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Summary Brucellosis, caused by Gram-negative Brucella , spreads in human and animal populations through contact with infected animals and products. Developing a rapid and sensitive detection technology for pathogen is crucial to reduce the risk of this disease transmitting between animal populations and to humans. We produced a monoclonal antibody LPS-6B5, which shows high affinity to LPS and limited cross-reactivity with other bacteria. Based on LPS-6B5, a colloidal gold immunochromatographic assay (GICA) was developed which demonstrates high sensitivity and specificity in detecting cultured B. melitensis , B. abortus and B. suis . The Gold Immunochromatographic Assay (GICA) strips exhibited the most sensitive detection limits, with a value of 7.8125 × 10 5 CFU/mL for Brucella melitensis , surpassing the sensitivity levels observed for Brucella abortus and Brucella suis . It is also suitable for clinical and field samples, providing a cost-effective and user-friendly alternative to traditional methods.

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate’s potential.Highlights• Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target• Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies• Molecular docking simulation method to rank based vaccine candidate and understand their binding modes

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2–3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species.

Swine brucellosis, caused by , is a worldwide infectious zoonotic disease. Currently, there are no available human or porcine vaccines to protect against infection, which is primarily acquired through the mucosa. We recently described MapB, the homologous protein of TamB, the inner membrane component of the TAM system. Our findings indicate that MapB is involved in bacterial cell envelope homeostasis. In this study, we characterize the outer membrane vesicles (OMVs) of 1330 (wt) and those of Δ (Δ ) mutant strain and evaluate their vaccine potential in mice. OMVs were isolated using the ultracentrifugation method and characterized through electron microscopy, Dynamic Light Scattering, SDS-PAGE and proteomics. Immunogenicity was assessed by intramuscular immunization of mice with wt OMVs or Δ OMVs, followed by the measurement of antigen-specific antibody levels and functional assays to evaluate the protective capacity of the antibodies. Cellular immunity was assessed by characterizing cytokine secretion through ELISA after stimulation of spleen cells with heat-killed . To determine the level of protection conferred by immunization, mice were challenged with virulent via intraperitoneal or intratracheal routes, and the bacterial load was quantified post-challenge. Dynamic Light Scattering of the OMVs from both strains revealed the presence of spherical structures of 90-130 nm. Proteomic analysis identified 94 and 95 proteins in the wt and Δ OMVs, respectively, including several known immunogens. Both OMVs showed immunoreactivity with sera from -infected pigs. Intramuscular immunization of mice with both OMVs induced antigen-specific IgG in serum, with the Δ OMVs group showing higher titers compared to the wt OMVs group. Serum antibodies from both OMVs groups reduced adherence and invasion of lung epithelial cells and enhanced its phagocytosis by macrophages. Upon antigen stimulation, spleen cells from mice immunized with Δ OMVs secreted higher levels of interleukin-17 and especially gamma interferon compared to cells from mice immunized with wt OMVs, suggesting the induction of a stronger T helper 1 response in the Δ OMVs group. While immunization with both wt and Δ OMVs achieved the same level of protection following intratracheal infection with (p<0.01), immunization with Δ OMVs provided higher levels of protection against intraperitoneal infection. Overall, these results demonstrate that the Δ OMVs are immunogenic and capable of inducing both cellular and humoral immune responses that protect against mucosal and systemic challenges.

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

Brucella is a genus of gram-negative bacteria that cause brucellosis . B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2Δ wbkF ) or only producing internal O-chain precursors (Bs2Δ wzm ) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2Δ wadB and Bs2Δ wadD ). We also investigated mutants in the pyruvate phosphate dikinase (Bs2Δ ppdK ) and phosphoenolpyruvate carboxykinase (Bs2Δ pckA ) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (10 8 and 10 5  CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2Δ wbkF , Bs2Δ wzm, Bs2Δ wadB and the Rev1 control (10 5  CFU). As described before for B. abortus , B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2Δ ppdK Δ pckA had the same metabolic phenotype as Bs2Δ ppdK and ppdK mutation was enough to generate attenuation. At 10 5  CFU, Bs2Δ ppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2Δ wbkF , the latter seems a better R vaccine candidate than Bs2Δ wzm . However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2Δ wbkF is advantageous over Bs2Δ wadB or Bs2Δ ppdK requires experiments in the natural host.

Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella , livestock’s reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene’s expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein.

Purpose The goal of this study was to develop a plasmid-based lux bio-reporter for use to obtain in vivo images of Brucella suis vaccine strain 2 ( B.suis S2) infection with high resolution and good definition. Procedures The pBBR- lux (pBBR1MCS-2-lxCDABE) plasmid that carries the luxCDABE operon was introduced into B. suis S2 by electroporation yielding B. suis S2- lux . The spatial and temporal transit of B. suis S2 in mice and guinea pigs was monitored by bioluminescence imaging. Results The plasmid pBBR-lux is stable in vivo and does not appear to impact the virulence or growth of bacteria. This sensitive luciferase reporter could represent B. suis S2 survival in real time . B. suis S2 mainly colonized the lungs, liver, spleen, and uterus in mice and guinea pigs as demonstrated by bioluminescence imaging. Conclusion The plasmid-based lux bioreporter strategy can be used to obtain high resolution in vivo images of B. suis S2 infection in mice and guinea pigs.

During a 4-year (2007–2010) survey, the presence of Brucella suis infection in domestic pigs in Sardinia was investigated. Serum samples were collected from breeding pigs located on 108 commercial farms with documented reproductive problems and analysed using the Rose Bengal (RBT) and complement fixation (CFT) tests for screening and confirmation of Brucella, respectively. Of the 1251 serum samples analysed by RBT, 406 sera, originating from 36 farms, were positive for B. suis. CFT was positive in 292/748 sera analysed, confirming positivity in all 36 pig herds. Pigs with international complement fixation test units per ml (ICFTU/ml) values ⩾160 were slaughtered, and their organs collected for bacteriological examination and testing by polymerase chain reaction (PCR). Brucella spp. strains were isolated in culture from 13/502 organs analysed, and subsequently identified as B. suis biovar 2. PCR detected positivity to Brucella spp. in 19/285 organs analysed. These results confirm the presence and emergence of B. suis infection in domestic pigs in Sardinia.

Brucellosis, in particular infections with Brucella abortus, Brucella melitensis or Brucella suis, remains a significant human health threat in many areas of the world. The persistence of pathogenic Brucella spp. in domestic livestock or free-ranging wildlife remains unresolved, despite decades of regulatory efforts worldwide. Although vaccination is probably the most economic control measure, administration of currently available vaccines alone is not sufficient for elimination of brucellosis in any host species. Complacency in brucellosis control programs usually results in failure, or at best, limited reductions in disease prevalence or incidence of human infections. New brucellosis vaccines with high efficacy and safety are needed that address the diversity in host species and can be more widely applied under field conditions. Development of safer and more efficacious vaccines alone, or combined with enhancements or increased emphasis on other regulatory program components, could have tremendous impact on reducing the worldwide prevalence of brucellosis and the associated zoonotic infections.