Brucellosis is a zoonotic infectious-allergic disease caused by bacteria of the Brucella genus.

Brucella is a Gram-negative, facultative intracellular bacterium renowned for its ability to evade the host immune system and adapt to a wide range of hosts. These bacteria can survive and replicate within the host’s mononuclear phagocyte system, interfering with immune responses through various mechanisms. In 1985, the World Health Organization classified Brucella into six species and 19 biovars based on host specificity and phenotypic characteristics: Brucella melitensis (biovars 1, 2, and 3), Brucella abortus (biovars 1, 2, 3, 4, 5, 6, 7, and 9), Brucella suis (biovars 1, 2, 3, 4, and 5), and Brucella species associated with sheep, dogs, and the new tumor type (5). In recent years, three additional Brucella species have been identified: Brucella pinnipedialis, Brucella ceti, and Brucella microti (6, 7). The primary species pathogenic to humans are B. suis, B. abortus, and B. melitensis (8). Human infection typically occurs through direct contact with infected animal tissues (such as placentas, aborted fetuses), blood, urine, or milk, or through the consumption of unpasteurized dairy products or undercooked meat (9). Additionally, farm workers, slaughterhouse personnel, veterinarians, and laboratory staff are at risk of infection through inhalation of aerosols or mucosal contact (10). Brucella most commonly enters the human body through the skin or mucous membranes or the gastrointestinal tract, where it is phagocytosed by macrophages and proliferates within them. However, it usually does not directly infect renal cysts but rather enters them incidentally after hematogenous dissemination to the kidneys (3). Therefore, Brucella-induced renal cyst infection is clinically rare and easily overlooked or misdiagnosed. Although the mortality rate of brucellosis is low, the persistence of the pathogen can lead to chronic infection and complications, significantly impacting the patient’s quality of life. Thus, understanding the pathogenesis of Brucella and current treatment strategies is crucial for the effective prevention and control of brucellosis (Table 1).

Brucellosis is one of the most common zoonotic diseases globally. The incidence of brucellosis varies significantly across different regions. According to the World Health Organization (WHO), the global incidence ranges from 0.01 to 200 cases per 100,000 population (11). Recent studies have indicated that the global incidence is higher than previously estimated, with approximately 1.6 to 2.1 million new human cases reported annually (12). The Eastern Mediterranean region is one of the highest-incidence areas for brucellosis, with some areas reporting an incidence as high as 200.41 cases per 100,000 population. In contrast, the incidence in the Americas is relatively low. In Asia, the Middle East and Central Asia are high-incidence areas for brucellosis. For example, the incidence in Iran is 20.33 cases per 100,000 population, while in Kyrgyzstan, it is 12.45 cases per 100,000 population (11). Currently, the average annual incidence rate of brucellosis in China is 3.0 cases per 100,000 population. In recent years, the incidence of brucellosis has shown a complex trend globally. Effective public health measures have led to a decrease in the global incidence, from 26.83 cases per 100,000 population in 2015 to 1.83 cases per 100,000 population in 2020 (12). However, in regions with limited surveillance and intervention, the incidence remains high or has even increased. For instance, the incidence in Saudi Arabia rose from 10.19 cases per 100,000 population in 2015 to 14.17 cases per 100,000 population in 2017 (13).

The pathogenicity of Brucella is closely linked to its ability to survive within host cells, with the cell wall playing a crucial role in this process. The Brucella cell wall consists primarily of an outer membrane and peptidoglycan. The outer membrane contains lipopolysaccharides (LPS), outer membrane proteins (such as lipoproteins and porins), and phospholipids (14). Compared to E. coli, Brucella LPS exhibits low endotoxin activity, strong resistance to macrophage degradation, and facilitates bacterial evasion of the host immune response. These properties enable Brucella to survive and proliferate within host cells, resulting in persistent infection (15). Figure 2 illustrates the pathogenesis of human brucellosis.

