Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

Abstract

Background: The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum.

Methods: Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene.

Results: Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases.

Conclusions: These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

Keywords: Ancylostoma caninum; Anthelmintics; Canine health; Hookworms; Resistance.

Fig. 1

Dose-response curves for the Egg Hatch Assay. Initial assays were performed singly using ETCR, Tara, Worthy and Worthy 1.1F. Subsequent assays were performed in triplicate using the Barrow 1.0 and Worthy 4.1F3P isolates with three replicates per concentration. Curves were generated using the variable slope nonlinear regression model analysis in GraphPad 8

Fig. 2

Dose-response curves for the Larval Development Assay. Initial assays were performed singly using ETCR 1.0 Lacy and Worthy 1.0. Subsequent assays were performed in triplicate using Barrow 1.0 and Worthy 4.1F3P isolates with two replicates per concentration. Curves were generated using the variable slope nonlinear regression model analysis in GraphPad 8

Fig. 3

Fecal egg counts (FEC) over the course of infection of a dog infected with the Tara isolate. Treatment with pyrantel was administered on day 66 (23 February 2018) and post-treatment FEC was performed on day 10 post-treatment

Fig. 4

Average of fecal egg counts (FEC) over the course of infection of two dogs infected with larvae from the second passage of the Worthy isolate, with a treatment event with fenbendazole on the first passage (Worthy 2.1F). Treatment with pyrantel was administered on day 55 (25 October 2018) and post-treatment FEC was performed on day 13 post-treatment