Brucellosis is a bacterial zoonotic disease, caused by various species of Brucella, primarily affecting domestic and wild animals with humans as accidental hosts (Corbel 2006). It remains a disease of major public health and economic significance, particularly in regions with extensive livestock production (Franc et al. 2018). The control of zoonotic diseases, including brucellosis, in human populations has largely depended on the management of animal diseases, including vaccination (Corbel 1997; Godfroid et al. 2005). For brucellosis, mainly caused by Brucella abortus, the most effective vaccine (B. abortus S19) is administered solely to calves, as vaccination of adults interferes with serological testing and can lead to reproductive complications, including orchitis and epididymitis in males, prolonged infection, and potential abortion in pregnant females (Lalsiamthara & Lee 2017).

Brucella abortus S19 and Brucella melitensis Rev 1 are established as effective vaccines for preventing B. abortus in cattle, and for protecting against B. melitensis and Brucella ovis in sheep and goats, respectively (Elberg 1996; Nicoletti 1990). Routine surveillance and control programmes largely focus on female animals, as they are the main source of brucellosis transmission through abortion and milk shedding. National surveillance programmes for bovine brucellosis generally focus on animals with the highest risk of transmitting infection through reproduction or milk, particularly adult females, and breeding stock, while males are tested selectively, particularly when used for breeding, linked to an infected herd, or required for movement compliance (Code of Federal Regulations [CFR] 2025; Khurana et al. 2021; United States Department of Agriculture [USDA] 2022). Although clinical signs such as orchitis and epididymitis may occur in males, serological testing and interpretation have historically been designed for herd-level detection, leading to a testing strategy centred on females and breeding animals rather than routine testing of all males (Godfroid, Nielsen & Saegerman 2010).

Serological testing, including the rose Bengal test (RBT), complement fixation test (CFT), and indirect enzyme-linked immunosorbent assay (iELISA), is therefore commonly targeted at females, both to identify infected individuals and to prevent spread within herds. Targeting females in diagnostic programmes allows for more effective detection of infection and supports vaccination strategies aimed at reducing reproductive losses and zoonotic risk (World Organization for Animal Health [WOAH] 2023). However, heifers born to infected cows often test negative before calving, with seroconversion typically occurring only after the birth of their first calf (Crawford, Huber & Sanders 1986). A major challenge in brucellosis control is diagnosing latent and chronic infections, which often escape detection by serological tests and even culture, particularly in endemic regions, where such infections contribute to underreporting and hinder eradication efforts.

Female animals typically present with abortion, stillbirth, or retained placenta, while males may develop orchitis, epididymitis, and seminal vesiculitis (Corbel 2006; Ducrotoy et al. 2017). Brucella transmission occurs primarily through reproductive fluids, aborted materials, and milk (Islam et al. 2023; Kiros, Asgedom & Abdi 2016). Following initial exposure to B. abortus, cows commonly abort once, while subsequent pregnancies are generally normal, although some may experience repeated abortions. Although non-pregnant cows and bulls are typically asymptomatic, they may still contribute to the maintenance and transmission of brucellosis within herds. Latent infections in heifers and vertical infections in calves further support disease persistence, while infected bulls may occasionally develop systemic illness (Neta et al. 2010; USDA 2022). A study in Southern India reported a high brucellosis prevalence among pigs, with 19 seropositive boars showing orchitis, suggesting that untested breeding males may have contributed to disease transmission within farms (Shome et al. 2019a). The transmission cycle associated with male animals, especially in breeding contexts, may be undervalued (Khurana et al. 2021). Brucella-infected bulls may have between 100 organisms/mL and 50 000 organisms/mL in their semen (Manthei, Detray & Goode 1950). These animals can spread the infection through both natural breeding and artificial insemination (Acha 2001; Khurana et al. 2021). In rams, brucellosis has been associated with reduced semen quality, including reduced total sperm output, poor motility, and a high percentage of morphological abnormalities (Cameron & Lauerman 1976). The reduced semen quality may affect the bacteriological examination of the specimen for the identification of Brucella spp. This highlights the importance of targeting females in surveillance programmes, while acknowledging the potential for males to act as silent carriers.

