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Brucella poses a great threat to animal and human health, and vaccination is a good way of controlling the bacterium. In this study, the immune response and protection ability of a recombinant adenovirus Ad-LL/BP containing L7/L12 and BCSP31 proteins of Brucella abortus (B. abortus) were evaluated in BALB/c mice model. Firstly, Adenovirus vector Ad-CMV and the recombinant adenovirus Ad-LL/BP were amplified in HEK 293AD cells. The TCID50 values of Ad-LL/BP and Ad-CMV were 10−8.68/0.1 mL and 10−8.35/0.1 mL, respectively. Mice were inoculated with 100 TCID50/mouse of Ad-LL/BP or Ad-CMV. Vaccination of mice with Ad-LL/BP vaccine was able to elicit higher IgG, IgG1 and IgG2a antibody levels when compared with Ad-CMV and PBS control animals (P0.05). Ad-LL/BP vaccine was able to reduce significantly the numbers of B. abortus in the spleens from the immunized mice. These results indicated that the recombinant Ad-LL/BP vaccine induced mainly cell-mediated immunity and partly humoral immunity, and provided moderately protection against B. abortus infection. Therefore, the vaccine could be further developed into a live-vector vaccine against B. abortus.

• Both OMP2b and BCSP31 proteins have several dominant T/B cell epitopes. • Each of OMP2b and BCSP31 protein has one T-B combined epitope. • T-B combined epitopes of OMP2b and BCSP31 have immunogenicity. • CTL epitopes of OMP2b and BCSP31 have immunogenicity. Brucella poses a serious threat to human health. High quality vaccines for Brucella are urgently needed to effectively reduce the incidence of brucellosis. OMP2b and BCSP31 are important component proteins of the Brucella outer membrane and are highly immunogenic. Here, we used the bioinformatics software ProtParam, SOPMA, SWISS-MODEL, Rasmol, BepiPred, SYFPEITHI and IEDB to analyze the structure of these two proteins and predict the epitopes of T cells and B cells. Through analysis, we predicted three Th cell epitopes, seven CTL epitopes, eight B cell epitopes, and one T-B combined epitope of OMP2b protein. Subsequently, we also obtained three Th cell epitopes, six CTL epitopes, nine B cell epitopes and one T-B combined epitope of BCSP31 protein. The T-B combined epitopes and CTL epitopes of OMP2b and those of BCSP31 were synthesized to detect their immunogenicity. The IFN-γ ELISPOT assay showed that the T-B combined epitope peptides of OMP2b and BCSP31 activated Th cell immune responses. ELISA analysis detected the specific antibodies against the T-B combined epitope peptide of OMP2b and BCSP31 in the serum of Brucellosis patients. Additionally, CTL epitope peptide of OMP2b and BCSP31 proteins promoted the secretion of soluble perforin and granzyme B in the culture supernatant. In conclusion, our study shows that the T-B combined epitopes and CTL epitopes of OMP2b and BCSP31 have immunogenicity and immunoreactivity. Our results may lay a theoretical foundation for the development of vaccines against Brucella. [ABSTRACT FROM AUTHOR]

This study identified and characterized Brucella species in the informal milk chain in Uganda. A total of 324 cattle bulk milk samples were screened for the genus Brucella by real-time PCR with primers targeting the bcsp31 gene and further characterized by the omp25 gene. Of the samples tested, 6.5% were positive for Brucella species. In the omp25 phylogeny, the study sequences were found to form a separate clade within the branch containing B. abortus sequences. The study shows that informally marketed cattle milk in Uganda is a likely risk factor for human brucellosis and confirms that B. abortus is present in the cattle population. This information is important for potential future control measures, such as vaccination of cattle.

