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 β-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 β-tubulin mutations were present in A. caninum, 49% and 67% for Q134H and F167Y, respectively. Mutation F167Y in the isotype-1 β-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 (IC