The detection of three independent benzimidazole resistance mutations in a single Ancylostoma caninum population from a Golden Retriever breeding kennel in Sao Paulo, Brazil

Multiple anthelmintic drug resistance (MADR) is widespread in the canine hookworm Ancylostoma caninum in kenneled greyhounds in the USA and Australia with benzimidazole resistance being associated with the isotype-1 β-tubulin SNPs Q134H(CAA > CAT) and F167Y(TTC > TAC) in both cases. These two SNPs have also been found to be widespread, often at high frequency, in A. caninum populations in pet dogs across the USA but the situation in domestic dogs in other countries is currently unknown. Here, we report the identification of a single A. caninum population, from a Golden Retriever breeding kennel in São Paulo Brazil, which contained three different benzimidazole resistance SNPs; Q134H(CAA > CAT), F167Y(TTC > TAC), and F200Y(TTC > TAC). These SNPs were confirmed by three different methodologies; allele-specific qPCR, Illumina and Oxford Nanopore Technologies (ONT) deep amplicon sequencing. This is the second report of the F200Y(TTC > TAC) SNP in A. caninum from Brazil, suggesting it may be established in this region and illustrating need to consider regional differences when applying molecular diagnostic tests of anthelmintic resistance in canine hookworms. Analysis of the ONT sequence data of the near full-length isotype-1 β-tubulin showed that these three separate canonical resistance SNPs were present on different haplotypes suggesting their genetic independence. The ONT sequencing also detected two additional, previously unreported, non-synonymous SNPs - S73N(AGT > AAT) and V368A(GTT > GCT) - whose potential role in benzimidazole resistance is currently unknown. There was a high degree of variance in the amplicon sequencing data between replicate PCRs illustrating the challenges for accurately determining allele frequencies from archived fecal samples. In conclusion, the identification of three independent benzimidazole resistance mutations in a single A. caninum population suggests a high risk of anthelmintic resistance emergence in high transmission and drug selection environments such as kennels. It also suggests regional differences in the suitability of specific molecular diagnostic tests to detect anthelmintic resistance in canine hookworms.