Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the genus Leptospira. In cattle, it can manifest as bovine genital leptospirosis (BGL), a chronic and often subclinical condition that compromises reproductive performance. The primary reproductive disorders associated with BGL include embryonic loss, infertility, and early fetal death (1). Leptospirosis represents not only a veterinary concern but also a major zoonotic threat within the One Health and Planetary Health frameworks, as it emerges at the interface between livestock, wildlife, and environmental systems. Understanding its persistence in animal hosts is, thus, critical for preventing transmission to humans and reducing the ecological burden of this neglected disease.

Previous studies have demonstrated the presence of pathogenic Leptospira spp. in various sites of the bovine reproductive tract, including the uterus (2), cervicovaginal mucus (3), ovaries and oviducts (4), follicular fluid (5), and cumulus–oocyte complexes (13). These findings support the hypothesis that genital colonization facilitates the silent persistence and dissemination of the pathogen within herds, especially when conventional tests based exclusively on urine samples are used, leading to underdiagnosis of BGL (6).

In addition to its epidemiological impact, uterine colonization by pathogenic Leptospira spp. may cause microenvironmental alterations that impair embryo development. Pedrosa et al. (7) detected pathogenic Leptospira DNA in uterine tissues and reported increased interleukin-6 (IL-6) concentrations in positive samples, suggesting that leptospiral colonization triggers a local inflammatory response capable of compromising uterine receptivity. Such chronic inflammation may contribute to embryo loss and irregular estrous cycles in affected females, as observed in the low fertility rates of infected cows.

Regarding diagnostic approaches, cervicovaginal mucus sampling and in vivo uterine biopsies have been proposed as effective and welfare-compatible alternatives for live animals (8). Later on, Aymée et al. (6) demonstrated that both cervicovaginal mucus and uterine fragments (UF) yield comparable results for detecting pathogenic Leptospira spp. in naturally infected cows, with positivity rates markedly higher than those observed in urine. These findings confirm that genital tract samples are more reliable for molecular diagnosis of BGL and highlight the diagnostic value of direct tissue sampling, which enhances test sensitivity and minimizes false negatives caused by intermittent bacterial shedding. Nevertheless, it remains unclear whether pathogenic Leptospira spp. are evenly distributed within the genital tract, which could influence the diagnostic sensitivity of tissue biopsies depending on the anatomical site. To date, no study has compared the detection of pathogenic Leptospira spp. among distinct genital regions or assessed whether a specific site better represents the infection status of the entire system.

Mapping the anatomical niches that sustain pathogenic Leptospira persistence contributes to integrated surveillance strategies, particularly in tropical regions where environmental and climatic drivers favor pathogen maintenance and dissemination. Understanding the spatial distribution of pathogenic Leptospira spp. within the female reproductive tract is therefore essential for optimizing sampling protocols and reducing false-negative results. Hence, the objective of this study was to investigate the presence of Leptospira spp. DNA in different anatomical regions of the genital tract—oviducts and distinct uterine sites—of naturally infected cows.

Considering the overall infection status, here defined as the detection of pathogenic Leptospira DNA in at least one of the analyzed anatomical sites, 55% (22/40) of the cows were classified as positive for BGL. Among the specific regions examined, pathogenic Leptospira DNA was detected in only one oviduct sample (2.5%; p = 0.550), whereas uterine fragments showed markedly different results: 50% (20/40; p < 0.001) of the UB samples were positive, while no positive results were found in the BUH; 0%; p = 1.0 and only 7.5% (3/40; p = 0.156) in the AUH (Figure 1). Statistical comparison among paired collection sites confirmed that the UB presented significantly higher positivity (p < 0.01) than all the other regions, which did not differ from each other (p = 1.0). The results obtained are presented in Table 1.

This study reinforces the relevance of BGL as a widespread reproductive infection in female cattle. The high overall positivity rate (55%) observed in slaughtered cows indicates a high prevalence of pathogenic Leptospira spp. in the reproductive tract, in agreement with previous reports identifying the pathogen in uterine tissue and cervicovaginal mucus of subfertile cows (2, 11). These findings support the notion that genital colonization represents an important mechanism of pathogen persistence within cattle populations and may contribute to underdiagnosis when surveillance relies solely on conventional clinical or urinary assessments. From a One Health perspective, the persistence of leptospires in reproductive tissues represents a potentially underestimated interface between livestock production, environmental contamination, and zoonotic risk.

