Benzimidazole (BZ) resistance in Haemonchus contortus, one of the most pathogenic blood-feeding gastrointestinal nematodes of small ruminants, represents a major challenge for sustainable parasite control. Benzimidazoles are proposed to bind to the isotype-1 β-tubulin subunit, inhibit microtubule polymerization, and disrupt essential cellular processes. Single nucleotide polymorphisms (F167Y, E198A, F200Y) in the isotype-1 β-tubulin gene are considered key markers of BZ resistance. To investigate the structural basis of BZ resistance, α/β-tubulin heterodimers from H. contortus (wild-type and BZ resistant variants) were modeled in silico and their interactions with albendazole (ABZ) were analyzed. Analyses revealed that ABZ weakens critical intradimeric contacts and prevents the transition from a curved to straight α/β-tubulin heterodimer conformation, whereas these interactions remain intact in resistant variants despite ABZ binding. To assess the functional effects of BZ binding, recombinant H. contortus isotype-1 α/β-tubulin heterodimers were expressed recombinantly in Leishmania tarentolae. Although the integration of the expression cassettes were confirmed, stable transgenic cell lines could not be established. The cells ceased proliferation, showed abnormal twitching, and expressed transcripts only transiently, indicating potential cytotoxic effects on the endogenous tubulin network. In parallel, deep-amplicon sequencing was used to quantify known and novel BZ-resistance associated polymorphisms in field samples from northeastern Germany. Alongside known polymorphisms, the novel E198G polymorphism in H. contortus and the F200Stop polymorphism in Trichostrongylus colubriformis were detected, both potentially associated with BZ resistance. Overall, these results integrate structural, functional, and genetic insights into BZ resistance in H. contortus and highlight the complexity of resistance development. The results underscore the importance of continuous monitoring, innovative control strategies, and polymorphism modeling. Together, these findings support the refinement of experimental models and contribute to improved strategies for the investigation and management of benzimidazole resistance in H. contortus.
