Multiple Drug Resistance in the canine hookworm Ancylostoma caninum: an Emerging Threat

In the past few years, diagnoses by veterinarians of recurrent canine hookworm infections have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in the parasite Ancylostoma caninum. To investigate this, we established three suspected-resistant and two susceptible A. caninum isolates in research dogs for further study. The egg hatch assay (EHA) and the larval development assay (LDA) were used for detecting resistance to benzimidazoles, and macrocyclic lactones, respectively. Resistance ratios ranged from 6.0 to >100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatments with fenbendazole, pyrantel and milbemycin oxime, reduction in fecal egg counts ranged from 64 to 86%, 0 to 2% and 58 to 92%, respectively. Deep amplicon sequencing of the isotype-1 beta-tubulin gene identified a high frequency of resistance-associated single nucleotide polymorphisms at codon 167 in the resistant isolates and clinical cases. These data conclusively demonstrate multiple anthelmintic resistance in A. caninum, and provide pivotal evidence that this is an emerging problem in the United States. Consequently, these findings should provide some concern to the global health community, as the scale-up of mass drug administration for soil-transmitted helminths (STH) is now placing similar selection pressures for benzimidazole resistance in human hookworms.