Angela M. Arenas-Gamboa

Brucellosis, a neglected zoonotic disease, is endemic in many sub-Saharan African countries, including Cameroon. Recent studies have shown that Brucella abortus infects multiple livestock species throughout Cameroon. While the presence and risk to humans have been identified at farms and abattoirs, the bacterium has never been detected in milk intended for human consumption. In Cameroon, milk is commonly sold at open-air markets for human consumption without regulation. This study investigates the presence of Brucella spp. in milk sold at these markets in three regions: the Far North, North, and West. Cow milk samples and surveys were collected from 380 merchants in each region, totaling 1,140 samples and surveys. Each sample underwent iELISA, culture, real-time PCR, and next-generation sequencing (NGS). Surveys assessed the knowledge, attitudes, and practices (KAP) of milk merchants. Results indicate that B. abortus is not only endemic in livestock but present in milk sold for human consumption. Culture results show a countrywide positivity of 1.3% (1.1% Far North, 1.1% North, 1.8% West). Real-time PCR results indicate a 4.0% positivity rate (2.4% Far North, 5.5% North, 4.0% West). iELISA indicates 21% of the milk samples contained anti- Brucella antibodies, with significant regional variations (38.4% Far North, 15.5% North, 8.9% West). Finally, NGS revealed that the bacterial isolates from milk are epidemiologically linked to those obtained from animals across the region. KAP analysis shows that only 26.4% of merchants boil and 91.2% mix the milk from multiple cattle before sale. All surveyed merchants selling culture-positive milk engaged in risky behaviors, resulting in the sale of contaminated milk to approximately 720 people each week. This study highlights the risk of human brucellosis extending beyond farms and abattoirs to the general public. Future research should investigate milk consumers’ habits and prevalence to better understand brucellosis risk in Cameroon.

Introduction: Brucellosis, a zoonotic disease, significantly impacts animal and public health, as well as agricultural economies reliant on livestock. This disease is endemic in many regions worldwide, with the highest risk of infection in sub-Saharan Africa. However, the true extent of the disease in Africa remains largely unknown. In Cameroon, a country in western Central Africa, Brucella abortus is endemic in livestock, and the bacteria have been found in milk sold at community markets throughout the country. Methods: This study aims to understand the knowledge, attitudes, and biosecurity practices of those closely working with animals throughout the livestock supply chain from farm to slaughter. Three KAP surveys were conducted among livestock owners, traders, and slaughter facility inspectors at live markets and abattoirs in the Far North, North, and West regions of the country due to their pivotal importance in the national and international supply chain. Results: Findings reveal minimal understanding of brucellosis and limited biosecurity practices across the network of owners, traders, and abattoir inspectors. Discussion: The lack of understanding and deficiencies in health infrastructure likely contribute to the persistence of B. abortus as endemic in the country and region as a whole. This study provides insights into animal and public health risks and aims to aid policymakers in developing interventions to reduce the disease burden.

Brucellosis is a zoonotic disease of nearly worldwide distribution. Despite its endemic presence in many regions, the true burden of the disease remains poorly defined. Armenia, a small country situated in the South Caucasus, exemplifies this lack of clarity. To better understand the epidemiological landscape of brucellosis in Armenia, we conducted a scoping review of studies addressing both human and animal brucellosis within the country. Comprehensive database and manual searches identified only 19 relevant articles published between 1982 and 2023. Reported individual seroprevalence estimates for animal brucellosis ranged dramatically from 0.26 to 18.04% (median—3.33%) in cattle and from 0.75 to 29.90% (median—1.94%) in small ruminants. Only one study investigated swine; however, the sample size was too small and precluded meaningful interpretation. None of the studies identified the Brucella species or strains involved, leaving unresolved the question of the primary livestock reservoirs. In humans, brucellosis was predominantly reported among individuals residing in rural areas, with an average incidence of approximately 8 cases per 100,000 population per year. Although constrained by limitations due to the scarcity of available data, this review highlights the neglected nature of brucellosis in Armenia underscoring the potential public health risk posed by this bacterium. Reporting inconsistencies, the absence of readily available diagnostic tools, and the lack of standardized testing procedures hinder an accurate assessment of the disease burden. This review summarizes and identifies critical gaps in research and control efforts, offering insight that can support the need for a comprehensive, long-term national surveillance and control strategy.

