| Sumario: | <i>Leptospira</i> are bacteria that cause leptospirosis in both humans and animals. Human <i>Leptospira</i> infections in Uganda are suspected to arise from animal–human interactions. From a nationwide survey to determine Leptospira prevalence and circulating sequence types in Uganda, we tested 2030 livestock kidney samples, and 117 small mammals (rodents and shrews) using real-time PCR targeting the lipL32 gene. Pathogenic <i>Leptospira</i> species were detected in 45 livestock samples but not in the small mammals. The prevalence was 6.12% in sheep, 4.25% in cattle, 2.08% in goats, and 0.46% in pigs. Sequence typing revealed that <i>Leptospira borgpetersenii</i>, <i>Leptospira kirschneri</i>, and <i>Leptospira interrogans</i> are widespread across Uganda, with 13 novel sequence types identified. These findings enhance the East African MLST database and support the hypothesis that domesticated animals may be a source of human leptospirosis in Uganda, highlighting the need for increased awareness among those in close contact with livestock.
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