Molecular Detection of Benzimidazole Resistance Associated with the F200Y Polymorphism in the β-Tubulin Gene of Ancylostoma caninum: First Report from India

Abstract

Purpose

The study aimed to detect benzimidazole (BZ) resistance in Ancylostoma caninum in dogs from Thrissur district, Kerala, India, using amplification refractory mutation system- polymerase chain reaction (ARMS-PCR). The primary objective was to determine the presence and frequency of mutations at codon 200 of the isotype-1 β-tubulin gene, associated with BZ resistance, in adult worms and infective larvae.

Methods

A total of 75 adult A. caninum worms were collected during post-mortem, and 280 infective larvae (L3) from 35 faecal samples positive for strongyle ova were obtained from various locations. Amplification of the isotype-1 β-tubulin gene at codon 200 was performed by ARMS PCR, followed by Sanger sequencing to confirm of single nucleotide polymorphisms (SNPs). Genotypes were analysed using EMBOSS and NCBI BLAST tools.

Results

All 75 adult worms exhibited the homozygous susceptible genotype at codon 200. Out of 280 larvae genotyped, two were homozygous resistant, one was heterozygous, and 277 were homozygous susceptible. The overall frequencies of the resistant (TAC) and susceptible (TTC) alleles were 0.01 and 0.99, respectively. Both homozygous and heterozygous resistant larvae were found in a household with recurring ancylostomosis. No mutations were observed at codon 198.

Conclusion

The study identified a low frequency of BZ-resistant alleles in A. caninum larvae in Thrissur, Kerala, marking the first such report in India. Although resistance remains at an early stage, ongoing monitoring is essential to mitigate its spread in the canine population.