Unveiling brucellosis risks in breeding herds: Case study on diagnostic failures and control gaps in South Africa.

Brucellosis is a zoonotic pathogen of livestock, wildlife and humans, with significant implications for reproductive losses and public health. The movement and introduction of breeding animals with unknown status into previously Brucella-negative herds pose a risk for disease introduction and sustained transmission. This case study documents two independent brucellosis outbreaks integrating epidemiological observations with serial serological testing, bacteriological culture and molecular confirmatory assays following abortion events. Diagnostic tests were performed, including serology, PCR and culture from diverse samples. On a cattle farm, animals previously certified as brucellosis-negative demonstrated progressive seroconversion from 2024 onwards. Brucella abortus was detected in the blood clots of tested animals using abortus-melitensis-ovis-suis PCR (AMOS-PCR), including a suspect bull that later tested seronegative on follow-up serology. A Brucella-positive human case occurred following direct exposure to the infected cattle farm, underscoring the zoonotic risk. On the sable antelope farm, B. abortus was detected in two seropositive females through culture of lymph nodes and amniotic fluid, while B. abortus was also isolated from the semen of a seronegative breeding bull, suggesting that the bull was a carrier and posing a transmission risk to the herd. The findings demonstrate that infected breeding males may remain seronegative while actively harbouring and shedding B. abortus. This study indicates that seronegative breeding males can undermine brucellosis control programmes, may act as undetected reservoirs of infection, thereby challenging current surveillance strategies. The findings highlight the diagnostic limitations of relying solely on serological tests, particularly in chronically infected cattle and wildlife, and support the integration of more sensitive assays, such as iELISA, alongside PCR methods, into diagnostic strategies on brucellosis-affected farms to improve detection and support eradication efforts. Contribution: Strengthened biosecurity, systematic testing of breeding males, and comprehensive vaccination programmes are critical to preventing ongoing spillover among livestock, wildlife, and humans in South Africa.