Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population

Abstract

Background: Anthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution.

Methods: Allele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined.

Results: The Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%.

Conclusions: This data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

Keywords: Ancylostoma caninum; Anthelmintic resistance; F167Y; Real-time PCR; US pet Dog population; β-tubulin isotype-1.

Fig. 1

Graphical overview of the validation processes for the qPCR assays.

Fig. 2

Standard curves for the A. caninum allele-specific qPCR tests using synthetic DNA constructs carrying either the resistant nucleotide ‘T' (A) or the susceptible nucleotide ‘A' (B) at beta-tubulin gene codon 167. The data points are the median of three replicates. The equation and r2 linearity value resulting from the linear regression analysis are shown in the graph.

Fig. 3

Box plots with pan-bacterial Cp values from two clinical stool sample populations (A) pre-treated with two different protocols. Protocol 1 included incubation for 10 min in a chaotropic lysis solution, protocol 2 included mechanical disruption using bead beating. (B) Box plots with Ancylostoma spp. Cp values from the same two sample populations.

Fig. 4

Sequencing trace chromatograms showing the F167Y codon and SNP in codon 167 (black arrows). A: ‘T’ at nucleotide position indicating presence of the susceptible allele. Note the smaller ‘A’ nucleotide peak underneath the larger ‘T’ peak indicating that both alleles are present in the sample. B: ‘A’ nucleotide in codon 167 the presence of the resistant allele. Note: No T peak is visible indicating a the majority of alleles are showing the resistant allele.

Fig. 5

Relative proportions of β-tubulin isotype-1 alleles with the polymorphism at locus F167Y conferring benzimidazole susceptibility or resistance as determined by allele-specific qPCR (C). The 55 samples with the detectable resistance allele were sourced from five states (B). Resistant hookworms were found in 14 different dog breeds including 8 Greyhounds (B).