Leptospirosis caused by the pathogenic serovars of Leptospira spp. is a zoonotic disease of global importance. Brown rats (Rattus norvegicus), due to their worldwide distribution and reservoir host status, are considered the most important reservoir for environmental leptospiral persistence and infections in animals. We aimed to isolate, identify, and characterize Leptospira spp. circulating among brown rat populations in Zaria, Kaduna state, Nigeria. Kidney samples from 305 brown rats from different locations in Zaria were collected April–June 2022 and cultured using bacteriologic methods. We also extracted DNA from 24 kidney tissue samples for molecular detection of pathogenic Leptospira spp. and for phylogenetic analysis. Leptospira spp. was detected in 57.7% of the samples by using culture methods and in 91.7% of the samples by using PCR. The phylogenetic tree revealed two distinct clusters among the reference sequences, aligning with the known P1 and P2 subclades. The sequences from this study all clustered together on the phylogenetic tree and were most similar to Leptospira interrogans serovar Pomona strain RZ 11. The high prevalence of Leptospira spp. detected in Zaria suggests that further surveillance and assessment of the public risk are warranted.

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