Brucellosis is a bacterial zoonotic disease caused by gram-negative bacteria of the Brucella genus, usually transmitted from livestock to humans via the consumption of raw and unpasteurized animal food products. The disease remains a major global public health concern, with an estimated 1.6 to 2.1 million new human cases annually (Laine et al., 2023).

The genus Brucella includes numerous species (LPSN: https://lpsn.dsmz.de/genus/brucella). For clarity, in this study, the term Brucella only includes a unique monophyletic clade (Leclercq et al., 2020), which maintains both IS711 and bcsp31 genes (Bounaadja et al., 2009; Aljanazreh et al., 2023; Sanjuan-Jimenez et al., 2017), that have been used for molecular diagnostics. Previously, the Brucellae carrying both IS711 and bcsp31 comprised 14 species of facultative intracellular bacteria. Six of these species were grouped as classical and recognized as members of the core clade basis on their pathogenicity, host preferences, and adherence to phenotypic characteristics, including B. abortus (cattle), B. melitensis (goats or sheep), B. suis (swine), B. ovis (sheep and goats), B. canis (dogs), and B. neotomae (desert rats) (Occhialini et al., 2022; Olsen and Palmer, 2014; Suárez-Esquivel et al., 2020; Whatmore and Foster, 2021). This clade additionally includes the species B. ceti and B. pinnipedialis, which have been described more recently and isolated from marine mammals (Cloeckaert et al., 2001; Foster et al., 2007; Orsini et al., 2022). In the last two decades, advances in field research, pathogen detection, and molecular typing have made it possible to identify new species in addition to the strains not yet classified (Occhialini et al., 2022; Suárez-Esquivel et al., 2020). Two new species, B. amazoniensis and B. nosferati, isolated from Brazilian gold miners working in the Amazon rainforest (About et al., 2023) and Costa Rican bats, respectively (Hernández-Mora et al., 2023), can be considered part of the core clade Brucella based on phylogenetic analyses. However, we still do not know how these species are transmitted, what the animal reservoirs are, or their evolution in relation to other Brucella species. Additionally, four Brucella species, including B. microti (prevalent in common voles, red foxes, and wild boars), B. inopinata (in humans), B. papionis (in baboons), and B. vulpis (in red foxes), were identified in a wide range of hosts. These species can be distinguished from classical Brucellae by atypical phenotype, which includes altered metabolism, higher metabolic activity, faster growth, different composition of lipopolysaccharide, and presence of flagella (Occhialini et al., 2022). Due to these differences, new strains can be classified as a typical Brucella species, thereby contributing to the increased diversity within this genus (Occhialini et al., 2022). At the same time, applying a second tier of classification based on genetic homology shows that B. microti and B. papionis group more closely with the classical core clade species owing to their higher genomic similarity, whereas other atypical species remain genetically distant and thus appropriately classified as non-core clade Brucella (Occhialini et al., 2022).

B. microti was the first atypical Brucella species to demonstrate a broad host spectrum, with notable occurrences in the common vole (Microtus arvalis) in the Czech Republic (Scholz et al., 2008), mandibular lymph nodes of red foxes (Vulpes vulpes) from Austria (Scholz et al., 2009), and wild boars (Sus scrofa) from Hungary (Rónai et al., 2015). In experimental infections, B. microti exhibited high pathogenic potential, causing death in murine models, similar to classical Brucella spp. known to affect humans, thereby highlighting zoonotic risks (Jiménez et al., 2010). To date, no confirmed human infections have been reported. However, there was a suspected case in which, based on the clinical course, a clear epidemiological link (a bite from an infected rodent), isolation of the pathogen from a sick vole, and a specific serological response in the human patient, led to the conclusion that the disease was likely caused by B. microti (Hubálek et al., 2023).

