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Introduction: Brucellosis, an infectious zoonotic disease caused by members of the genus Brucella , results in chronic multi-organ injury. Improving the specificity and sensitivity of serological methods for diagnosing brucellosis necessitates the development of novel diagnostic antigens. The twin-arginine translocation (Tat) pathway is responsible for transporting folded proteins across the cytoplasmic membrane and has been implicated in the virulence of Brucella. Three Tat substrate proteins—L,D-transpeptidase ErfK (A0577), linear amide C-N hydrolase YxeI (A1479), and thioesterase domain-containing protein EntF (B0249)—contribute significantly to Brucella virulence. However, the roles of these Tat substrate proteins in diagnosing brucellosis remain unclear. Methods: In this study, ErfK, YxeI, and EntF were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera from bovines and sheep diagnosed with brucellosis were analyzed using indirect ELISA with these proteins. Results: For bovine serum, the combined protein group (ErfK + YxeI + EntF) and YxeI demonstrated the highest diagnostic accuracy of 94.23% and 93.58%, respectively. Meanwhile, the combined protein group showed the strongest ability to detect Brucella in sheep serum, achieving an accuracy of 88.10%. Both the combined protein group and YxeI displayed no cross-reactivity with rabbit serum immunized against Yersinia enterocolitica O9, Escherichia coli O157:H7, Mycobacterium tuberculosis , Vibrio cholerae , Legionella , and Salmonella , indicating relatively good specificity. Conclusion: The findings of this study suggest that Tat substrate proteins serve as promising candidate antigens with significant potential value for the clinical diagnosis of brucellosis. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an idiopathic and chronic autoimmune disease for which there are currently no effective treatments. Oxypeucedanin hydrate (OXH) is a natural coumarin known for its potent anti-inflammatory properties. However, further investigations are needed to determine its therapeutic efficacy in treating RA. In this study, we evaluate the anti-inflammatory activity of OXH by treating LPS-induced RAW264.7 macrophages. Our results show that OXH treatment reverses the changes in iNOS, COX-2, IL-1β, IL-6, and TNF-α levels. Additionally, OXH reduces ROS production. Further analysis reveals that OXH suppresses the activation of the NF-κB/MAPK pathway. CETSA results show that OXH competes with LPS for binding to the TLR4/MD2 complex. MST experiments demonstrate the specific affinity of OXH for the TLR4/MD2 complex, with a Kd value of 33.7 μM. Molecular docking analysis suggests that OXH binds to the pocket of the TLR4/MD2 complex and interacts with specific amino acids, such as GLY-343, LYS-388, and PHE-345. Molecular dynamics simulations further confirm this conclusion. Finally, we investigate the potential of OXH in treating RA using a collagen-induced arthritis (CIA) model in rats. OXH effectively ameliorates the symptoms of CIA, including improving body weight, reducing swelling and redness, increasing talus volume, and decreasing bone erosion. OXH also decreases the mRNA levels of pro-inflammatory factors in synovial tissue. Transcriptome enrichment analysis and western blot analysis confirm that OXH suppresses the NF-κB/MAPK pathway, which is consistent with our in vitro findings.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guo, Sun, Guo, Wu, Yan, Liu, Li, Sun, Fan, Zhang, Sun, Wang and Yin.)

Introduction: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis , M. bovis , and BCG, poses a potential challenge., Methods: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively., Results: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis , 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica , Mycoplasma bovis , Haemophilus parasuis , Escherichia coli , Pasteurella multocida , Ochrobactrum anthropi , Salmonella choleraesuis , Brucella melitensis , and Staphylococcus aureus , and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 10 3 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis , M. bovis , and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively., Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis , M. bovis , and BCG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qu, Liu, Sun, Liu, Liu, Hu, Jiang, Qi, Nan, Yan, Sun, Shao, Li, Sun, Zhang and Fan.)