In South Africa, Trichard et al. (1982) reported a case of unilateral orchitis in a bull caused by B. abortus biotype 1. Serological testing detected positive antibody titres while cultures remained negative a month before the onset of clinical signs; subsequent cultures confirmed infection. This finding demonstrated that brucellosis in bulls can be detected serologically prior to the development of orchitis, emphasising both the diagnostic value of serology in males and their potential role as sources of infection before clinical manifestation. Similarly, a study in South Dakota investigated seven seropositive bison bulls aged 3–4 years, from which B. abortus biovar 1 was isolated in six animals (Rhyan et al. 1997). One bull exhibited severe necrotising and pyogranulomatous orchitis, while four others presented with mild seminal vesiculitis. Routine herd screening typically focuses on females; however, evidence from published abattoir surveys (Djangwani et al. 2021; Kolo et al. 2019; Madzingira et al. 2023), case reports of orchitis (Rhyan et al. 1997; Trichard et al. 1982), and laboratory records from eradication programmes (Govindasamy et al. 2021) indicate that seropositive bulls also occur. In most of the studies mentioned, infected males were identified through slaughterhouse monitoring, movement, or export testing or follow-up investigations in herds showing clinical signs. Collectively, these studies illustrate that male animals can harbour and shed Brucella organisms even before overt clinical signs appear, underscoring their epidemiological importance in disease transmission.

Despite the introduction of the bovine brucellosis control scheme in South Africa in the 1980s, the disease continues to rise, particularly among livestock, due to limited resources, inadequate vaccination, poor husbandry, and unrestricted animal movement (De Boni et al. 2022). A recent study on brucellosis in South Africa reported a seroprevalence of 1.73% among male livestock (Mazwi et al. 2023). In addition, Brucella culture-positive cases (11/200; 5.5%) from slaughtered animals included three males, with the younger males testing seropositive on both RBT and CFT, while the older male was RBT-positive but CFT-negative (Kolo et al. 2019). These findings raise concerns about the persistence of brucellosis and the challenges of its potential eradication within herds. Similarly, in Namibia, a slaughterhouse-based survey reported four CFT-positive males among five CFT-positive animals overall (Madzingira et al. 2023). In South Africa, outbreaks of B. melitensis in sable antelope have repeatedly involved males, with bulls identified as index cases, testing seropositive, and yielding isolates from bull testes, underscoring their potential role in persistence of infection within herds (Glover et al. 2020). Serological testing, including RBT and CFT, is performed by accredited laboratories in South Africa for cattle and the African buffalo (Syncerus caffer). In addition, B. melitensis and B. suis serology is available but non-accredited tests, and no serological tests are permitted for wildlife unless accredited. This diagnostic gap impairs early detection and hampers effective outbreak containment.

Despite evidence of Brucella infection in males, there is limited documentation of seronegative yet infectious breeding males contributing to transmission in the field. This represents a critical gap in current surveillance strategies, which rely heavily on serological testing. This study documented brucellosis on two farms: a cattle farm in Gauteng and a sable antelope farm in the Limpopo province of South Africa. The study aimed to: (1) report brucellosis outbreaks in cattle and sable antelope herds, (2) evaluate the performance of serological and molecular diagnostics, and (3) assess the role of seronegative breeding males in disease transmission.

Across both farms, brucellosis outbreaks were associated with the presence of infected breeding animals, particularly bulls, thus highlighting their role in herd-level transmission. On the Gauteng cattle farm, the transition from a brucellosis-negative status in 2022 to multiple abortion events and confirmed B. abortus infection in 2024 strongly suggests a recent introduction of the pathogen. Similarly, in the Limpopo sable antelope herd, routine screening in 2024 identified seropositive females, and B. abortus was confirmed by culture and molecular diagnostics. The detection of B. abortus in both female antelopes and a seronegative breeding bull indicates sustained transmission within the herd following initial spillover, consistent with evidence that breeding males may harbour and shed Brucella despite negative serology (Junqueira Junior et al. 2013; Khamesipour, Doosti & Taheri 2013; Rhyan et al. 1997).

Bulls infected with Brucella can shed the organism in semen, representing a recognised and biologically plausible route of venereal transmission during natural service (Acha & Szyfres 2001; Nicoletti 1990). Although the cow’s vaginal tract possesses natural defensive mechanisms, such as resident microbiota, local immune responses, and an acidic environment, these barriers do not completely prevent infection when Brucella organisms are introduced during mating (Quinn et al. 2011). Brucella is highly adapted to invade mucosal surfaces, including the genital tract, and even low numbers of organisms may penetrate the mucosa and establish infection (Corbel 2006). The risk is heightened in pregnant cows, as the gravid uterus contains erythritol and related polyols that support rapid bacterial replication (Jain et al. 2011). While venereal transmission is less common than exposure to aborted materials or contaminated environments, it remains epidemiologically relevant in brucellosis control programmes (WOAH 2023). In this study, infected breeding males, including seronegative individuals, likely contributed to within-herd transmission, posing a significant risk to the effectiveness of test-and-slaughter eradication strategies.

