Stocker, T. | bioRxiv Science

bioRxiv2025

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 {beta}-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 {beta}-tubulin isotype-1 gene; however, the {beta}-tubulin genes of U. stenocephala have not been fully characterised. We aimed to identify {beta}-tubulin genes and confirm the coding sequences for key residues (Q134, F167, E198, and F200) in the {beta}-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 {beta}-tubulin isotype-1 (10 exons) and isotype-2 (9 exons), both homologous to {beta}-tubulins from other hookworms (A. caninum, A. ceylanicum, A. duodenale and Necator americanus). The {beta}-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 {beta}-tubulin isotype-1 protein sequence. Characterisation of the complete coding regions of {beta}-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{beta}-tubulin isotypes-1 and -2 were fully characterised in Uncinaria stenocephala Key benzimidazole-susceptible residues were confirmed in {beta}-tubulin isotype-1 {beta}-tubulin isotypes-1 and -2 enables SNP screening and resistance surveillance efforts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/664546v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1598c5corg.highwire.dtl.DTLVardef@df5f0org.highwire.dtl.DTLVardef@8d8ec7org.highwire.dtl.DTLVardef@198b768_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Widespread occurrence of benzimidazole resistance SNPs in the canine hookworm, Ancylostoma caninum, in Australia

Canine hookworm (Ancylostoma caninum), a gastrointestinal nematode of domestic dogs, principally infects the small intestine of dogs and has the potential to cause zoonotic disease. In greyhounds and pet dogs in the USA, A. caninum has been shown to be resistant to multiple anthelmintics. We conducted a molecular survey of benzimidazole resistance in A. caninum from dogs at veterinary diagnostic centers in Australia and New Zealand. First, we implemented an internal transcribed spacer (ITS)-2 rDNA deep amplicon metabarcoding sequencing approach to ascertain the species of hookworms infecting dogs in the region. Then, we evaluated the frequency of the canonical F167Y and Q134H isotype-1 {beta}-tubulin mutations, which confer benzimidazole resistance, using the same sequencing approach. The most detected hookworm species in diagnostic samples was A. caninum (90%; 83/92); the related Northern hookworm (Uncinaria stenocephala) was identified in 11% (10/92) of the diagnostic samples. There was a single sample with coinfection by A. caninum and U. stenocephala. Both isotype-1 {beta}-tubulin mutations were present in A. caninum, 49% and 67% for Q134H and F167Y, respectively. Mutation F167Y in the isotype-1 {beta}-tubulin mutation was recorded in U. stenocephala for the first known time. Canonical benzimidazole resistance codons 198 and 200 mutations were absent. Egg hatch assays performed on a subset of the A. caninum samples showed significant correlation between 50% inhibitory concentration (IC50) to thiabendazole and F167Y, with an increased IC50 for samples with >75% F167Y mutation. We detected 14% of dogs with >75% F167Y mutation in A. caninum. Given that these samples were collected from dogs across various regions of Australia, the present study suggests that benzimidazole resistance in A. caninum is widespread. Therefore, to mitigate the risk of resistance selection and further spread, adoption of a risk assessment-based approach to limit unnecessary anthelmintic use should be a key consideration for future parasite control.

microbiology↗