Molecular detection of a Leptospira lineage with high similarity to Leptospira kirschneri in human, environmental, and rodent samples from Gilan Province, Iran: an integrated field and laboratory investigation

Background: Leptospirosis remains an undercharacterized public health threat in Iran's Caspian coastal regions, despite high exposure risks within agricultural communities. Therefore, the circulation of Leptospira (L.) spp. in the Gilan Province was investigated in a prospective, integrated field and laboratory study from January 2020 to December 2023. Method: 182 clinical specimens from febrile patients and 120 environmental samples (water, soil, wildlife tissues) were collected. Samples were analyzed using microscopic agglutination test (MAT), real-time PCR (qPCR), gene sequencing (secY), bacterial culture, and whole-genome sequencing (WGS). Statistical analysis identified risk factors. Results: Among 41 cases, confirmed by the microscopic agglutination test (MAT), Leptospira DNA was detected by qPCR in 37 (90.2%). Sequencing of the secY gene identified sequences with 98.1–98.9% identity to L. kirschneri in 12 clinical and 7 environmental samples. Culture from rodent kidneys yielded three viable isolates. Whole-genome sequencing revealed these isolates shared 97.2% average nucleotide identity with L. kirschneri serovar grippotyphosa, firmly placing them within this species. A notable finding was the discrepancy between MAT serology, which showed high titers against L. interrogans serovars, and molecular data, which consistently indicated the presence of a L. kirschneri-lineage. Identical secY sequences were detected in human, water, and rodent samples, suggesting a potential transmission chain, though definitive culture confirmation was limited to rodents. Multivariate analysis identified rice farming (aOR=2.7, 95% CI: 1.5-4.8), daily floodwater exposure (aOR=2.1, 95% CI: 1.2-3.7), and proximity to drainage ditches (aOR=2.9, 95% CI: 1.6-5.2) as significant risk factors. Conclusion: The findings provide compelling molecular and epidemiological evidence that L. kirschneri-lineage organisms are a likely cause of leptospirosis in northern Iran and should be included in future surveillance and diagnostic frameworks.