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Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway.

Since Brucella infection mostly occurs through the mucosal surfaces, immune response induced by vaccine that is delivered by a way of mucosal route can be drastically enhanced to control the brucellosis. Omp31is the major outer membrane protein of Brucella , and is considered as a protective antigen against Brucella infection. Accordingly, Lactococcus lactis has been used as an antigen-delivering vector to develop a vaccine-induced mucosal response for having a safer vaccination against brucellosis. A designed omp31 gene fused to the usp45 signal peptide and M6 cell wall anchor was sub cloned in the pNZ7021 expression vector, and a recombinant L . lactis displaying Omp31 was constructed. Omp31 protein expression was confirmed using Western blotting and immunofluorescence analysis. Animals were orally and intraperitoneally immunized with live or killed L . lactis expressing Omp31, respectively. The humoral and cellular immune responses were evaluated by measuring the specific cytokines and antibodies. sIgA, serum IgA, IgM, and total IgG antibodies significantly increased in the mice immunized with live recombinant L . lactis expressing Omp31 and also serum IgM, and total IgG antibodies significantly increased in mice immunized with killed recombinant L . lactis expressing Omp31. Among IgG subtypes, IgG2a response was significantly higher in both groups compared to IgG1. In mice groups immunized with recombinant L . lactis , the IFN-γ and IL-10 level elevated; however, there was no change in the level of IL-4. These results indicated that recombinants L . lactis induce both humoral and cellular immune responses in mice, and also vaccines based on L . lactis -derived live carriers are promising interventions against Brucella melitensis infections.

Outer membrane proteins (OMPs) of microorganisms play important roles in directly interacting with host cells. Brucella species inhibit the apoptosis of host cells to benefit their own intracellular survival and replication. However, the association between OMP31 of Brucella and host cell apoptosis, and the underlying mechanism are unclear. In this study, an OMP31 gene deletion mutant based on B. melitensis 16M was constructed. Following the infection of RAW264.7 cells with B. melitensis 16M or the mutant strain, colony formation, apoptosis, tumor necrosis factor (TNF)-α levels and the levels of key downstream factors of the apoptosis pathways triggered by TNF-α, namely caspase-3, −8 and −9, cytochrome c, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected. The mutant strain was shown to have the same phenotype as the parent strain using traditional microbiological tests. However, the mutant strain had impaired intracellular survival, with higher levels of apoptosis and TNF-α expression in infected RAW164.7 macrophages than the parent strain. The downstream factors of apoptosis triggered by TNF-α, including increased caspase-8, −3 and −9, cytochrome c and Bax, and decreased Bcl-2, indicated that the classical and mitochondrial cell death pathways were involved. It may be concluded that OMP31 from Brucella inhibited apoptosis and benefitted the intracellular survival of this microorganism. Furthermore, TNF-α may have served as a switch triggering classical death and mitochondrial cell death pathways.

Vaccination is one of the most effective means to protect humans and animals against brucellosis. Live attenuated Brucella vaccines are considered effective in animals but they may be potentially infectious to humans, so it is vital to improve the immunoprotective effects and safety of vaccines against Brucella. This study was designed to evaluate the immunogenicity of DNA vaccines encoding outer membrane proteins (Omp25 and Omp31) against Rev1 in a mouse model. For this propose, Omp25 and Omp31 genes were cloned (individually and together) into the eukaryotic expression vector pcDNA3.1/Hygro (+). Expressions of recombinant plasmids were confirmed by SDS-PAGE and Western blot analysis. Six groups of BALB/c mice (seven mice per group) were intramuscularly injected with three recombinant constructs, native pcDNA3.1/Hygro (+) and phosphate-buffered saline (PBS) as controls and subcutaneous injection of attenuated live vaccine Rev1. Results indicated that DNA vaccine immunized BALB/c mice had a dominant immunoglobulin G response and elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) compared to the control groups. Collectively, these finding suggested that the pcDNA3.1/Hygro DNA vaccines encoding Omp25 and Omp31 genes and divalent plasmid were able to induce both humoral and cellular immunity, and had the potential to be a vaccine candidate for prevention of infections.