Brucella enters host cells by interacting with macrophage membranes via lipid rafts, forming a Brucella-containing vacuole (16). After entry, Brucella initially localizes to early phagosomes, where it avoids fusion with late endosomes and lysosomes, a crucial step in evading the host immune response. The acidic environment of the phagosome specifically induces the expression of the VirB operon, which regulates genes associated with the Type IV Secretion System (T4SS) (17). Through T4SS, Brucella delivers effector proteins to the host cell, altering the endosomal environment to promote bacterial survival and replication. Additionally, Brucella modifies its intracellular trafficking, transitioning from early phagosomes to autophagosomes and eventually reaching the endoplasmic reticulum (ER), where it fuses in a Sar1- and Rab2-dependent manner (18). Within the ER, Brucella replicates extensively without disrupting the host cell’s essential functions or inducing apoptosis. The ER supports Brucella replication within a single vacuole through continuous membrane production, leading to the formation of multiple Brucella replicons enclosed by the ER membrane, which are released through both lytic and non-lytic mechanisms (19). Brucella has specific survival mechanisms within macrophages and is protected by immune evasion mechanisms such as blocking macrophage apoptosis, inhibiting Th1-specific immune responses, and suppressing the production of tumor necrosis factor-α (TNF-α), making the pathogen difficult to eliminate.

Another mechanism by which Brucella survives within host cells is by altering the lipid composition of the phagosome membrane. Brucella expresses and secretes cyclic glucan synthase (CGS), which disrupts the lipid raft microdomains of the surrounding cell membrane, inhibiting phagosome maturation and preventing fusion with lysosomes, thus ensuring bacterial survival. This process occurs independently of the VirB operon (20). Additionally, oxidative killing is a key defense mechanism by which host phagocytes control intracellular pathogen replication, and Brucella has developed various strategies to counteract free radicals. It expresses two superoxide dismutases (SOD), SODA and SODC, with SODA involved in neutralizing free radicals and SODC protecting against superoxide anions produced during macrophage respiratory bursts (21). Brucella also expresses alkyl hydroperoxide reductase C to reduce peroxides, preventing oxidative killing, while its O-polysaccharide (O-PS) resists host cationic peptides and oxidative metabolites (22, 23). In summary, Brucella precisely regulates enzymes and secretory proteins at various stages to evade oxidative killing, allowing it to migrate to the endoplasmic reticulum and Golgi apparatus for replication.

The clinical manifestations of human brucellosis are diverse, most commonly presenting with fever, fatigue, headache, and muscle or joint pain. The severity and nature of symptoms vary depending on the Brucella species, the disease progression, and the affected organ systems. Infections caused by Brucella melitensis and Brucella suis tend to be more severe, while those due to Brucella abortus are generally milder. Due to the nonspecific symptoms, misdiagnosis is common, and if left untreated for more than 6 months, the disease can progress to chronic brucellosis, affecting multiple organ systems. The bones and joints are frequently involved, often presenting as sacroiliitis or spondylitis, though these symptoms are not specific. Digestive system involvement may manifest as hepatosplenomegaly (24). Brucella melitensis is particularly associated with orchiepididymitis, which may require surgery in severe cases (25). Gynecological conditions such as oophoritis and endometritis can also occur, potentially impacting fertility. Renal involvement may result in proteinuria. Neurobrucellosis, though rare, can present with neurological symptoms including meningitis and encephalitis (26). Cardiovascular involvement, while uncommon, may lead to serious complications and is the leading cause of morbidity and mortality in chronic brucellosis. Respiratory and endocrine involvement are less frequent but may present as pneumonia or thyroiditis. In conclusion, the clinical features of brucellosis are often nonspecific, complicating accurate diagnosis and treatment.

In recent years, the diagnosis of brucellosis has emerged as a topic of increasing interest and significance. The current diagnostic and main examination methods for brucellosis in WHO and China are shown in Tables 2, 3. The diagnosis of brucellosis primarily depends on the patient’s history of exposure in endemic areas, medical history, clinical presentation, routine laboratory tests, bacterial culture, and serological results. Laboratory tests often reveal normal or low white blood cell counts with relatively increased lymphocytes, though these findings lack diagnostic specificity. Bacterial culture remains the gold standard for diagnosing brucellosis, but it is time-consuming and may produce false negatives. Serological tests, which detect antibodies against Brucella lipopolysaccharides, face challenges such as cross-reactivity and the absence of standardized protocols. Molecular diagnostics, including PCR, are rapid and sensitive but similarly suffer from a lack of standardization. Immune cell markers, such as Toll-like receptors (TLRs), macrophages, T cells, and neutrophils, are involved in immune responses but have limited diagnostic utility (27–29). Protein markers like recombinant Brucella outer membrane protein 2b (Omp2b) demonstrate diagnostic potential (30). In terms of genetic markers, qPCR detection of specific Brucella gene fragments offers rapid, sensitive, and specific diagnosis. Additionally, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are involved in the immune pathogenesis and may serve as diagnostic markers (31, 32). Advanced genetic testing methods, such as cgMLST and SNP analysis, improve the resolution of Brucella strain identification and hold promise for clinical diagnosis (33).

