合的“抗性”突变，没有一个在两个位点均显示纯合的“抗性”基因型。在FBZ-rS和FBZ-R1群体之间未观察到β微管蛋白1型和2型mRNA水平的显著差异。
