Leptospirosis is a neglected zoonotic disease in the world. Leptospirosis is rarely recognized in U.S. and is associated with individuals living in economically disadvantaged urban inner-city environments, recreational exposures, and occupational exposures to infected animals. Transmission to humans and animals occurs by direct contact with reservoir animals or indirect exposure through contact with environmental water or moist soil contaminated with pathogenic Leptospira spp. Leptospira can be maintained in soils and waters for weeks to months. The leptospirosis epidemiology is complex and is a paradigm of a One Health disease. To understand the diversity of strains that occupy water environments in Central Iowa and the evolutionary dynamics of Leptospira, we isolated strains and conducted whole-genome sequencing of 82 Leptospira isolates in this study. The study was conducted in Iowa, a midwestern U.S. state during the summer from June to September, 2021. Water samples were processed for culture and lipL32 rt-PCR. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera. A total of 105 water samples were collected in nine counties in Iowa state, 5/105 (4.7%) were positive by lipL32 rt-PCR. Molecular typing indicated that three isolates were L. montravelensis, two were L. kemananensis, five were L. bourretii, six were L. bouyouniensis, six were L. bandadrabouensis, one was L. abararensis, two were L. chreensis, four were L. bourretti, six were L. terpstrae, one was L. yanagawae, two were L. ellinghausenii and twenty-two were possible new species; the remaining twenty-five comprised mixed species. We were able separate one L. interrogans from one mixed species.
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