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

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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.