Amitriptyline hydrochloride (AMT), a tricyclic antidepressant, is known to exhibit antimicrobial effects against a wide range of bacterial species. This study aims to evaluate the effect of AMT on Brucella (B.) abortus infection in RAW 264.7 cells and ICR mice, which has not yet been clearly characterized. The results showed that all tested concentrations of AMT had no direct bactericidal effect on B. abortus survival at any incubation time point. Interestingly, RAW 264.7 cells pre-treated with a non-toxic high concentration of AMT before B. abortus infection showed a significant reduction in the phagocytosis of B. abortus at 20 min post-infection, compared to untreated cells. However, AMT treatment did not affect the intracellular replication of B. abortus compared to the control cells. Based on the reduced bacterial uptake observed in-vitro, an in-vivo experiment was conducted to assess whether daily oral administration of AMT at a dose of 20 mg/kg could inhibit B. abortus growth in ICR mice. The results showed that AMT treatment slightly increased both organ weights and bacterial loads, suggesting possible systemic effects of prolonged AMT exposure. In summary, these preliminary results provide initial insight into the potential effects of AMT on B. abortus infection both in-vitro and in-vivo. Therefore, further study should focus on dose optimization in-vivo and exploration of the underlying cellular mechanisms involved in AMT-mediated inhibition of phagocytosis during Brucella infection.

The primary therapeutic approach for Brucella species infections has mainly been based on antibiotic treatment. However, the development of vaccines for brucellosis control remains controversial. Furthermore, there is currently no licensed vaccine available for human brucellosis. This study aims to evaluate the effect of a combination of recombinant protein vaccines against Brucella (B.) abortus infection using a mouse model. Two B. abortus genes, namely dapB and gpm, were cloned and expressed in competent Escherichia (E.) coli DH5α using the pCold-TF vector. Successfully cloned vectors were subjected to PCR amplification using specific primer pairs. The apparent sizes of dapB and gpm were detected at 807 bp and 621 bp, respectively. Besides, the purified recombinant proteins dapB and gpm were detected using SDS-PAGE electrophoresis with correct sizes of 82.86 kDa and 87.61 kDa, respectively. These recombinant proteins were used to immunize mice as a combined subunit vaccine (CSV) to elicit host immunity against B. abortus infection. Mice immunized with CSV exhibited increased proliferation of CD4+ and/or CD8+ T cells at week 7th and 9th before sacrifice, in comparison to the control group. Notably, CSV immunization showed a significant decrease in bacterial burden in the spleen compared to the control group. Altogether, CSV using dapB and gpm induced host adaptive immune response against Brucella infection, suggesting its potential as an effective new subunit vaccine candidate.

Extensive research and testing continue to be conducted for the development of vaccines targeting zoonotic diseases such as brucellosis. In this study, the potential of the DapB as a recombinant protein vaccine to effectively combat Brucella abortus 544 infection in BALB/c mice was evaluated. Western blotting assay results showed that recombinant protein DapB reacted with Brucella-positive serum, indicating its potential immunoreactivity. In vivo results showed that the peripheral blood CD4+ and CD8+ T cell population significantly increased in the DapB-immunized mice group after the first, second and third blood collection, compared to the control group that received PBS. Additionally, at the fourth blood collection, an increase in CD4+ T cell activation was observed in three vaccination groups compared to PBS negative control group. These results indicate the potential of DapB in stimulating cellular immunity. Fourteen days after infection, the bacterial load in the spleen was evaluated. The reduction in bacterial replication in the spleen by both DapB and RB51 highlights their protective efficacy against Brucella infection. These findings contribute to the ongoing efforts in developing effective vaccines against brucellosis and provide valuable insights for further research in this field.

Subunit vaccines are being developed as a potential therapy for preventing microbial pathogen infection. In this study, the immunogenicity of recombinant Brucella (B.) abortus Fe/Mn superoxide dismutase (rFe/Mn SOD) protein as a subunit vaccine against B. abortus was investigated in BALB/c mice model. Brucella Fe/Mn SOD gene was cloned into a pcold-TF DNA vector. The bacterial recombinant protein was expressed using the Escherichia coli DH5α strain with a size of 82.50 kDa. The western blotting assay showed that rFe/Mn SOD reacted with Brucella-positive serum, indicating the potential immunoreactivity of this recombinant protein. After the second and third vaccinations, the peripheral CD4+ T cell population was increased significantly in the rFe/Mn SOD-immunized mice group compared to the PBS control group. Moreover, immunization of this recombinant protein increased the CD4+ T cell population from the first vaccination to the third vaccination. Meanwhile, the CD8+ T cells were slightly enhanced after the second vaccination compared to the first vaccination and compared to control groups. Fourteen days after the bacterial infection, the splenomegaly and the number of bacteria in the spleen were evaluated. The result showed that both rFe/Mn SOD and positive control RB51 decreased the bacterial replication in the spleen and the splenomegaly compared to control groups. Altogether, these results suggested that rFe/Mn SOD could induce host immunity against B. abortus infection.