Despite repeated documentation of high B. abortus bacterial loads in the semen of infected bulls, natural venereal transmission is frequently described as inefficient or of limited epidemiological importance in the literature. Reported microbial concentrations ranging from 10² to > 10⁴ organisms/mL of semen demonstrate clear biological plausibility for transmission during natural service; however, field observations seldom isolate transmission events solely attributed to mating (Acha 2001; Manthei et al. 1950; Nicoletti & Winter 1990). Several biological and epidemiological factors may explain this apparent discrepancy. During coitus, much of the ejaculate is expelled or lost from the reproductive tract (see Figure 2), and the bovine vaginal environment, characterised by resident microbiota, mucosal immune responses, and transiently acidic conditions, may reduce bacterial survival or limit ascending infection (Quinn et al. 2011). In addition, venereal exposure is typically brief and intermittent compared to prolonged environmental exposure to heavily contaminated aborted material, placentas, or uterine discharges, which are widely recognised as the dominant transmission pathways (Corbel 2006). These factors have contributed to a perception that venereal transmission plays only a secondary role in herd-level epidemiology, leading some surveillance frameworks to deprioritise routine testing of breeding males. However, this interpretation may underestimate the cumulative risk posed by infected bulls that service multiple females over extended breeding seasons. Even if the per-mating probability of transmission is low, repeated exposure events can substantially increase the likelihood of infection, particularly in pregnant females, whose gravid uterus provides a favourable environment for Brucella replication due to the presence of erythritol (Corbel 2006). Furthermore, venereal transmission may occur silently, without immediate clinical signs, delaying detection until abortions occur months later. From an epidemiological perspective, the limited emphasis on males in control programmes may therefore reflect diagnostic bias rather than true biological insignificance. Surveillance systems centred on abortion events and female serology inherently detect transmission only after amplification has occurred, while infected males, especially those that remain seronegative, escape early identification. Consequently, the contribution of breeding bulls may be systematically underestimated, reinforcing the view that venereal transmission is rare. The findings of this study challenge this assumption by demonstrating active infection in seronegative breeding males and providing field-based evidence that such animals may act as reservoirs, sustaining low-level, undetected transmission within herds over time.

The infection likely originated from a wildlife–livestock interface, given the historical presence of brucellosis-infected African buffalo on the farm, a transmission route repeatedly documented in Southern Africa’s multispecies systems, where buffalo are recognised maintenance hosts for B. abortus with the capacity to spill over to sympatric wildlife and livestock populations (Cossu et al. 2025; Ducrotoy et al. 2017; Godfroid et al. 2010). Although direct contact between buffalo and sable was not reported, indirect transmission through environmental contamination or shared interfaces cannot be excluded, consistent with previous reports demonstrating Brucella persistence in contaminated pastures, water resources, and fomites at wildlife-livestock interphases (Corbel 2006; Glover et al. 2020). The detection of B. abortus in both female antelopes and a seronegative breeding bull from which Brucella was isolated in semen indicates active infection and suggests sustained transmission within the herd following initial spillover. These findings align with published evidence in cattle, bison, and wildlife species showing evidence that breeding males may harbour and shed Brucella despite negative serology, thereby acting as reservoirs that are rarely detected through routine surveillance (Junqueira Junior et al. 2013; Khamesipour et al. 2013; Rhyan et al. 1997). Continued detection of seropositive females despite routine testing and bull replacement further supports ongoing transmission dynamics as previously described in endemic settings; here, undetected carriers contribute to long-term persistence even under test-and-slaughter strategies (Godfroid et al. 2010; Govindasamy et al. 2021). Collectively, these findings demonstrate that infected males can sustain and disseminate Brucella within herds, emphasising the need for rigorous, routine testing of breeding bulls in both livestock and wildlife systems, a recommendation increasingly supported by studies highlighting the epidemiological significance of male-mediated transmission and the limitation of female-centred surveillance frameworks (Khurana et al. 2021; WOAH 2023).

The diagnostic findings from both case studies highlight a critical limitation of serological testing, particularly in males. Several studies have shown that infected bulls often fail to mount detectable antibody responses, resulting in false-negative interpretations despite active infection (Corbel 2006; Junqueira Junior et al. 2013; Khamesipour et al. 2013). This phenomenon was evident in both the cattle and sable herds, in which B. abortus was detected in bulls by PCR and culture, respectively, despite negative or inconsistent serological results. Such animals represent silent carriers that may remain undetected during routine surveillance while continuing to shed the pathogen. From an epidemiological perspective, these undetected reservoirs complicate eradication efforts and necessitate the incorporation of complementary diagnostic approaches, including bacteriological culture and molecular assays. Bacteriological culture and molecular assays, particularly PCR targeting species-specific markers such as IS711, have been recommended to overcome the shortcomings of serology and improve detection in breeding males (Hinić et al. 2008; Legesse et al. 2023). In endemic areas, the use of serological tests such as RBT and CFT, detecting mainly IgM and IgG1, may fail to identify latent or chronic infections due to reduced sensitivity (Corbel 2006; Nielsen 2002; WOAH 2023). In contrast, the iELISA detects a broader range of IgG, including IgG2, exhibits higher sensitivity for chronic infections (Corbel 2006; Khurana et al. 2021; WOAH 2023). Complementary diagnostics such as bacteriological culture, PCR targeting species-specific markers (e.g. IS711), and iELISA, which detects IgG2 in addition to IgG1, are essential to overcome these shortcomings.

