Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

Brucellosis is a globally prevalent zoonotic disease that imposes considerable economic burdens on the livestock industry and remains a significant threat to public health. Although lipopolysaccharide (LPS)-based serological assays are widely used in routine diagnosis, their inherent limitations—particularly cross-reactivity with other Gram-negative bacteria—underscore the need for the development of diagnostic approaches based on non-LPS antigens. In this study, we developed a trivalent nanobody–horseradish peroxidase (3Nbs-HRP) fusion protein targeting Brucella OMP16 and established a cELISA for the serological detection of bovine brucellosis. The diagnostic performance of the assay was assessed using 204 Brucella antibody-negative and 123 Brucella antibody-positive bovine serum samples. ROC curve analysis yielded a sensitivity of 87.7% and a specificity of 89.4%, with no significant cross-reactivity observed. By employing a recombinant antigen and a 3Nbs-HRP probe, this assay enhances biosafety and demonstrates strong potential for standardization and large-scale application, serving as a complementary tool to conventional LPS-based assays for the surveillance, diagnosis, and control of bovine brucellosis.

(1) Background: One method of eradicating brucellosis is to cull cattle that test positive for antibodies 12 months after being vaccinated with the 19-strain vaccine. Variations in immunization regimens and feeding practices may contribute to differences in the rate of persistent antibodies. We conducted this study to investigate the real positive rate of Brucella antibody in field strains of Brucella spp. after immunization over 12 months in dairy cows. This research aims to provide data to support the development of strategies for preventing, controlling, and eradicating brucellosis. (2) Method: We employed the baseline sampling method to collect samples from cows immunized with the A19 vaccine for over 12 months in Lingwu City from 2021 to 2023. Serological detection was conducted using the RBPT method. An established PCR method that could distinguish between 19 and non-19 strains of Brucella was utilized to investigate the field strains of Brucella on 10 dairy farms based on six samples mixed into one using the Mathematical Expectation strategy. (3) Results: We analyzed the rates of individual seropositivity and herd seropositive rates in dairy cattle in Lingwu City from 2021 to 2023 and revealed that antibodies induced by the Brucella abortus strain A19 vaccine persist in dairy herds for more than 12 months. We established a PCR method for identifying both Brucella A19 and non-A19 strains, resulting in the detection of 10 field strains of Brucella abortus from 1537 dairy cows. By employing a Mathematical Expectation strategy, we completed testing of 1537 samples after conducting only 306 tests, thereby reducing the workload by 80.1%. (4) Conclusions: There was a certain proportion of cows with a persistent antibody titer, but there was no evidence that all of these cattle were naturally infected with Brucella. The established PCR method for distinguishing between Brucella abortus strain 19 and non-19 strains can be specifically utilized for detecting natural Brucella infection in immunized cattle. We propose that relying solely on the detection of antibodies in cattle immunized with the A19 vaccine more than 12 months previously should not be solely relied upon as a diagnostic basis for brucellosis, and it is essential to complement this approach with PCR analysis to specifically identify field Brucella spp. Brucella abortus was the predominant strain identified in the field during this study. Detection based on the Mathematical Expectation strategy can significantly enhance detection efficiency.

Brucellosis is an important zoonotic disease caused by Brucella spp., posing significant impacts on animal health, livestock production, and public health. Infected dairy animals can shed the pathogen in milk, making milk a noninvasive and readily accessible sample for disease surveillance. Therefore, detection of Brucella in milk can be used for monitoring brucellosis in dairy herds. In this study, polyclonal antibodies were produced by immunizing camels with lipopolysaccharide (LPS) derived from Brucella abortus vaccine strain 19 (A19). These antibodies were subsequently conjugated to magnetic beads to develop immunomagnetic beads (IMBs). The IMBs were combined with a sandwich ELISA for the enrichment and detection of Brucella in milk samples. The IMB-sELISA showed a limit of detection (LOD) of 1 × 10⁴ CFU/mL. The method reduced matrix interference and showed satisfactory analytical performance. This approach can be applied for on-farm sample screening and herd-level surveillance to support the diagnosis and control of brucellosis in dairy animals.