We investigated the effect of a synthetic complement peptide C3a on the outcome of Brucella abortus 544 infection in a murine macrophage cell line RAW264.7 cell. First, we determined the highest non-cytotoxic concentration of the peptide in the cell line. We also found that the peptide significantly increased the growth of the bacteria at 8 and 24 h. Although the number of bacterial CFU was also elevated at 48 and 72 h, the increases were not significant as compared to controls. We further investigated the effect of C3a peptide on the growth of Brucella by pre-incubating the peptide at various temperatures and found that the effect was reversed at 24 h post-incubation suggesting that incubation of peptide at high temperatures including 65°C or 95°C could inactivate its action. This also could indicate the beneficial effect of high temperature during infection. Although several studies reported the inhibitory effect of different antimicrobial peptides including C3a, the present study preliminarily revealed that it had no positive contribution on the control of B. abortus 544 infection in vitro and indirectly to its receptor, CD88, which belongs to GPCR. Moreover, the encouraged further exploration of the effect of other similar peptides would be performed for the purpose of finding Brucella-host cell interaction for the control of disease progression.

This study aims to investigate the effects of exogenous succinic acid (SCA) on Brucella (B.) abortus infection in macrophage RAW 264.7 cells and ICR mice. Firstly, the in vitro experiment was conducted by MTT cytotoxicity and bacterial internalization assay to evaluate the uptake of B. abortus into macrophage cells. Two non-cytotoxic concentrations of SCA demonstrated attenuated invasion of Brucella into macrophages at 30 and 45 min post- infection (pi). Secondly, ICR mice were treated with SCA and infected with B. abortus. On day-14 pi, spleen and blood serum were collected to evaluate the bacterial burden and total spleen weight as well as the production of cytokine/chemokine, respectively. The results showed that SCA treatment promoted bacterial growth and reduced the total spleen weight in mice. Furthermore, SCA treatment increased the level of IL-10 cytokine in the sera, while dampening the production of MCP-1 chemokine compared to the control. The results of bacterial load in spleen and spleen weight together with cytokine/chemokine production profile in the sera indicated that SCA induced the host anti-inflammatory response which is beneficial for the survival of Brucella. Therefore, these findings suggest that SCA contributed to host immunity against Brucella infection and the emerging potential topic-immunometabolism should be invested for further investigations.

This study investigated the efficacy of four Brucella (B.) abortus recombinant proteins, namely adenylate kinase (Adk), nucleoside diphosphate kinase (Ndk), 50S ribosomal protein (L7/L12) and preprotein translocase subunit (SecB), as a combined subunit vaccine (CSV) against B. abortus infection in BALB/c mice. Immunoblotting assay showed that these four recombinant proteins as well as pcold-TF vector reacted individually with Brucella-positive serum, but not with Brucella-negative serum. The peripheral blood CD4+ T cell population was increased in CSV-immunized mice compared to PBS and pcold-TF vector groups. In addition, CSV and pcold-TF groups displayed induced IgG1 and IgG2a antibodies production compared to PBS and RB51 group, whereas IgG2a titer was higher than IgG1 titer in CSV group. The secretion profiles of IgG1 and IgG2a production together with an enhancement of CD4+ T cell population suggested that CSV did not only induce T helper 1 (Th1) T cell immunity but also humoral immunity. Therein, Th1 T cell immunity is more predominant in eliminating intracellular bacteria B. abortus. Furthermore, CSV immunization significantly reduced the bacterial burden in the spleen as well as the spleen weight in comparison to PBS and pcold-TF groups. Altogether, combination of these antigens could be potential to induce protective immunity against B. abortus infection in animals.