The most important outcome of the study was the markedly high detection rate observed in the UB (50%), supporting the hypothesis that this region constitutes a key anatomical site of pathogenic Leptospira colonization in BGL. Similar observations have been reported by Pires et al. (12) and Pedrosa et al. (7), who identified that uterine infection activates local inflammatory pathways, increasing cytokine levels and possibly compromising embryo viability. Together, these data reinforce the biological relevance of uterine colonization beyond its diagnostic implications.

In contrast, no positive samples were found in the BUH, and only low detection rates occurred in the AUH (7.5%) and OVID (2.5%). This uneven distribution suggests site-specific differences in susceptibility to colonization within the female genital tract, likely driven by a combination of anatomical and physiological factors. In addition to the higher vascularization of the UB, luminal fluid dynamics, hormonal fluctuations across the estrous cycle, and regional differences in epithelial architecture and receptivity may modulate bacterial persistence and detection, as previously suggested (7). From a diagnostic perspective, this anatomical asymmetry implies that sampling restricted to distal uterine sites may result in false-negative outcomes, reinforcing the UB as the most reliable anatomical site for molecular detection.

The interpretation of these findings must consider the inherent limitations associated with the use of slaughterhouse-derived samples. The cows included in this study were slaughtered for unknown reasons, and no clinical, reproductive, or serological histories were available. Consequently, it was not possible to distinguish between asymptomatic animals, cows with subclinical reproductive impairment, or animals removed from production for reasons unrelated to reproduction. While this limitation precludes definitive conclusions regarding the clinical expression of infection at the individual level, the frequent detection of pathogenic Leptospira spp. in reproductive tissues underscores the potential for genital colonization to occur without overt clinical information at the time of slaughter, highlighting a critical gap in routine surveillance strategies.

Although the UB was identified as the most consistent site for molecular detection, it is important to acknowledge that not all tissues analyzed in this post-mortem study are readily accessible under field conditions. In live animals, uterine sampling is feasible primarily through transcervical biopsy, a technique previously described as applicable for the diagnosis of BGL (6). Cervicovaginal mucus remains the least invasive and most commonly used alternative in routine practice (6, 11). In this context, the present findings suggest that, whenever uterine sampling is performed, prioritizing regions anatomically closer to the UB may improve diagnostic sensitivity and reduce false-negative outcomes associated with the asymmetric distribution of leptospires within the uterus.

There are additional limitations to be considered. Although the overall prevalence observed in this study falls within the range reported in previous investigations conducted in the same geographical region (4, 5, 11), the sample size remains limited for drawing broader epidemiological inferences. In particular, the low number of positive samples in specific anatomical sites may have reduced the statistical power to detect subtle regional differences. Moreover, information on breed composition, farm management practices, and/or the level of technological development was not available, limiting the generalizability of the findings to other production systems. In a previous study, crossbred pluriparous cows from medium- to high-technification commercial dairy herds were used, and the presence of pathogenic Leptospira spp. was detected in approximately 27% of the animals (4). Finally, the detection threshold of the qPCR assay was not experimentally determined, precluding quantitative comparisons of bacterial load among samples, including cases in which multiple anatomical sites tested positive.

In conclusion, this study demonstrates that pathogenic Leptospira spp. are unevenly distributed within the bovine female reproductive tract, with a marked predominance in the UB. These findings suggest that BGL may frequently remain undetected under field conditions, particularly in the absence of overt clinical or reproductive data, although the lack of individual animal histories limits definitive conclusions regarding subclinical infection. Collectively, these results strengthen our understanding of BGL pathophysiology and reinforce the importance of targeted sampling strategies to improve diagnosis, surveillance, and control in cattle production systems. These results not only enhance diagnostic precision in bovine leptospirosis but also contribute to a broader understanding of how pathogen persistence in livestock can influence zoonotic risks and ecosystem health under the One Health framework.