Targeted molecular screening for protozoan parasites in wild platypuses (Ornithorhynchus anatinus) and associated risk factors for infection

The platypus (<i>Ornithorhynchus anatinus</i>) is a unique monotreme endemic to eastern Australia, yet little is known about the prevalence and impact of protozoan infections in wild populations. This study investigated the occurrence of protozoan parasites and associated health parameters in wild platypuses from New South Wales (NSW), Australia, with a focus on seasonal variation and demographic risk factors. A total of 90 sampling events (79 individuals and 11 recaptures) yielded 72 blood and 81 faecal samples for parasitological screening through targeted molecular techniques. <i>Theileria ornithorhynchi</i> was detected in 100 % of blood samples (95 % CI: 95.01-100), confirming its widespread distribution and high prevalence in NSW platypuses. <i>Trypanosoma</i> spp. were identified in 40.3 % of blood samples (95 % CI: 28.88-52.50), and sequencing confirmed the presence of <i>Trypanosoma binneyi</i>, reported here for the first time in NSW. <i>Toxoplasma gondii</i> was detected in one sub-adult male (1.39 %, 95 % CI: 0.04-7.50). No blood samples tested positive for <i>Plasmodium</i> spp. (0 %, 95 % CI: 0-4.99). In addition, all faecal samples tested negative for <i>Cryptosporidium</i> spp. and <i>Giardia</i> sp. (0 %, 95 % CI: 0-4.45). Boosted Regression Tree (BRT) analysis identified season as the primary risk factor for <i>Tr. binneyi</i> infection (RI 89.5 %), with higher prevalence in summer. Males were also at increased risk (RI 10.5 %), while age class and location (river catchment) had no measurable influence. Season also significantly affected physiological parameters, including tail volume index (TVI) and packed cell volume (PCV). These findings expand the known distribution of protozoan parasites and provide new insights into the seasonal dynamics of parasitic infections in wild platypuses. This study contributes to the understanding of platypus health ecology and highlights the importance of long-term, seasonally informed monitoring programs.