Abstract This study aimed to develop direct PCR methods, which enable the diagnosis of brucellosis agents from ruminant aborted fetus samples at species and genus levels, and determine the applicability of the newly developed methods. For this purpose, 137 lung, 137 liver, and 52 fetal stomach fluid samples belonging to 166 ruminant aborted fetuses (326 samples in total) were examined. Firstly, agent isolation and identification were performed and species-specific multiplex PCR (m-PCR) from the culture was applied to the samples. In addition, the Mayer-Scholl m-PCR method was modified and termed 'modified Mayer-Scholl', and genus specific Bcsp31 PCR was also modified with minor changes. Four different methods were applied to direct examination samples and the obtained results were compared. The conventional culture method was set as the standard method to which sensitivities and specificities of the molecular methods were calculated. According to the assessments on the basis of fetus (n = 166), sensitivity and specificity values for modified Mayer-Scholl m-PCR method were 94.11% and 98.76%, and the same indicators for the modified Bcsp31 PCR were 95.29% and 98.76%, respectively. When all organ samples were taken into account (n = 326), sensitivity and specificity values for the modified Mayer-Scholl m-PCR method were 85.38% and 98.06%, and for the modified Bcsp31 PCR, they were 83.62% and 98.06%, respectively. As a result, it was found that the diagnostic power of the tests were 'high' when results were evaluated at fetus level. On the other hand, it was found to be 'clinically useful' when evaluated at organ level. We concluded that species level identifications can be made through the modified Mayer-Scholl method, which is a direct m-PCR method, with a high diagnostic power by specifying DNAs belonging to Brucella species directly from clinical samples. Highlights • Multiplex PCR is applicable to the direct diagnosis of brucellosis from clinical samples • The sensitivity and specificity values of the PCR are considered as satisfactory • For the diagnosis of brucellosis, first, the fetal stomach fluid should be preferred • Then, lung and liver tissues should be opted for diagnosis, respectively [ABSTRACT FROM AUTHOR]

Background & Objective: One of the most common diseases between zoonosis - especially in developing countries – is brucellosis. Identification of Brucella cell antigen combinations in terms of the amount and type of immune response in infected hosts, are important in vaccine design. 31kDa Brucella cell surface protein (BCSP31) is shared among all Brucellae. We aimed to define specific immune responses to BCSP31 which are elicited during infection of murine host with B. abortus. Surface protein of 31 kDa (BCSP31) is present in all strains, and here, the host immune response during murine infection with Brucella abortus which are formed in proportion to the proteins are studied. Materials & Methods: Recombinant BCSP31 (rBCSP31) of B. abortus was produced and purified. One group of BALB/c mice were infected with pathogenic B. abortus 544 and maintained for 60 days; a no-infected group of mice also was considered. Specific serum antibodies directed to rBCSP31 was evaluated through ELISA. Splenocytes of mice were cultured and stimulated with rBCSP31; thereafter, IL-4, IL-12 and IFN-γ cytokine responses of spleen lymphocytes were assessed. Results: We detected a remarkable IgG titer along with IgG1 and IgG2a specific to recombinant BCSP31 in serum samples from infected mice. Significant titers of IgG, IgG1 and IgG2a antibodies than BCSP31 in the the serum of mice infected with the virulent strain were observed. The Evaluation of Splenocytes responses to rBCSP31 also showed a significant IL-12 and IFN-γ production along with remarkable IL-4 production in infected mice. All responses were significantly different from that of non-infected mice (p

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10 CFU and 2.28 × 10 CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen. [ABSTRACT FROM AUTHOR]

This study identified and characterized Brucella species in the informal milk chain in Uganda. A total of 324 cattle bulk milk samples were screened for the genus Brucella by real-time PCR with primers targeting the bcsp31 gene and further characterized by the omp25 gene. Of the samples tested, 6.5% were positive for Brucella species. In the omp25 phylogeny, the study sequences were found to form a separate clade within the branch containing B. abortus sequences. The study shows that informally marketed cattle milk in Uganda is a likely risk factor for human brucellosis and confirms that B. abortus is present in the cattle population. This information is important for potential future control measures, such as vaccination of cattle. [ABSTRACT FROM AUTHOR]