Brucellosis due to Brucella melitensis remains a high-priority zoonosis worldwide, with substantial health and economic consequences driven by bacterial shedding in livestock during abortions and in milk. Historically, surveillance and control efforts have been constrained by diagnostic limitations: serological assays often yield false positives due to cross-reactivity, while bacteriological culture, despite being definitive, is slow, labor- intensive, susceptible to false negatives, and requires high-level biocontainment measures. These challenges obstruct effective surveillance, accurate prevalence estimation, and timely response measures thereby prolonging economic losses. This study validates a highly sen- sitive and specific real-time PCR assay targeting the IS711 insertion sequence and the B. melitensis-specific gene BMEII0636, assessing the diagnostic performance in experimen- tally infected goats. Tissue samples (retropharyngeal, parotid, and submandibular lymph nodes), placenta, milk, and vaginal swabs were collected at the time of abortion from goats conjunctively inoculated with 1.1×10 CFU of B. melitensis 16M (n=20). Brucella presence was confirmed by culture. Control samples were obtained from naive goats from a certified brucellosis-free herd (n=11). All tissue samples were blinded and directly tested via real-time PCR. This assay demonstrates high sensitivity — 93.4% for IS711 and 94.2% for BMEII0636— with no false positives in culture-negative controls. Limits of detection were 8.75×10¹ gene copies for IS711 and 1.25×10 for BMEII0636. Furthermore, the time required to obtain results was shortened from several days with bacteriological culture to a few hours using this method. These findings support the integration of this validated PCR assay into brucellosis surveillance frameworks, enabling accurate prevalence estima- tion, timely prevention and control interventions, and reduced zoonotic transmission risk. Notably, placenta, milk, and vaginal swabs, which are easily collected and yield a high bacterial load, showed strong diagnostic performance, underscoring their utility for field surveillance and rapid-response protocols. This approach is especially critical for enhanc- ing One Health-aligned control strategies worldwide.

Brucellosis is a neglected zoonotic disease impacting agricultural economies, as well as animal and human health globally. In sub-Saharan Africa, brucellosis is considered endemic in many countries based on serological evidence, although the presence and distribution of specific Brucella species remain unverified due to limited bacteriological confirmation. Due to the economic importance and national/international trade routes, this cross-sectional study investigates Brucella prevalence in 4612 animals (cattle, sheep, goats, and pigs) from slaughterhouses in the Far-North, North, and West regions of Cameroon sampled between February 2021 and May 2023. The analysis includes serology (Rose Bengal Test and indirect Enzyme-Linked Immunosorbent Assay), culture, real-time PCR, and next-generation sequencing (NGS). Results show only Brucella abortus is present, primarily affecting cattle (8.3%) and goats (7.3%). NGS reveals the local B. abortus strain is related to the clade from Uganda and Sudan, indicating it is endemic to Africa, rather than introduced from outside the continent. These results confirm the presence of a genetically distinct African lineage and reinforce brucellosis as a major concern for both animal and public health. The study emphasizes the critical need for coordinated surveillance systems to support evidence-based control strategies, enhance disease monitoring, and reduce the risk of transboundary transmission.

