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Simple Summary: The platypus (Ornithorhynchus anatinus) is a monotreme, a rare kind of mammal that lays eggs and suckles its young. It is amphibious, lives in freshwater, and is found only in Australia, where it is closely protected. During prior conservation studies, several pathogens had been discovered in the platypus, including leptospires, which are a kind of bacterium that can cause severe disease in animals and people. We evaluated the available data concerning this infection in platypuses, tested blood samples in the Platypus Serum Bank, and combined the results with historical records. We found evidence of leptospirosis in 50% of platypuses from 14 river basins in southeastern Australia. Leptospiral infection did not stop females from breeding and juvenile platypuses were recruited to the population each year. The high seroprevalence, evidence of ongoing exposure to leptospires, lack of disease, mild kidney pathology, and visible leptospires in the kidney tubules suggest that the platypus may be a reservoir host for leptospires rather than an accidental host that suffers from the disease. Cattle on adjacent farms also had leptospirosis, but the evidence was not convincing that cattle caused the infection in platypuses. A landscape-wide study is recommended to determine the actual infection pathway. The platypus (Ornithorhynchus anatinus) is an amphibious, egg-laying mammal of high conservation value that is found only in Australia. The zoonotic bacterium Leptospira interrogans serovar Hardjo was discovered in platypuses in prior studies, but little is known about its epidemiology. Samples in the Platypus Serum Bank were tested in 2023 and the results were combined with historical records. Antibodies against L. interrogans serovar Hardjo were found in 50% of 464 serum samples from 411 platypuses collected from 14 river basins in southeastern Australia between 1981 and 2012; prevalence remained high over three decades in the Shoalhaven River population. Seroprevalence increased with age, suggesting environmental exposure. Individual platypuses had persistent titres, some for six years. Seropositive females lactated, juveniles were recruited into the population, and there were no reports of clinical leptospirosis. Three necropsied platypuses were seropositive and had mild nephritis with leptospires in the renal tubules. The high seroprevalence, persistent titres, lack of disease, mild renal lesions, and renal colonisation suggest the platypus may be a maintenance host. Sympatric cattle had L. interrogans serovar Hardjo titres, but the spatial association with seropositive platypuses was statistically weak. Other mammalian wildlife species and sheep also have L. interrogans serovar Hardjo titres; therefore, a complex ecological network must be considered. A landscape-wide study is recommended to properly assess transmission pathways and confirm who is infecting whom. [ABSTRACT FROM AUTHOR]