First isolation of Leptospira interrogans from follicular fluid of naturally infected cows

The presence of leptospires in the follicular fluid has only been confirmed through molecular techniques, as culturing leptospires is extremely challenging. The lack of studies demonstrating the viability of leptospires in this site limits a deeper understanding of pathogenesis. Therefore, this study aimed to cultivate and molecularly characterize Leptospira spp. from follicular fluid and uterine tissue samples collected from naturally infected cows. A total of 85 cows from herds with leptospirosis were selected and 53 follicular fluids (FF) and 85 uterine fragments (UF) were collected after slaughter. The samples were seeded into T80/40LH, and evaluated by Darkfield Microscopy (DFM). Positive cultures were tested by lipL32-PCR to confirm the presence of pathogenic Leptospira spp. Positive tubes were submitted to serogrouping and genotyping by sequencing of the secY gene. A maximum likelihood (ML) tree was constructed. A total of 33/85 (39 %) cows were positive, 16/53 (30.2%) only in FF, 14/85 (16.4 %) only in UF, and three in both samples. It was possible to obtain one isolate from FF, serogrouped as Icterohaemorrhagiae. Six samples were sequenced by secY. All of them were identified as L. interrogans, with > 99 % identity. The ML tree revealed that all sequences belong to a group with strains close to serovar Hardjo. Herein, we highlight the presence of live L. interrogans in follicular fluid, emphasizing it as an important site of infection, as leptospires could impair embryo production. The similarity of the strains involved to highly virulent strains in humans raises concerns, posing a potential zoonotic risk.