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bioRxiv · 10.1101/2025.08.16.670690

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

Yi-Jou, C.·Li, V.·Suwandy, M.·Mitrea, I. B.·Hayward, D.·Jaensch, S.·Francis, E. K.·Slapeta, J.
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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

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Yi-Jou, C., Li, V., Suwandy, M., Mitrea, I. B., Hayward, D., Jaensch, S., Francis, E. K., Slapeta, J.. 2025-08-17. Ambient temperature storage in DESS supports molecular studies of benzimidazole resistance from canine hookworm eggs. https://doi.org/10.1101/2025.08.16.670690

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