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

血绦虫是反刍动物中致病性最高的胃肠道线虫属，其食性为食血。它存在于绵羊、山羊和牛的胃中。几十年来，在世界各地的集约化反刍动物生产系统中，反复的驱虫治疗一直是维持畜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产生耐药性，包括50多年前使用的苯并咪唑（BZ）类药物。抗bz虫种群和大部分弯曲血蜱高度流行。H. contortus和其他GIN的β -微管蛋白同型1基因编码序列的特异性变化与bz -抗性（BZ-R）有关。通过cDNA序列分析，确定了弯叶鼠β -微管蛋白同型基因Phe167Tyr、Glu198Ala和Phe200Tyr三个位点的氨基酸变化与BZ-R相关。然而，最近，我们和其他人在bz抗性人群中观察到其他交换，包括Glu198Leu。到目前为止，仅证实了Phe200Tyr单核苷酸多态性（SNP）的功能相关性，而其他SNP的重要性仅从与表型的相关性中得出结论。GIN β -微管蛋白的药物结合特性以及与BZ-R相关的snp相关的结构变化尚未阐明。因此，我们将研究所有已知的BZ-R相关β -微管蛋白snp的功能和结构后果。这将通过异种表达（酵母和/或秀丽隐杆线虫），通过微管聚合抑制研究，药物靶点亲和力分析和H.扭曲β -微管蛋白SNPs的x射线晶体学结构表征来完成。通过这个项目，我们将为Haemonchus spp中BZ-R相关多态性的实际功能和表型相关性提供先进和详细的见解。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的实际患病率是未知的。我们将确定这些遗传变化在当地血蜱种群中的流行程度，并通过体内和体外抗性测试提供这些各自种群的表型特征。为了开发适合莫桑比克农业和畜牧业条件的适用于田间的可持续蠕虫防治方法，将在马普托省的7个小型反刍动物养殖场长期采用一种与传统整体处理方法相比的有针对性的选择性处理方法。这将伴随着目前Haemonchus种群BZ-R相关β -微管蛋白snp的分子监测。这些调查将使我们全面了解处理和农场管理对BZ-R演变的相互依赖性，以及如何在田间条件下最好地预防BZ-R。

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.