Within South Africa’s bovine brucellosis control scheme, new herds entering the programme must undergo two consecutive negative tests before a certified-area 3 (CA3) or brucellosis status declaration is issued by the state veterinarian. However, reliance on RBT and CFT alone may fail to detect all infected animals, particularly those with chronic or latent infection (Legesse et al. 2023; Shome et al. 2019b). The findings of this study demonstrate that breeding bulls not consistently identified as positive through serology may have contributed to bacterial transmission within the herd. Inter-herd transmission is commonly associated with the introduction of an infected animal (Radostits Otto et al. 2006), reinforcing the importance of pre-movement testing and biosecurity. These findings support the inclusion of iELISA and PCR-based assays as complementary diagnostic tools, particularly for detecting infection in animals that test negative on conventional serological tests. PCR assays targeting species-specific regions such as IS711 further enhance detection by identifying Brucella DNA in blood and tissue samples (Ahmadi et al. 2021; Hinić et al. 2008; Legesse et al. 2023). This was demonstrated in this study by detecting B. abortus in blood clots from cattle and in semen and tissues from sable antelope.

Brucella infections in sable antelope have historically been associated with B. melitensis, including the identification of a sable-associated clade in South Africa (Mazwi et al. 2024). In contrast, this study reports B. abortus infection in sable antelope on a farm with a known history of B. abortus infection in African buffalo. Despite the absence of direct contact, indirect transmission through environmental contamination, fomites, or an unidentified intermediate host remains plausible. This finding underscores the need for enhanced biosecurity and systematic surveillance in multispecies game farms where Brucella can circulate silently across wildlife species. Overall, both timelines highlight delayed detection, the role of reproductive events as sentinel indicators, and the under-recognised contribution of breeding males in maintaining transmission, particularly in complex livestock–wildlife systems.

Currently, the bovine brucellosis control scheme in South Africa is voluntary, leading to limited compliance among livestock owners and inadequate enforcement of relevant legislation nationwide (De Boni et al. 2024). This study encourages the need for a national mandatory vaccination programme in livestock as a major control strategy for brucellosis in South Africa, where the infection is endemic. As reported by Zhang et al. (2018), to prevent and eradicate bovine brucellosis, breeding herds must have a vaccination rate of at least 80%. As of 2020, an overall vaccination of 36% was reported in Gauteng (De Boni et al. 2024), indicating that more vaccination programmes are required to achieve the recommended 80% vaccination rate. Vaccination lowers the incidence of abortions in livestock and will prevent the spillover to other hosts such as wildlife, prevent the spread of disease, and help protect herds from the illness. A coordinated national strategy that includes vaccination, movement control, mandatory bull testing, and farmer education is urgently needed.

Importantly, a confirmed case of human brucellosis in a young male, linked to exposure to an aborted foetus on the cattle farm, highlights the significant zoonotic risk associated with undetected infection in livestock. Human brucellosis remains underdiagnosed in South Africa, despite documented cases such as the first molecularly characterised infection reported in a 27-year-old male from the Western Cape Province (Wojno et al. 2016). Subsequent investigations in that case identified two additional human infections associated with goat handling, alongside a high proportion of seropositive animals in the affected herd, illustrating the close link between animal infection and human disease. Furthermore, genomic analysis has demonstrated close relatedness between B. melitensis isolates from humans and livestock in South Africa, supporting evidence of interspecies transmission (Mazwi et al. 2024). These findings, together with reports of human brucellosis across multiple African countries (Akakpo, Têko-Agbo & Koné 2009), underscore the importance of strengthening surveillance, improving diagnostic capacity, and increasing awareness among high-risk groups to reduce zoonotic transmission.

This study was limited by the small sample size, the inability to purify isolates for complete typing, and the reliance on blood clot DNA for PCR, which may reduce sensitivity. However, the findings provide critical evidence of hidden transmission risks, particularly the potential for seronegative male animals to infect females, as well as the shortcomings of current screening practices. In addition, heifers testing seronegative before sexual maturity and seronegative bulls that are unknowingly retained in breeding programmes pose a significant ongoing risk for perpetuating undetected transmission within the herd. Overall, the evidence demonstrates that infected males can sustain and disseminate Brucella within herds, reinforcing the need for routine bull testing and integrated diagnostics in South Africa.