Arafa Waleed M, Holman Patricia J, Craig Thomas M
Department of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, 62512, Egypt.
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, 77843-4467, USA.
Parasitol Res. 2017 Feb;116(2):797-807. doi: 10.1007/s00436-016-5357-y. Epub 2016 Dec 28.
Haemonchus contortus isolates were evaluated for benzimidazole (BZ) resistance or susceptibility by allele-specific PCR based on β-tubulin isotype 1 gene polymorphisms at the F167Y, E198A, and F200Y sites. Two isolates, one presumed susceptible from wild pronghorn antelope (PH) and one known to be resistant from goats (VM), were also assayed phenotypically for BZ resistance or susceptibility in the larval development assay (Drenchrite®). The BZ EC50 was 0.198 μM (intermediate between susceptible and weak resistant) for PH with critical well 5 (intermediate between susceptible and weak resistant) and 1.456 μM (intermediate weak resistant and resistant) for VM with critical well 8.5 (resistant). Genotypically, DNA extracted from pooled VM L3 larvae in the Drenchrite® wells with the highest BZ concentration was homozygous susceptible (SS) at the F167Y and E198A sites and homozygous resistant (RR) at the F200Y site by PCR, and sequence analysis bore this out. PH L3 larvae DNA from a control well (no BZ) was SS at all three sites by PCR, confirmed by sequence analysis. All single adult worm samples (N = 21) from PH, VM, Egypt goat (EG), and a Texas llama were SS at F167Y and E198A by PCR; however, only 3 PH worms and 1 EG worm were SS at F200Y. Three additional PH worms were RS and upon cloning two clones were identified as resistant by sequencing and two as susceptible. Clones from single adult worms VM, llama, and EG samples that were RR by PCR at F200Y were sequence verified as resistant. In this study, F200Y was the most frequently found genotypic marker for BZ resistance or susceptibility in the different Haemonchus isolates.
基于β-微管蛋白同型1基因在F167Y、E198A和F200Y位点的多态性，通过等位基因特异性PCR评估捻转血矛线虫分离株对苯并咪唑（BZ）的抗性或敏感性。还通过幼虫发育试验（Drenchrite®）对两个分离株进行了BZ抗性或敏感性的表型分析，一个是来自野生叉角羚（PH）的假定敏感株，另一个是来自山羊的已知抗性株（VM）。PH的BZ半数有效浓度（EC50）为0.198 μM（介于敏感和弱抗性之间），临界孔为5（介于敏感和弱抗性之间）；VM的BZ EC50为1.456 μM（介于中等弱抗性和抗性之间），临界孔为8.5（抗性）。从基因型上看，在Drenchrite®试验中，从BZ浓度最高的孔中收集的VM L3幼虫提取的DNA，通过PCR检测在F167Y和E198A位点为纯合敏感型（SS），在F200Y位点为纯合抗性型（RR），序列分析证实了这一点。来自对照孔（无BZ）的PH L3幼虫DNA通过PCR检测在所有三个位点均为SS，序列分析也证实了这一点。来自PH、VM、埃及山羊（EG）和德克萨斯骆驼的所有单个成虫样本（N = 21）通过PCR检测在F167Y和E198A位点均为SS；然而，在F200Y位点，只有3条PH虫和1条EG虫为SS。另外3条PH虫为RS，克隆后经测序鉴定，两个克隆为抗性，两个为敏感。通过PCR在F200Y位点为RR的单个成虫VM、骆驼和EG样本的克隆经序列验证为抗性。在本研究中，F200Y是不同捻转血矛线虫分离株中最常发现BZ抗性或敏感性的基因型标记。
