Conference Agenda

Bovine Trichomonosis, caused by the protozoan parasite Tritrichomonas foetus, poses significant economic threats to the global cattle industry. In Australia, disease control relies heavily on accurate diagnosis followed by culling infected bulls. However, conventional diagnostic methods, including culture and quantitative PCR (qPCR), are limited by the complex microbial environment of the bull prepuce, which leads to false-negative and positive results. These arise from T. foetus low abundance and cross‑reactive trichomonad species.

To address these limitations, we developed and tested a diagnostic test using Oxford Nanopore Technologies (ONT) long‑read sequencing to simultaneously detect multiple venereal-transmitted organisms, T. foetus and Campylobacter fetus subsp. venerealis. Using artificially spiked DNA samples and field samples, our pipeline improved sensitivity and specificity, achieving detection limits for T. foetus down to 0.4 ng and identifying 50.0% more positives (n =9) than qPCR (n =6) across samples from persistently infected bulls. Simultaneously detecting C. fetus subsp. venerealis down to 0.04 ng, lower than its 0.1 ng qPCR limit.

These findings highlight the potential of ONT long-read sequencing as a robust, single-test alternative for accurately detecting T. foetus and C. fetus subsp. venerealis in bulls. Its adoption would enhance disease surveillance and help safeguard the Australian cattle industry.

Cyathostomin infections caused by small strongyle worms are among the most prevalent and clinically significant parasitic diseases affecting horses worldwide. Although considerable research has focused on identifying host–parasite interaction markers in serum and faecal samples, the salivary proteome remains largely unexplored. Saliva represents a non-invasive biofluid that may enable monitoring of infection status and immune responses, while providing access to both host and parasite derived proteins.

In this study, the salivary proteome of four naturally infected horses was analysed at two timepoints using mass spectrometry. Two preparation approaches were evaluated to determine the feasibility of saliva for parasite protein detection: direct digestion of neat saliva using S-Trap protocol, and on-bead enrichment method employing hydrophilic interaction liquid chromatography (HILIC) beads to concentrate polar and extracellular vesicle-associated proteins. Samples were analysed by data-independent acquisition (DIA) on an Orbitrap Astral mass spectrometer, searched in Spectronaut using DirectDIA, and analysed in the DIA Analyst platform.

Across all samples, 2,535 proteins (2,448 groups) were identified. Among these, 34 Cylicocyclus nassatus proteins were detected with more than one unique peptide, including 28 proteins with no detectable homology to the host proteome. These findings demonstrate the potential of equine saliva for detecting parasite and host immune proteins.

Gastrointestinal (GI) nematodes are a major constraint to dairy productivity and heifer growth globally and are of particular concern in subtropical Queensland. In this study, we collected fresh faecal samples from the paddock of 43 dairy farms, categorised by age into six groups: calves, weaners, heifers, springers, milking cows, and dry cows. Faecal egg counts (FEC) were performed using the modified McMaster technique, and species identification was conducted through larval culture, with morphological characterization of third-stage (L₃) larvae. All 43 farms had at least one age group positive for GI parasites, indicating widespread endemic infection throughout the region. The overall parasite prevalence was 78.7%, with infection burden varying by age: heifers had the highest FEC (mean EPG of 226.7; 26 to 553), followed by weaners (235.7; 31 to 651), and milking cows (75.8; 5 to 180). Calves were mostly uninfected. Egg counts did not differ significantly between farms (Kruskal–Wallis, p > 0.05) but varied strongly within age groups (H=107.5, p<0.001), with a clear age‑dependent gradient (p < 0.001). Haemonchus spp. (47.2%) and Cooperia spp. (43.1%) dominated, comprising ~90% of larvae, underscoring the need for regular, age‑based faecal monitoring to guide targeted parasite control on dairy farms in subtropical Queensland.