Brucellosis is an infection caused by bacteria that has the potential to impact a wide range of animal species [1]. The microorganisms are Gram-negative coccobacilli capable of facultative intracellular growth [2]. Presently, there are a total of 12 species that have been officially documented, with the majority of them displaying a strong preference for specific hosts [3]. Brucella abortus mainly affects cattle, Brucella melitensis primarily affects goats, and Brucella canis primarily affects dogs. Brucella infections are often spread among animals by transferring semen and discharge [4]. However, they may also be acquired through inhalation and oral ingestion. Infected animals often do not exhibit symptoms, but when they are pregnant, there is a possibility of fetal abortion, which may be accompanied by persistent vaginal discharge [5].

Certain Brucella species, such as B. abortus, possess a significant capacity to transmit from animals to humans and are often responsible for causing human infections [6]. Bang's illness, also known as human brucellosis caused by B. abortus, is characterized by a persistent and fluctuating fever that may last for a prolonged period if left untreated. The infection can potentially endure in the brain or bone marrow [7]. The death rate is relatively low; however, the management of the disease is complex, and there is now no preventive medication available. The minimum number of bacteria required to cause infection is 10–100 for B. abortus [8]. B. abortus may endure in tap water for a maximum of 114 days, according to the temperature [9]. The primary modes of transmission for human brucellosis include inhalation, direct contact with infected livestock or their bodily fluids through skin wounds or mucous membranes, and consumption of infected food, particularly unpasteurized dairy products [10]. The bacteria are very selective in their growth requirements and can survive in the environment for extended durations. Although brucellosis may be treated with antibiotics, it significantly debilitates the human body and often necessitates prolonged recuperation for many people [11]. Furthermore, since Brucella prefers infecting cells, the range of drugs that may effectively treat this infection is restricted [12]. It is worth mentioning that in 5–40 % of patients, antibiotic therapy leads to the recurrence of the illness, necessitating prolonged treatment with various combinations of antibiotics [13], [14]. This fact and the absence of an authorized anti-brucellosis vaccination for humans pose a significant challenge for regions where brucellosis is prevalent. Hence, it is crucial to have robust strategies to avoid this illness in livestock and its subsequent transfer to people via dairy products [14].

In the past, several nations extensively used live attenuated vaccines derived from B. abortus 19 BA or B. melitensis 104M microorganisms for human immunization [15]. Typically, in individuals who have had vaccinations, these vaccines provided a temporary immune system defense and were followed by a strong reaction and heightened sensitivity, particularly when many vaccine doses were given [16]. A crucial approach in creating secure and efficient vaccinations against brucellosis is employing live genetically altered vectors, which are harmless microbes that generate brucellosis antigens [10]. Currently, lactic acid bacteria, Escherichia coli, and Semliki Forest virus (SFV) are employed as carriers for expressing brucellosis antigens in living organisms [15], [17]. Experimental evidence has shown that the investigated bacterial (intracellular) and viral vectors could infect various kinds of cells and produce brucellosis antigens inside the infected cell [18]. In addition to being a common gram-positive bacterium in probiotic product manufacturing, Lactococcus lactis is well-known for being the first live genetically modified organism utilized in the treatment of human illness [19]. Dr. Doosti's research group previously used Lactococcus lactis for enhancing vaccination and effectively modified Brucella immune outer membrane proteins (omp25) [20] and Brucella lumazine synthase (BLS) protein [21].

Although significant progress has been achieved in developing recombinant vaccines, their use is hindered by drawbacks such as instability, restricted storage requirements, and high manufacturing and purification expenses [21], [22]. Hence, the use of novel systems is very crucial. Multi-epitope vaccines, which are generated employing reverse vaccinology, are a novel kind of vaccination that does not possess the drawbacks described [23]. Multi-epitope vaccines are free from biological contamination because they are produced by chemical synthesis. The multi-epitope vaccines possess water solubility and can maintain stability under essential circumstances [24]. The peptides may be engineered explicitly for selectivity. Reverse vaccinology entails the replication and manifestation of all the proteins inside an organism's genomic sequence that are forecasted, using computational methods, to be located on the surface or secreted [25]. Subsequently, every protein undergoes high-throughput vaccination to assess its capacity to induce antibodies in mice that can effectively eliminate or neutralize the target microorganism [26].

The first use of reverse vaccinology focused on N. meningitidis serogroup B (MenB). This disease lacks a widely effective vaccination due to the resemblance of its capsular polysaccharides to a self-antigen and the high diversity of its primary outer membrane protein antigens. This method resulted in the discovery of 29 new antigens that can stimulate the production of antibodies that can kill the bacteria in a controlled laboratory setting [27].

This work investigates the feasibility of using L. lactis as a therapeutic agent for immunizing mice against B. abortus OMPs. We used the recombinant L. lactis inducible expression pNZ8124 vector to create a Brucella multi-epitope OMPs antigen. This antigen was then administered orally to mice using L. lactis-pNZ8124 and recombinant L. lactis-pNZ8124-OMPs. Next, we examined the immunological response. Hence, we aimed to create a mucosal vaccine using the lactic acid bacteria L. lactis as a live bacterial vector for delivering antigens. This approach was a favorable alternative and safer immunization method against B. abortus infections.