Furthermore, B. microti was the only Brucella species to be isolated from soil (Scholz et al., 2008), indicating its environmental persistence, which diverges from the facultative intracellular evolution of classical core clade Brucella spp. Additionally, B. microti was also isolated from aquatic environments (Jaÿ et al., 2020), and the first strain was recovered from frogs (Pelophylax ridibundus) raised for human consumption (Jaÿ et al., 2018), further emphasizing its ability to persist in diverse ecological niches. The innate ability of B. microti to withstand acidic environments, ranging from moderate to extreme pH levels (Occhialini et al., 2022; Damiano et al., 2015; Freddi et al., 2017; de la Garza-García et al., 2021; Occhialini et al., 2012), along with its adaptation to anoxic conditions (Freddi et al., 2023), as well as accelerated metabolism, is associated with enhanced nutrient utilization and enzymatic activities (Al Dahouk et al., 2010; Al Dahouk et al., 2012). Furthermore, heterogeneity in LPS genes may explain its adaptability to environmental conditions. This environmental resilience, alongside its presence in a wide range of mammalian hosts, underscores its open lifestyle, making it distinct from more host-restricted Brucella species. However, to date, to the best of our knowledge, no identification of B. microti has been reported in cattle, small ruminants, or pigs, highlighting a gap in understanding its potential to colonize livestock and raising questions about its true zoonotic potential. With this study, the ability of B. microti to infect small ruminants is confirmed, shedding light on the potential risks and the need for proper management and identification of this bacterium in animals, especially from epizootic regions.

Brucellosis is a highly contagious bacterial zoonosis, primarily transmitted to humans through the consumption of contaminated, unpasteurized dairy products, undercooked meat, or direct contact with infected livestock and their reproductive materials. The major species responsible for brucellosis in humans, B. melitensis and B. abortus, are typically associated with livestock, making comprehension of transmission paramount for control measures and public health protection. No B. microti human cases have been reported worldwide, even if a strong exposure to this bacterial species occurred in French farmed frogs destined for human consumption (Jaÿ et al., 2020; Jaÿ et al., 2018). In the case of B. microti, the transmission pathways are unknown. Given the growing recognition of B. microti as a pathogenic species and the presence of all 53 Brucella spp. known virulence genes, the absence of confirmed human infection remains surprising. Indeed, B. microti appears highly pathogenic in common voles and other rodents (Hubálek et al., 2007), as confirmed by experimental infections in murine models (Jiménez et al., 2010; Hubálek et al., 2007; Hanna et al., 2011; Ouahrani-Bettache et al., 2019). The absence of human cases might be partly explained by a lower pathogenicity in non-rodent species (Rudolf et al., 2024). In frogs, only a low percentage of animals showed clinical symptoms (Jaÿ et al., 2020; Jaÿ et al., 2018), as well as in foxes (Scholz et al., 2009) and wild boars (Rónai et al., 2015). According to these data, B. microti is likely to behave as an opportunistic soil bacterium that occasionally infects mammals (other than rodents) through the ingestion of contaminated products, with limited potential for sustained transmission in these hosts (Rudolf et al., 2024).

Our study reports for the first time that B. microti is capable of infecting livestock, particularly small ruminants, thereby expanding the known host range of this emerging pathogen. B. microti exhibits several physiological characteristics, in particular, expanded metabolic activity, compared to classical zoonotic Brucella spp. This enhanced metabolic flexibility suggests a greater ability to survive and proliferate in environmental reservoirs. This may also contribute to the capacity of B. microti to infect a broader range of hosts. Therefore, host hopping could represent a key mechanism driving the spread of these epizootic species (Occhialini et al., 2022). While B. microti has been primarily considered as a pathogenic bacterium for rodents and wildlife, the potential for transmission to livestock has not been thoroughly investigated (Rudolf et al., 2024). The detection of B. microti in livestock may represent a previously overlooked link in the potential transmission pathways, bridging the gap between environmental reservoirs, wildlife, and domestic animals. Cross-species spill-over transmission is strongly influenced by the frequency, duration, and nature of contacts. Opportunities for human intervention strongly impact this parameter (Lloyd-Smith et al., 2009). In Europe, due to the increased size of some wildlife populations in agricultural areas, such as ungulates or suidae, together with a progressive reduction of the usable agricultural area (Perpiña et al., n.d.), the probability of contact between domestic and wild species has increased during the past decades (Ledger et al., 2022). This may lead to potential emergence of wildlife pathogens, as previously observed in domestic dogs infected with B. suis biovar 2, strains presenting high genomic similarities with those circulating in hares and wild boars (Girault et al., 2023).

