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

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 primarily 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 cox1 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.