Cyathostomin infections, caused by small strongyle worms, are among the most prevalent and clinically significant parasitic diseases affecting horses worldwide. While extensive research for useful host-parasite injection markers has occurred for serum and faecal sample types, the salivary proteome remains largely unexplored. Saliva offers a non-invasive alternative for monitoring infection and immune activity, with potential to reveal both host and parasite-derived proteins.
This study used mass spectrometry analyses of the salivary proteome of four horses at two timepoints that were naturally infected with cyathostomins. Two methods were used to assess the feasibility of saliva for parasite protein detection: direct digestion of neat saliva biofluids using the S-Trap protocol, and an on-bead enrichment method using hydrophilic interaction liquid chromatography (HILIC) beads to concentrate polar and extracellular vesicle (EV) associated proteins. Samples were analysed using data-independent acquisition (DIA) on an Orbitrap Astral mass spectrometer (Thermo), searched in Spectronaut using DirectDIA and analysed using the DIA Analyst platform.
We identified 2,535 proteins (2,448 protein groups) across all samples. Of these, 37 Cylicocyclus nassatus proteins were detected with >1 unique peptide, including 28 Cylicocyclus proteins with no sequence homology to the host proteome. These results demonstrate the suitability of equine saliva for a non-invasive tool for detecting low-abundance parasite and host immune proteins, providing dual opportunities for advancing our understanding of equine parasitism and host responses.