At the same time, the fact that only one animal per farm was infected may indicate that either these infections were accidental, suggesting the opportunistic nature of B. microti, or that the current surveillance system can identify early onsets of farm infection and therefore protect against greater spread and economic losses. It is important to note that no total culling of the herds was carried out as recommended for control of zoonotic Brucella (B. abortus, B. melitensis, and B. suis), but only the positive animal was removed, and at present, no other positive serological results have been observed. These data suggest that the B. microti infections observed in small ruminants are accidental, indicating the opportunistic nature of infection, similar to Ochrobactrum spp. (Ryan and Pembroke, 2020; Thoma et al., 2009), rather than indicative of widespread transmission within herds, like those of classical Brucella species (Corbel et al., 2006; Godfroid et al., 2013). This raises the need to evolve current regulations by considering all Brucella species, beyond just the controlled zoonotic ones, and defining the appropriate measures based on the bacterial species-host combination. Nevertheless, the performance characteristics of the CFT and RBT tests in ruminants infected with B. microti have not been established. Therefore, the absence of detectable antibodies or intermittent and short-term seropositivity in seronegative animals cannot be excluded. Experimental infection studies in ruminants with B. microti would be valuable to generate validation data for these serological assays, as well as to explore pathogenicity in these hosts, since no clinical symptoms have been observed in these three cases.

The detection of B. microti in small ruminants underscores a significant challenge in current brucellosis monitoring strategies. Traditional serological tests, commonly used to detect classical Brucella infections in livestock, rely on the detection of antibodies against the lipopolysaccharides (LPS), specifically the O-polysaccharide (OPS) component, which links to the outer core and extends into the extracellular environment (Erridge et al., 2002; Mancilla, 2015). These OPS components are found in all smooth Brucella spp., where the relative abundance and distribution of a homopolymeric linear chain of N-formyl-perosamine residues, linked via α1,2 and/or α-1,3 glycosidic bonds, are responsible for the structure and antigenicity (Bundle et al., 1989; Moriyón and López-Goñi, 1998; Zygmunt et al., 2015). Although there are slight structural variabilities in the OPS, they are not crucial in the indirect diagnosis of Brucella infections, since anti-OPS antibodies recognize stable terminal sugar residues. However, this homogeneity in the OPS structure between B. microti and the surveyed highly zoonotic Brucella (B. abortus, B. melitensis, and B. suis) poses a significant challenge for diagnostics, as they share similar antigenic profiles, leading to cross-reactivity in serological tests. As a result, control plans based solely on immuno-serology may fail to accurately identify B. microti-infected animals. Currently, in the EU, ruminants infected with highly zoonotic Brucella sp. have to be slaughtered (Regulation EU 2020/689; ELI: http://data.europa.eu/eli/reg_del/2020/689/oj). In some member states like France, the whole cattle and small ruminant farm has to be slaughtered, which leads to significant economic losses for farmers and low acceptability for citizens. Therefore, the misdiagnosed infection with B. microti may lead to unnecessary culling of healthy livestock. Moreover, spontaneous mutations in B. microti can lead to the conversion of smooth LPS to a truncated rough form as previously identified (Ouahrani-Bettache et al., 2019). The absence of OPS in these rough strains makes the use of classical smooth Brucella sp. antigens ineffective for diagnostics, as is the case with rough strains of B. canis and B. ovis (Djokic et al., 2023). As envisaged by Ouahrani-Bettache et al. (Moriyón et al., 2004) the rough B. microti strain could serve as an interesting candidate for brucellosis vaccination in addition to existing vaccines, since it does elicit an antibody response that is distinguishable from that induced during active infection when complete OPS is present.

France has been officially recognized as brucellosis-free since 2005, following the European regulation (EFSA, 2023). In both departments affected by B. microti infection in small ruminants, no brucellosis outbreaks in cattle, sheep, or goats have been identified since 2003. Only sporadic cases of B. suis biovar two infection have been reported in suidae (Wendling et al., 2020). However, no data are available concerning B. microti prevalence and distribution in France, nor have accidental isolations been reported to the national reference laboratory aside from the previously described frog cases (Jaÿ et al., 2020; Jaÿ et al., 2018). The detection of B. microti in sheep and goats from three geographically distinct French farms highlights the potential for localized outbreaks. Moreover, the fact that these outbreaks occurred in different regions within the same timeframe suggests that B. microti may be more widespread than initially thought. Building on the previous study (Jaÿ et al., 2020), which reported a significant presence of B. microti-like strains in both domestic frogs (Pelophylax ridibundus) and surrounding environments, including water and soil, this study suggests that infection of small ruminants may be linked to environmental reservoirs of this pathogen, even in geographically distant areas, as the affected farms were located in different regions of France. The widespread environmental presence of B. microti in amphibian habitats raises the possibility that similar reservoirs may exist in agricultural environments, potentially facilitating transmission of the pathogen to livestock. The broad-spectrum wgSNP analysis linked the isolated strains from small ruminants in the same subclade with all known B. microti strains (Figure 2). However, there is a greater genetic similarity of these strains with the reference B. microti CCM 4915 isolated in Central Europe (Audic et al., 2009), compared to the frog isolates found in France. Interestingly, compared to the B. microti CCM 4915 reference strain, the two B. microti isolates from sheep in two different French departments exhibited no difference and variations between seven SNPs when aligned with B. melitensis 16 M and B. microti CCM 4915, respectively. In contrast, one and eight SNPs were detected in the goat isolate. These minimal genetic variations among the livestock isolates suggest a high degree of genetic similarity within this population. However, when compared to the frog isolate 17–2,122-4144, a more pronounced divergence was observed, with at least 10 and 134 SNPs distinguishing it from new isolates and B. microti CCM 4915. This genetic difference is also evident in MLVA-16 analysis, where the frog isolate (colored in yellow) clusters separately from the small ruminant strains (colored in blue), which group with B. microti CCM4915 (colored in turquoise) (Figure 1 and Supplementary Figure 1). This indicates that the frog isolate represents a possible distinct lineage, potentially reflecting ecological adaptation or host-specific evolution. The considerable variation of SNPs between the frog and livestock isolates underscores the genetic diversity within B. microti. It highlights the need for further investigation into the ecological and host-associated factors contributing to this diversity.