Brucellosis is an infectious disease that affects practically all species of mammals, including human, and is a major zoonosis worldwide. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in phagocytic and nonphagocytic cells such as trophoblast and epithelial cells. Among the six recognized species of the genus Brucella, Brucella melitensis is the main etiological agent involved in goat brucellosis and is also the most pathogenic for human. It causes significant losses in livestock production as a result of abortions, metritis, infertility, and birth of weak animals. Outer membrane proteins (OMPs) are exposed on the bacterial surface and are in contact with cells and effectors of the host immune response, whereby they could be important virulence factors of Brucella species. To evaluate this hypothesis, the gene encoding for the major outer membrane protein Omp31 was amplified, cloned into pUC18 plasmid, and inactivated by inserting a kanamycin cassette, rendering pLVM31 plasmid which was transformed into B. melitensis wild-type strain to obtain LVM31 mutant strain. The Outer membrane (OM) properties of the mutant strain were compared with B. melitensis Bm133 wild-type and B. melitensis Rev1 vaccine strains, in assessing its susceptibility to polymyxin B, sodium deoxycholate, and nonimmune serum. The mutant strain was assessed in vitro with survival assays in murine macrophages J774.A1 and HeLa cells. Our results demonstrate that LVM31 mutant is more susceptible to polymyxin B, sodium deoxycholate, and nonimmune serum than control strains; moreover, Omp31 mutation caused a decrease in the internalization and a significant decrease in the intracellular survival compared with the reference strains in both cell lines. These results allow us to conclude that Omp31 is important for maintaining OM integrity, but also it is necessary for bacterial internalization, establishment and development of an optimal replication niche, and essential for survival and intracellular multiplication.

Background Designing a potent recombinant vaccine, using the appropriate subunits with the greatest effect on stimulating the immune system, especially in the case of intracellular pathogens such as gram negative Brucella Melitensis bacteria, is of great importance. In this study, three repeats of 27 amino acids of the immunogenic epitope derived from OMP31 antigen (3E) from the Brucella melitensis , in a protective manner against Brucellosis have been used. To fortify the delivery system of recombinant antigens, IL-2 cytokine as a molecular adjuvant was fused to recombinant constructs. Recombinant proteins were evaluated for immunological studies in a mouse model (BALB/c). Results The results showed that all recombinant proteins could stimulate the immune system to produce Th1 cytokines and antibodies in compare to the negative control treatments. 3E-IL2 and then OMP31-IL2 proteins stimulated higher levels of IFN-γ and IL-2 compared to the other treatments ( p  < 0.05). Also, the results indicated that experimental treatments produced a higher level of IgG2a isotype than IgG1 isotype. In addition, the findings of the experiment showed that the presence of chemical adjuvant (IFA) along with molecular adjuvant can play a significant role in stimulating the immune system. After determining the potency of recombinant structures, their efficacy in stimulating the immune system were also evaluated. B. melitensis M16 strain was used to challenge 30 days after last immunization. The microbial load of the splenocyte in the treatments receiving chimeric proteins were significantly lower. Also, Wright serological test confirmed that these treatments had the lowest agglutination rate, as well as the positive treatment, while in the negative treatments in excess of blood serum dilutions, agglutination rate were more than 2 + . Conclusions 3E-IL2 treatment showed the best performance compared to other recombinant proteins and could be considered as the suitable candidate for further research on the production of recombinant vaccine against Brucella.

Brucellosis is a zoonotic disease causes by Brucella bacteria. So far, there is not any efficient treatment for this infectious disease in animals, although subunit vaccines can be a safe alternative. To this aim, we have designed a new chimeric OMP25‐OMP31 antigen formulated in Chitosan nanoparticles and studied its protective efficiency in vivo. OMP25–OMP31 was produced using spliced overlap extension by polymerase chain reaction and the 3D protein structure and antigenic ability were predicted using computational tools. In addition, the humoural and cellular immune responses were measured by ELISA in six different experimental groups. The immune response showed chimeric rOMP25–OMP31 antigen‐induced higher titers of IFN‐γ and TNF‐α cytokines, while the lowest amount of IL‐4 was dedicated to itself. Also, rOMP25–OMP31 stimulated higher titer of IgG than individual injection of rOMP25 and rOMP31 treatments and the cell proliferation assay demonstrated the vaccination with rOMP25–OMP31 elicits a vigorous antigen‐specific cell proliferative. In addition, the challenge experiment showed immunised mic stimulated a higher level of protection than negative controls. Overall, the results of rOMP25–OMP31 could be promising for considering chimeric constructs as a feasible vaccine candidate for further investigations against brucellosis. Chimeric antigen induced better immune response.

