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Characterisation of β-tubulin isotypes in Uncinaria stenocephala and implications for benzimidazole resistance in hookworms

Thomas Stocker,J. Šlapeta

Published 2025 in bioRxiv

ABSTRACT

Uncinaria stenocephala is a widespread hookworm of dogs across Europe, Canada, southern Australia, and other temperate regions, where it often outnumbers infections caused by Ancylostoma caninum. Although a putative β-tubulin isotype-1 mutation associated with resistance has been detected in U. stenocephala, clinical resistance to benzimidazoles has not yet been confirmed. Benzimidazole resistance is linked to single nucleotide polymorphisms (SNPs) in the β-tubulin isotype-1 gene; however, the β-tubulin genes of U. stenocephala have not been fully characterised. We aimed to identify β-tubulin genes and confirm the coding sequences for key residues (Q134, F167, E198, and F200) in the β-tubulin isotype-1 gene of the U. stenocephala genome. Two U. stenocephala specimens were subjected to Illumina sequencing, and species identity was confirmed through morphological and molecular analysis using ITS rDNA and cox-1 markers. Genome assembly revealed the presence of β-tubulin isotype-1 (10 exons) and isotype-2 (9 exons), both homologous to β-tubulins from other hookworms (A. caninum, A. ceylanicum, A. duodenale and Necator americanus). The β-tubulin isotype-1 protein sequence of U. stenocephala contained two variable residues (S37Q and G441A) compared to other hookworm sequences. While the isotype-2 protein sequence was conserved among Ancylostoma species, U. stenocephala exhibited six distinct polymorphisms (E39D, T40S, N115S, L130I, A287S, T439G). The benzimidazole-susceptible residues (Q134, F167, E198, F200) were present in the β-tubulin isotype-1 protein sequence. Characterisation of the complete coding regions of β-tubulin isotypes 1 and 2 enables population-level screening for benzimidazole resistance-associated SNPs and provides a foundation for future epidemiological studies in U. stenocephala. Highlights Uncinaria stenocephala outnumbers Ancylostoma caninum in temperate regions globally β-tubulin isotypes-1 and -2 were fully characterised in Uncinaria stenocephala Key benzimidazole-susceptible residues were confirmed in β-tubulin isotype-1 β-tubulin isotypes-1 and -2 enables SNP screening and resistance surveillance efforts

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