Multiple drug resistance in hookworms infecting greyhound dogs in the USA

Abstract

Ancylostoma caninum is the most prevalent nematode parasite of dogs. We confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the USA. Cases of MDR hookworms appear to be highly overrepresented in greyhounds. The aims of this study were to evaluate the drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from greyhounds of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite treatment with anthelmintics, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA), respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 54 animals. Mean and median IC₅₀ and IC₉₅ values for the EHA were 5.3 μM, 3.6 μM, and 24.5 μM, 23.4 μM, respectively. For the LDA, the median IC₅₀ value was >1000 nM. These values ranged 62-81 times higher than our susceptible laboratory isolate. Only post-treatment samples were available. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools. Deep sequencing of the isotype 1 β-tubulin gene only revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples. These clinical, in vitro, and genetic data provide strong evidence that greyhound dogs in the USA are infected with MDR A. caninum at very high levels in prevalence and infection intensity.

Keywords: Ancylostoma caninum, hookworms; Deep-amplicon; Greyhounds; Multiple-drug resistance (MDR).

Graphical abstract

Fig. 1

Scatter dot plots of greyhound samples showing the log transformed Egg Hatch Assay (EHA) IC₅₀ (A) and IC₉₅ (B) values, and the Larval Development Assay (LDA) IC₅₀ (C) values for the benzimidazoles (BZs) and macrocyclic lactones (MLs), respectively. Each dark blue and light blue dot represent an assay performed on an individual or a pooled sample, respectively. The black dot represents the value of our susceptible laboratory isolate for reference (Jimenez Castro et al., 2019). Dose-responses were analyzed using the variable slope nonlinear regression model analysis contained in GraphPad 9.0.2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

The relative proportions of isotype-1 β-tubulin alleles encoding resistance conferring polymorphisms at F167Y vs wild type susceptible as measured by deep-amplicon sequencing in 70 samples from greyhounds that originated from 16 different locations in 8 different states (C). Only four samples had more than 3000 eggs, so we set the max at 3000 eggs because those outliers with large numbers would distort the overall chart (A). The average read depth is also reported (B).

Fig. 3

Scatterplots of EHA (A) IC₅₀ values (r = 0.48) or (B) IC₉₅ values (r = 0.54) vs. F167Y SNP frequency based on deep-amplicon sequencing. Only 15 samples had results from both assays. The gray dot represents a pooled sample. The highest concentration tested in the EHA was 40 μM, therefore the four values with IC₉₅ of 40 μM likely would have been greater if higher concentrations were tested.