Altered larval activation response associated with multidrug resistance in the canine hookworm Ancylostoma caninum

Abstract

Parasitic gastrointestinal nematodes pose significant health risks to humans, livestock, and companion animals, and their control relies heavily on the use of anthelmintic drugs. Overuse of these drugs has led to the emergence of resistant nematode populations. Herein, a naturally occurring isolate (referred to as BCR) of the dog hookworm, Ancylostoma caninum, that is resistant to 3 major classes of anthelmintics is characterized. Various drug assays were used to determine the resistance of BCR to thiabendazole, ivermectin, moxidectin and pyrantel pamoate. When compared to a drug-susceptible isolate of A. caninum, BCR was shown to be significantly resistant to all 4 of the drugs tested. Multiple single nucleotide polymorphisms have been shown to impart benzimidazole resistance, including the F167Y mutation in the β-tubulin isotype 1 gene, which was confirmed to be present in BCR through molecular analysis. The frequency of the resistant allele in BCR was 76.3% following its first passage in the lab, which represented an increase from approximately 50% in the founding hookworm population. A second, recently described mutation in codon 134 (Q134H) was also detected at lower frequency in the BCR population. Additionally, BCR exhibits an altered larval activation phenotype compared to the susceptible isolate, suggesting differences in the signalling pathways involved in the activation process which may be associated with resistance. Further characterization of this isolate will provide insights into the mechanisms of resistance to macrocyclic lactones and tetrahydropyrimidine anthelmintics.

Keywords: Ancylostoma caninum; benzimidazole; dog hookworm; macrocyclic lactone; multiple anthelmintic drug resistant; pyrantel.

Graphical abstract

Figure 1.

Dose–response curves of Ancylostoma caninum isolates to thiabendazole (TBZ) using the egg hatch assay (EHA). Each point represents a minimum of 6 replicates from 3 independent experiments (WMD n = 59, KGR n = 60, BCR n = 62). The curves were analysed using the sigmoidal dose–response (variable slope) function in GraphPad Prism (ver. 9) to generate the IC₅₀ values reported in Table 1.

Figure 2.

Dose–response curves of Ancylostoma caninum isolates WMD (sensitive), KGR (double resistant) and BCR (triple resistant) using a larval development assay (LDA) and a larval activation inhibition assay (LAIA). Dose–response curves to (A) ivermectin (IVM), (B) moxidectin (MOX) and (C) pyrantel (PYR) determined by LDA. The curves were analysed using the sigmoidal dose–response (variable slope) function in GraphPad Prism (ver. 9) to generate the IC₅₀ values reported in Table 1. Each point represents a minimum of 6 replicates from 3 independent experiments. (D) In vitro larval activation inhibition assay (LAIA) for PYR resistance. Third-stage infective larvae (iL3) of susceptible WMD and resistant BCR isolates were incubated with 100 μg mL⁻¹ PYR overnight at 37°C, then assayed for activation. The mean of 4 (WMD) or 6 (BCR) replicates from 2 or 3 independent experiments were analysed by 1-tailed t-test in GraphPad Prism. **P < 0.01.

Figure 3.

Fecal egg counts (FEC) in BCR and KGR isolates. (A) FEC were performed over the course of infection for a dog infected with the BCR hookworm isolate and (B) the KGR isolate to test for pyrantel resistance. The red arrow marks when pyrantel treatment was administered. BCR and KGR were treated at 101- and 37-days post-infection, respectively. Blank areas marked with red asterisks indicate days when the FEC was not measured.

Figure 4.

Sequences of β-tubulin isotype 1 cDNAs from 4 Ancylostoma caninum isolates. (A) DNA sequences. The non-synonymous mutation in codon 167 associated with benzimidazole resistance is shaded grey. A synonymous mutation in codon 165 of the BAL sequence is underlined. There were no mutations in codons 134, 182, 198 and 200, which have been associated with resistance to benzimidazoles, detected using this technique. (B) Protein translation of the relevant region of the β-tubulin isotype 1 sequence. The F167Y mutation is highlighted. BAL, Baltimore isolate reference genome (DQ059758; Bioproject PRJNA72585); WMD, thiabendazole-susceptible isolate (MH253570); KGR, thiabendazole-resistant isolate (MH253569); BCR, thiabendazole-resistant isolate (MZ889668).

Figure 5.

Altered feeding response in pyrantel-resistant BCR isolate. (A) General activation pathway. (B) Hookworm iL3 from each isolate were incubated in medium alone (NA), serum stimulus (ACT) or 15 mm 8-bromo-cGMP for 24 h, after which the number of activated (feeding) iL3 was determined. Experiments were done in triplicate and repeated twice, so that each value is the mean ± s.d. of 9 replicates, and analysed by pairwise ANOVA. ****P < 0.0001. mGC, membrane guanylyl cyclase; MuscR, muscarinic receptor; InsR, insulin receptor, FOXO, forkhead transcription factor.