Association of the Novel Benzimidazole Resistance Marker Q134H with F167Y in Dogs with Ancylostoma caninum

Abstract: Background: Three single nucleotide polymorphisms (SNP) in the highly conserved -tubulin gene were identified to confer benzimidazole resistance in nematodes. Recently, a novel fourth marker, Q134H, was associated with functional benzimidazole resistance.

Objective: The objectives were to validate a Q134H specific real-time PCR (qPCR), to determine frequency for the Q134H mutation and association with the F167Y genetic marker, in dogs naturally infected with Ancylostoma caninum.

Animals: A group of 143 fecal samples positive for Ancylostoma spp. by routine zinc sulfate centrifugal flotation (ZCF) and confirmed by Ancylostoma spp. qPCR were collected. Subsequently, samples were screened for the presence of both the Q134H and F167Y resistance markers.

Methods: Ancylostoma spp. detected by ZCF were confirmed by qPCR. Benzimidazole resistance markers Q134H and F167Y were detected by allele-specific qPCR. The Q134H mutation was confirmed using conventional Sanger sequencing and nanopore amplicon deep sequencing protocols.

Results: Canine hookworm benzimidazole resistance (F167Y or Q134H) was detected in 39/143 (27.3%) of the hookworm confirmed samples. The F167Y allele resistance marker was detected in 31/143 (21.7%) samples. Nineteen (13.3%, 19/143) samples were positive for both markers while 12/143 (8.4%) were positive only for F167Y and 8/143 (5.6%) were positive only for the Q134H marker.

Conclusions and clinical importance: Anthelmintic resistance in hookworms is an emerging concern in veterinary medicine. This study confirms the utility of the F167Y marker for benzimidazole resistance identification in Ancylostoma positive samples, and highlights the importance in expanding testing to incorporate the novel Q134H marker.