Background Brucellosis is a severe zoonotic disease worldwide. Detection and identification of Brucella species are essential to prevent or treat brucellosis in humans and animals. The outer membrane protein-31 (Omp31) is a major protein of Brucellae except for B. abortus, while the Omp31 antigenic epitopes have not been extensively characterized yet . Results A total of 22 monoclonal antibodies (mAbs) were produced against Omp31 of Brucella ( B. ) melitensis , of which 13 recognized five linear epitopes, 7 reacted with semi-conformational epitopes and 2 reacted with conformational epitopes, respectively. The mAb isotypes were 11 (50%) IgG2a, 5 (23%) IgG1 and 6 (27%) IgM. On the basis of epitope recognition and reactivity levels, 8 mAbs including 3 IgM and 5 IgG clones were considered as highly reactive and potentially diagnostic antibodies. Among these mAbs, 7A3 (IgG1), 5B1 (IgG2a), 2C1 (IgG2a) and 5B3 (IgG2a) reacted with differently conserved linear epitopes of B. melitensis , B. ovis , B. suis and B. canis strains, while 5H3 (IgG2a) highly reacted with a conformational epitope of Omp31 when tested with several immunoassays. Conclusions These potent monoclonal antibodies can be used for identifying Omp31 antigens or detecting B. melitensis and other Brucella species beyond B. abortus in vitro or in vivo.

•An attenuated Brucella melitensis vaccine M5-90 has been mostly used for animals in China.•Immune response to BP26 and OMP31 of the vaccine was evaluated in sheep.•Specific antibody and IFN-γ response were detected at low levels in vaccinated sheep.•Protective efficacy of the vaccine was overall weak and shortlived.•These data are helpful to design a safer and more effective B. melitensis vaccine. In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P=0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.

Brucellosis is one of the leading causes of abortion in domestic animals that imposes costs on both economy and society. The disease is highly zoonotic and poses risk to animal handlers due to its zoonotic nature. It causes stillbirth, loss of kids and abortion in last term of pregnancy. Reproductive damage includes infertility in does and orchitis and epididymitis in breeding bucks, which result in high financial losses to farmers and the agriculture industry as a whole. It requires highly sensitive and specific assays to diagnose the disease at field level. In the current study, a visual loop-mediated isothermal amplification (LAMP) assay and the TaqMan® real-time PCR were developed with high sensitivity and specificity. For the TaqMan® probe, real-time PCR primers were developed using Omp31 gene as target and primers were designed using discontiguous conserved sequences of Omp31 gene. The Omp31 probes were designed by attaching 6-FAM reporter dye at the 5′ end and BHQ-1 quencher at the 3′ end. Published primers were used for visual LAMP assay targeting the Omp25 gene. Sensitivity of the standardized visual LAMP assay and TaqMan® real-time PCR assay was determined by serial dilution of positive Brucella melitensis DNA (10 2 to 10 −4  ng) obtained from standard culture. The TaqMan® probe real-time assay can detect as low as 100 fg of B. melitensis DNA, whereas culture from vaginal swab washings has a limit of detection (LOD) of only 1 cfu/ml. Similarly, the visual LAMP assay can detect as low as 10 fg of B. melitensis DNA as compared to an LOD of 30 cfu/ml from culture of vaginal swab washings. Both assays were compared with serological tests (serum tube agglutination test (STAT) and indirect enzyme-linked immunosorbent assay (iELISA)) for diagnostic sensitivity and specificity. Diagnostic sensitivities and specificities for TaqMan® real-time PCR vs. LAMP assays were 98 and 100% vs. 100 and 97.8%, respectively. Results of visual LAMP assay indicated that LAMP is a fast, specific, sensitive, inexpensive and suitable method for diagnosis of B. melitensis infection under field conditions. On the other hand, Omp31 TaqMan® probe real-time assay can be used in conjunction with the other field-based diagnostic tests due to its high specificity.