Functional analysis and evidence based prevention approaches of Benzimidazole-resistance in Haemonchus spp.

血矛线虫是反刍动物胃肠道线虫中致病性最强的一种。它存在于绵羊、山羊和牛的皱胃中。几十年来，在世界各地的集约化反刍动物生产系统中，反复驱虫治疗一直是维持羊群健康和生产力的基础。这导致蠕虫种群对最常用的驱虫药（包括超过50年历史的苯并咪唑（BZ）类药物）产生耐药性。GIN和大多数捻转血矛线虫的BZ抗性蠕虫种群高度流行。H. β-微管蛋白同种型1基因编码序列的特异性变化。扭转和其他GIN与BZ抗性（BZ-R）相关。根据cDNA序列分析，β-微管蛋白1基因的三个氨基酸位置，即Phe 167 Tyr，Glu 198 Ala和Phe 200 Tyr的变化已被确定为与H中BZ-R相关。扭曲然而，最近其他人和我们在BZ抗性种群中观察到了包括Glu 198 Leu在内的其他交换。到目前为止，只有Phe 200 Tyr单核苷酸多态性（SNP）的功能相关性已被证明，而其他SNP的重要性仅从与表型的相关性中得出。GIN的β-微管蛋白中与BZ-R相关的SNP的药物结合特征以及结构变化尚未阐明。在此，我们将研究所有目前已知的BZ-R相关β-微管蛋白SNP的功能和结构后果。这将通过异源表达（在酵母和/或C. elegans），通过微管聚合抑制研究、药物-靶标亲和性分析和X射线晶体学结构表征H.捻转β-微管蛋白SNP。通过这个项目，我们将提供先进的和详细的洞察力的实际功能和表型相关的BZ-R相关的多态性在血矛线虫属。对于大多数地理区域，包括马普托省，BZ-R相关β-微管蛋白SNP的实际流行率是未知的。我们将确定这些遗传变化在当地血矛线虫种群中的患病率，并使用体内和体外耐药性试验提供这些相应种群的表型表征。为了开发适合莫桑比克农业和畜牧业条件的可持续蠕虫防治技术，将在马普托省的7个小型反刍动物养殖场长期采用与传统全草处理相比的有针对性的选择性处理方法。这将伴随着BZ-R相关β-微管蛋白SNP的分子监测，目前的血矛线虫种群。这些调查将使我们全面了解BZ-R演变的治疗和农场管理相互依赖性，以及如何在田间条件下最好地预防。

Due to its hematophagous feeding habit, Haemonchus is the most pathogenic ruminant gastro-intestinal nematode (GIN) genus. It resides in the abomasum of sheep, goats and cattle. For decades, repeated anthelmintic treatments have been the basis to maintain the flock health and productivity in intensive ruminant production systems all around the world. This has led to the evolution of worm populations resistant to the most often used anthelmintics including the over 50 years old benzimidazole (BZ) drug class. BZ-resistant worm populations of GINs and most of all of Haemonchus contortus are highly prevalent. Specific changes at the coding sequence of the beta-tubulin isotype 1 gene of H. contortus and other GIN are associated with BZ-resistance (BZ-R). Based on cDNA sequence analyses, amino acid changes at three positions, i.e. Phe167Tyr, Glu198Ala and Phe200Tyr, of the beta-tubulin isotype 1 gene have been identified to correlate with BZ-R in H. contortus. However, recently additional exchanges, including Glu198Leu, have been observed in BZ-resistant populations by others and us. So far only the functional relevance of the Phe200Tyr single nucleotide polymorphism (SNP) has been demonstrated while importance of other SNPs is only concluded from correlation with the phenotype. The drug-binding characteristics as well as the structural changes correlated with the BZ-R associated SNPs in the beta-tubulin of GIN have not been elucidated. Herein, we will thus investigate the functional and structural consequences of all currently known BZ-R associated beta-tubulin SNPs. This will be done by heterologous expression (in yeast and/or C. elegans), through microtubule polymerization inhibition studies, drug-target affinity analyses and by X-ray crystallographic structural characterization of H. contortus beta-tubulin SNPs. Through this project we will provide advanced and detailed insight of the actual functional and phenotypic relevance of the BZ-R associated polymorphisms in Haemonchus spp..For most geographical regions, including the Maputo province, the actual prevalence of BZ-R associated beta-tubulin SNPs is unknown. We will establish the prevalence of these genetic changes in the local Haemonchus populations and provide a phenotypic characterization of these respective populations using in vivo and in vitro resistance testing. To develop field applicable sustainable worm control, adapted to the Mozambican farming and husbandry conditions, a targeted-selective-treatment approach in comparison to classical whole heard treatments will be employed over an extended time period on 7 small ruminants farms each in the Maputo province. This will be accompanied by molecular monitoring of BZ-R associated beta-tubulin SNPs the present Haemonchus populations. These investigations will provide us with a comprehensive understanding of treatment and farm management interdependencies on BZ-R evolution and how it can best be prevented under field conditions.