B. microti was isolated from reproductive tissues, including genital and retromammary lymph nodes, which raises the possibility of bacterial excretion in milk. Although milk samples were not available for direct testing, the presence of the bacterium in these tissues suggests a potential downstream risk associated with the consumption of unpasteurized dairy products from the implicated farms. This underscores the need for strict food safety practices and enhanced public health surveillance in such settings. A further limitation of this study is the inability to conduct environmental sampling in the surrounding areas of the farms or in wildlife habitats to trace the potential source of infection. Given that B. microti has been isolated from wild animals and soil, the possibility of environmental reservoirs contributing to the transmission cycle is high. Interestingly, the first two farms have 40 and 80 cattle, respectively, and no positive serological results were found during the prophylaxis campaigns before and after slaughter of infected ruminants. Additional annual monitoring of these herds has not shown any positive serological results. This suggests that, potentially, B. microti, at least for now, is more adapted to small ruminants. Identification of B. microti in domestic animals emphasizes the importance of monitoring Brucella infections across diverse populations, applying “One Health” approaches at the wildlife-farm-human interface (Lloyd-Smith et al., 2009). Future studies incorporating environmental sampling and broader surveillance of wildlife populations are essential to elucidate the transmission dynamics of this emerging pathogen and to devise effective control measures. Furthermore, the ability of B. microti to persist in wildlife and spill over into domestic animals suggests that targeted strategies are needed to prevent its transmission between these populations. Biosecurity is essential in livestock farming to prevent the spread of diseases, ensure animal welfare, and maintain farm sustainability (Bellini, 2018). In practice, to prevent various infectious diseases, it is recommended to maintain the surroundings of the farm double-fenced, which greatly limits the contact of wild animals with livestock. At the same time, it is recommended to avoid watering in ponds or rivers accessible to wildlife or downstream from other farms. Take precautions when distributing feed in pastures and restrict access to manure in the fields by using a tarpaulin or electric fencing. Domestic carnivores, suidae, and poultry can be sources of many infectious agents for cattle. In extensive and small-scale ruminant farms, biosecurity implementation may be impaired by inadequate premises infrastructure and uncontrolled contacts among different species (Alavedra et al., 2025). Compliance, in these cases, was influenced by farmers’ age, education level, herd size, and production.

In particular, dairy farms showed better biosecurity practices, probably because of improved management and infrastructure. This study highlights the challenges of implementing biosecurity measures on small-scale, extensive farms and shows the ineffectiveness of standardized plans. Biosecurity management in cattle farms consists of so-called protective measures to avoid the introduction of pathogens into the farm and limit the spread and the clinical expression of conditions already present in the farm. It includes a forward flow, management of introductions (animals, feed, workers, and instruments). Their presence should be prohibited in breeding and professional areas where feed is stored. This is true of dogs about neosporosis and poultry about botulism and salmonellosis. Finally, the installation of nets must prevent bird access to open-air feed storage to reduce the risk of contamination of milk by pathogens [Salmonella, HP STEC (highly pathogenic Shiga toxin-producing Escherichia coli), etc.], particularly in the case of raw milk production.

In conclusion, current results in conjunction with previous findings showed that B. microti is a potential pathogen at the interface of livestock, wildlife, and the environment. This highlights the need to improve screening tools for ruminants and to establish surveillance in wildlife and environmental reservoirs to better detect atypical Brucella species.