Background & Objective: One of the most common diseases between zoonosis - especially in developing countries -- is brucellosis. Identification of Brucella cell antigen combinations in terms of the amount and type of immune response in infected hosts, are important in vaccine design. 31kDa Brucella cell surface protein (BCSP31) is shared among all Brucellae. We aimed to define specific immune responses to BCSP31 which are elicited during infection of murine host with B. abortus. Surface protein of 31 kDa (BCSP31) is present in all strains, and here, the host immune response during murine infection with Brucella abortus which are formed in proportion to the proteins are studied. Materials & Methods: Recombinant BCSP31 (rBCSP31) of B. abortus was produced and purified. One group of BALB/c mice were infected with pathogenic B. abortus 544 and maintained for 60 days; a no-infected group of mice also was considered. Specific serum antibodies directed to rBCSP31 was evaluated through ELISA. Splenocytes of mice were cultured and stimulated with rBCSP31; thereafter, IL-4, IL-12 and IFN-γ cytokine responses of spleen lymphocytes were assessed. Results: We detected a remarkable IgG titer along with IgG1 and IgG2a specific to recombinant BCSP31 in serum samples from infected mice. Significant titers of IgG, IgG1 and IgG2a antibodies than BCSP31 in the the serum of mice infected with the virulent strain were observed. The Evaluation of Splenocytes responses to rBCSP31 also showed a significant IL-12 and IFN-γ production along with remarkable IL-4 production in infected mice. All responses were significantly different from that of non-infected mice (p<0.05). Conclusion: These findings suggest that specific humoral and cell-mediated responses to BCSP31 is formed during murine host infection with B. abortus. Based on these findings, rBCSP31 can be used in further design of immunogenic strategies for vaccination against brucellosis. [ABSTRACT FROM AUTHOR]

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis. [ABSTRACT FROM AUTHOR]

Abstract: Culture is considered as the reference standard assay for diagnosis of Brucella spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan® probes and targeting the 3 following specific genes: (i) the insertion sequence IS711, (ii) bcsp31 and (iii) per genes for the detection of Brucella at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 Brucella strains, including all species and biovars. The analytical specificity was evaluated on a collection of 68 clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of Brucella ovis, B. melitensis bv. 1, B. abortus bv. 1 and B. canis reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All Brucella species DNA were amplified in the three tests. However, the earliest signal was observed with the IS711 real-time PCR, where it varied according to the IS711 copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS711 presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella. [Copyright &y& Elsevier]

The aim of this study was to develop a LightCycler-based real-time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Brucella DNA in serum samples. Following amplification of a 223-bp gene sequence encoding an immunogenetic membrane protein (BCSP31) specific for the Brucella genus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. The intra- and inter-assay variation coefficients were 1.3% and 6.4%, respectively, and the detection limit was 5 fg of Brucella DNA (one genome equivalent). After optimisation of the PCR assay conditions, a standard curve was obtained with a linear range (correlation coefficient = 0.99) over seven orders of magnitude from 107 to 10 fg of Brucella DNA. The LC-PCR assay was found to be 91.9% sensitive and 95.4% specific when tested with 65 negative control samples and 62 serum samples from 60 consecutive patients with active brucellosis. The assay is reproducible, easily standardised, minimises the risk of infection in laboratory workers, and has a total processing time of < 2 h. It could therefore form a promising and practical approach for the rapid diagnosis of human brucellosis. [ABSTRACT FROM AUTHOR]

As part of a program to support the USDA Animal Plant Health Inspection Service Bovine Brucellosis Eradication Program, the Brucellosis Research Unit of the National Animal Disease Center (NADC) sought to develop a bovine brucellosis vaccine that would allow vaccinated animals to be distinguished from virulent field infected animals. In order to meet that goal, several avenues of research were undertaken to construct and test candidate vaccines, including Brucella abortus RB51. In early vaccine development studies, a subunit preparation obtained by extracting B. abortus with salts was studied as a candidate subunit vaccine. Later, molecular biological techniques were used both to clone genes encoding products found in the salt extract (BCSP31 and Cu–Zn SOD) and genes encoding proteins of B. abortus that were antigenic (HtrA) or possibly essential (two-component systems) for full virulence of B. abortus. In vitro systems using mammalian cells lines such as HeLa and macrophage-related were used along with the mouse model and host animal models. Results obtained at NADC and in other Brucellosis research laboratories, using survival in mammalian cell lines and the mouse model to access pathogenicity and virulence of genetically engineered strains, do not necessarily identify loci that are essential for full virulence or pathogenicity in the natural host, the bovine. Studies at NADC and other brucellosis laboratories showed that antigenicity was not a predictor of the effectiveness of a protein as a subunit vaccine. [Copyright &y& Elsevier]

The article discusses a study that examined the molecular characterization of the contagious disease Brucella abortus from cattle and buffaloes. Topics covered include the inoculation of 84 samples of vaginal mucus, placenta, cotyledons and fetal stomach contents from aborted cattle and buffaloes using Brain Heart Infusion as the selective media, and the effectiveness of the use of polymerase chain reaction in the diagnosis of brucellosis.