The current treatment regimen for human brucellosis remains based on the World Health Organization’s 1986 guidelines, which recommend doxycycline for 6 weeks combined with streptomycin for 2–3 weeks, or doxycycline with rifampin for 6 weeks (34). If streptomycin is unavailable or contraindicated due to allergic reactions, gentamicin may be used as an alternative (35). Studies indicate that combining doxycycline with streptomycin or rifampin reduces the minimum inhibitory concentration (MIC) of antibiotics within macrophages, achieving bacteriostatic levels. In cases complicated by arthritis, spondylitis, or endocarditis, a triple therapy consisting of streptomycin or gentamicin, doxycycline, and rifampin is typically prescribed for a minimum of 8 weeks, along with symptomatic treatment as needed (35). However, despite adherence to these regimens, the recurrence or clinical failure rate remains significant, ranging from 5 to 15% (34, 36).

The treatment of brucellosis mainly faces challenges such as antibiotic cell permeability, the bacterial replication niche within host cells, drug resistance, and the presence of antibiotic-resistant strains (37). To enhance antibiotic cell permeability, researchers have loaded antibiotics onto nanocarriers, adjusting surface charge to regulate circulation time and utilizing the high surface-area-to-volume ratio of nanocarriers for slow intracellular drug release, ultimately boosting antimicrobial efficacy (38–40). However, studies have shown that even increasing intracellular antibiotic concentrations does not completely suppress Brucella replication in macrophages. The bacteria remain viable and virulent after antibiotic treatment, posing a risk of re-infection (37). This persistence is likely linked to Brucella’s ability to establish a specialized replication niche within host cells, evading the phagolysosomal pathway and proliferating in a hostile microenvironment. Modifying this microenvironment, such as using hydroxychloroquine to raise the pH, has been shown to inhibit bacterial proliferation.

Bacterial drug resistance and the emergence of antibiotic-tolerant strains are additional factors contributing to treatment failure. Reports of antibiotic-resistant Brucella strains are increasing globally. In China, research on Brucella isolates has found that while most strains remain sensitive to commonly used therapeutic agents, some exhibit resistance to specific drugs. Prolonged use of antibiotics may also lead to the development of antibiotic-tolerant strains, which can survive repeated antibiotic exposure without acquiring a formal resistance mechanism. These strains may contribute to the chronic nature and recurrence of brucellosis.

Brucellosis not only causes substantial economic losses in the livestock industry but also poses a serious threat to human health. Despite prevention and control strategies proposed by the World Health Organization and the World Organization for Animal Health (OIE), brucellosis remains a major public health issue in many developing countries (41). Drawing from the experiences of developed regions like the European Union, brucellosis can be controlled and eradicated in stages at the source. Effective control measures include: (1) Enhanced monitoring and control: Monitoring helps reduce animal infections, identify infected groups, and prevent disease transmission through isolation (42). (2) Strict border quarantine: Controlling livestock movement and implementing rigorous border quarantine procedures, including testing imported animal products, prevents the introduction of infected animals (42). (3) Vaccination and test-slaughter strategy: Vaccination plays a crucial role in controlling brucellosis. Regular testing, combined with the immediate slaughter of infected animals, helps limit the spread of the disease (43). (4) Public education and legal measures: Raising awareness, particularly among farmers and herders, about proper protective practices through media campaigns, as well as implementing legal measures and financial compensation policies, is vital for effective disease control (44). (5) Laboratory quality management and biosafety: Ensuring proper management of laboratory personnel, equipment, sample handling, and testing procedures is essential, with attention to biosafety protocols. Given that Brucella species are among the pathogens that can be acquired in laboratory settings, strict adherence to biosafety level 3 (BSL-3) practices is necessary when handling Brucella cultures and samples. This includes the use of personal protective equipment (PPE), such as gloves, gowns, and eye protection, as well as the implementation of engineering controls like biological safety cabinets. Regular training of laboratory personnel on biosafety and biosecurity measures is also crucial to minimize the risk of laboratory-acquired infections. Global cooperation and localized implementation of these strategies are necessary for effectively controlling and ultimately eradicating brucellosis.