Diagnosis of brucellosis is not a straightforward task, with over 40 different tests available. Accurate diagnosis requires a series of diagnostic testing with proper interpretation of results. The World Organization for Animal Health (WOAH) provides guidelines describing the different assays including their recommended use, protocols, and interpretation. PubMed, Embase and Web of Science databases were searched without restrictions and original work describing cross-sectional studies focusing on livestock species (cattle, buffaloes, sheep, goats and swine) were included while reviews, case reports, and case-control studies were excluded. In this study, we systematically reviewed the literature and critically assessed the findings from 349 research studies to provide an overview of the different diagnostic methods used worldwide in livestock, and compared the tools and strategies used against the WOAH recommendations. A total of 232 studies (66.5%) focused on cattle followed by goats (34.1%), sheep (31.5%), buffaloes (14.6%), and swine (5.2%). Of these studies, 171 were from Africa (48.9%), 132 from Asia (37.8%), 36 from the Americas (10.3%), and 10 from Europe (2.8%). The most utilized immunological assayswere Rose Bengal test and indirect ELISA (63.9 and 36.7%, respectively). Interestingly, 73 studies (20.9%) used a single immunological assay to report on the status of animals. Direct methods such as culture and PCR were performed in 100 studies (28.7%) with culture being the most utilized (19.8%). Strikingly, we found that only 16% of included studies followed WOAH recommendations in terms of sample chosen, diagnostic assay utilized, protocol employed and results interpretation. In countries that reported the presence of B. abortus, B. melitensis, and B. suis to the WOAH, only 4 of 28, 2 of 19, and 1 of 6 countries (reporting these strains, respectively), contained studies that followed guidelines and confirmed the presence of the pathogen. This highlights, not only significant gaps in currently available literature leading to an inaccurate picture of brucellosis in livestock, but most importantly raises significant issues regarding the accuracy of data reported by countries. These findings are concerning due to the significant consequences of not adhering to these guidelines including inaccurate diagnosis, delayed disease control, and increased zoonotic risk for exposed individuals.

Brucellosis is a neglected and re-emerging zoonotic disease of nearly worldwide distribution. Recently, a new model utilizing publicly available data, voluntarily provided by countries to the World Organization for Animal Health (WOAH), estimated a global annual incidence of at least 1.62–2.10 million cases. However, global and regional knowledge gaps, partially attributed to lack of diagnostic capability and disease unawareness, result in misdiagnosis. This study incorporated misdiagnosis to assess the potential impact on incidence estimates. Specifically, we examined the potential impact of misdiagnosing brucellosis as malaria. Incidence rates modeled from WOAH data comprised a baseline estimate for brucellosis rates. A range of potential misdiagnosis rates based on World Bank malaria data and scientific literature were utilized to estimate the number of misdiagnosed cases. We assumed a conservative misdiagnosis range of 0.25-4.00% based on clinical studies that reported a 4–11% misdiagnosis rate. We show that malaria misdiagnosis can significantly impact global annual incidence estimation of brucellosis. Rates of 0.25% increase brucellosis incidence by 0.34-5.4 M cases, while rates of 4% increase these estimates by 2.43-7.45 M cases. Human brucellosis misdiagnosed as malaria can significantly impact global annual incidence estimates. Enhanced efforts are needed to identify misdiagnosed cases in countries where both diseases are endemic.

Global Human Brucellosis Incidence

Brucella species are considered a significant cause of reproductive pathology in male and female animals. Importantly, Brucella melitensis can induce reproductive disease in humans. Reproductive pathogenesis and evaluation of newly developed countermeasures against brucellosis studies have traditionally utilized female animal models. However, any potential, new intervention for use in humans would need to be evaluated in both sexes. Therefore, animal models for male reproductive brucellosis are desperately needed to understand disease progression. Accordingly, we evaluated guinea pigs and mice using B. melitensis 16 M in an intratracheal model of inoculation at different stages of infection (peracute, acute, and chronic) with an emphasis on determining the effect to the male reproductive organs. Aerosol inoculation resulted in colonization of the reproductive organs (testicle, epididymis, prostate) in both species. Infection peaked during the peracute (1-week post-infection [p.i.]) and acute (2-weeks p.i.) stages of infection in the mouse in spleen, epididymis, prostate, and testicle, but colonization was poorly associated with inflammation. In the guinea pig, peak infection was during the acute stage (4-weeks p.i.) and resulted in inflammation that disrupted spermatogenesis chronically. To determine if vaccine efficacy could be evaluated using these models, males were vaccinated using subcutaneous injection with vaccine candidate 16 MΔvjbR at 10⁹ CFU/100 μl followed by intratracheal challenge with 16 M at 10⁷. Interestingly, vaccination efficacy varied between species and reproductive organs demonstrating the value of evaluating vaccine candidates in multiple models and sexes. Vaccination resulted in a significant reduction in colonization in the mouse, but this could not be correlated with a decrease in inflammation. Due to the ability to evaluate for both colonization and inflammation, guinea pigs seemed the better model not only for assessing host-pathogen interactions but also for future vaccine development efforts.