Ambient temperature storage in DESS supports molecular studies of benzimidazole resistance from canine hookworm eggs

Abstract

Emerging benzimidazole resistance in canine hookworms poses a growing concern for veterinary and public health. Molecular diagnostics targeting {beta}-tubulin gene mutations are essential for resistance surveillance but traditionally rely on refrigerated faecal samples. This study evaluates dimethyl sulfoxide, EDTA, and saturated NaCl (DESS) as a room-temperature preservation medium for canine faeces. Using ITS-2 rDNA and isotype-1 {beta}-tubulin gene (tubb-1) amplicon metabarcoding, we compared DNA integrity and diagnostic performance of DESS-preserved samples (4, 28, and 106 days) to refrigerated controls. No significant differences in PCR amplification or sequencing outcomes were observed. DESS enabled reliable detection of hookworm species and resistance-associated SNPs, including F167Y in tubb-1, with mutation frequencies consistent across treatments. Therefore, DESS can preserve samples from remote settings without cold chain logistics. Our findings validate DESS as a robust alternative for sample collection for molecular parasitology, facilitating expanded surveillance of anthelmintic resistance in field conditions. HighlightsO_LIDESS preserves canine faeces for DNA analysis without refrigeration. C_LIO_LIHookworm species and resistance SNPs detected reliably from DESS samples. C_LIO_LIPCR and NGS results from DESS samples match refrigerated controls. C_LIO_LIF167Y mutation found in A. caninum and U. stenocephala samples. C_LIO_LIDESS enables remote hookworm surveillance without cold chain logistics. C_LI