BALB/c mice were vaccinated with Brucella (B.) abortus recombinant protein L27 (50S ribosomal protein L27) cloned into a pMal vector system. L27 was induced, purified and injected intraperitoneally (IP). Mice were vaccinated on 0-, 15- and 35-day. Serum cytokines were evaluated on 36- and 49-day from first vaccination. Mice were intraperitoneally infected with 5×104 CFU of virulent B. abortus 544 on day-50 and sacrificed after two weeks from infection. Bacterial burden from the spleen was quantified and showed a 0.7- and 0.9-log reduction in vaccinated mice in comparison to PBS and MBP (maltose binding protein) groups respectively. Cytokines in the serum demonstrated increased interferon-gamma (IFN-γ) and other pro-inflammatory cytokines such as macrophage chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6). On the other hand, interleukin 10 (IL-10) was attenuated in the sera of vaccinated mice. This cytokine profile is indicative of a cell-mediated type of immune response which is favorable for the eradication of intracellular infections. The current study showed the potential of another B. abortus ribosomal protein in inducing protective immunity against B. abortus infection.

In this study, we examined the protective immunity of a combination of seven Brucella abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), 50S ribosomal protein (50s rL7/L12), nucleoside diphosphate kinase (rNdk), malate dehydrogenase (rMDH), arginase (rRocF), and elongation factor (rTsf) cloned in a pMal vector system and expressed in DH5α. Mice groups were immunized thrice with a combined subunit vaccine (CSV-7) at 0, 2, and 5 weeks and subsequently challenged with B. abortus at 5 × 104 CFU at 6 weeks. At four weeks post-infection, the mice were sacrificed and the bacterial burden in their spleens was quantified. Results revealed bacterial log reductions of 0.63 and 0.34 in comparison to PBS and maltose-binding protein (MBP), respectively. Cytokine profiling revealed a marked increase in IFN-γ (interferon-gamma), MCP-1 (macrophage chemoattractant protein-1) and IL-6 (interleukin 6) cytokines at 5-weeks post-immunization. On the other hand, only TNF was heightened at 7-weeks post-immunization. In general, this cytokine profile is consistently reflective of a Th1 immune response, which is beneficial for host immunoresistance.

This study evaluated the protective effects of a combination of eight B. abortus recombinant proteins that were cloned and expressed into a pMal vector system and DH5α: nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12), malate dehydrogenase (rMDH), DNA starvation/stationary phase protection protein (rDps), elongation factor (rTsf), arginase (rRocF), superoxide dismutase (rSodC), and riboflavin synthase subunit beta (rRibH). The proteins were induced, purified, and administered intraperitoneally into BALB/c mice. The mice were immunized three times at weeks 0, 2, and 5 and then infected intraperitoneally (IP) with 5×104 CFU of virulent B. abortus 544 one week after the last immunization. The spleens were collected and the bacterial burden was evaluated at four weeks post-infection. The results showed that this combination produced a significant reduction of the bacterial burden in the spleen with a log reduction of 1.01 compared to the PBS group. Cytokine analysis revealed induction of the cell-mediated immune response in that TNF (tumor necrosis factor) and proinflammatory cytokines IL-6 (Interleukin 6) and MCP-1 (macrophage chemoattractant protein-1) were elevated significantly. In summary, vaccination with a combination of eight different proteins induced a significant protective effect indicative of a cell mediated immune response.

We investigated the effects of two Brucella proteins expressed in a pMAL expression system, RocF and EF-Ts, as subunit vaccines on immune modulation and protective efficacy using a mouse model. Mice vaccinated with MBP-RocF and MBP-EF-Ts displayed increased production of TNF, IFN-, MCP-1, IL-10 and IL-6, and TNF and MCP-1, respectively. Furthermore, mice vaccinated with MBP-EF-Ts showed decreased induction of IFN- and Th2-related cytokines, IL-10 and IL-6. Higher proportions of CD4+ and CD8+ T cells were observed in the blood of mice vaccinated with MBP-RocF than in the PBS-vaccinated group, although the increases were not significant. Furthermore, significantly reduced Brucella proliferation in the spleens of the MBP-RocF and MBP-EF-Ts groups were observed, but inflammation of these organs was not attenuated. Overall, these results indicate that RocF and EF-Ts could be potential subunit vaccine candidates against animal brucellosis.

Treatment of dextran sodium sulfate(DSS) on HeLa cells results to an enhanced susceptibility to Brucella(B.) abortus infection. An increase in the adherence, invasion and intracellular replication of B. abortus was observed in DSS-treated cells. Furthermore, a marked elevation in the intensity of F-actin fluorescence was also observed in DSS-treated cells compared with untreated B. abortus-infected cells. An upregulation of phagocytic signaling proteins by Western blot analysis demonstrated an apparent activation of ERK, p38α and JNK phosphorylation levels in B. abortus-infected DSS-treated cells compared with the control. Colocalization with LAMP-1 proteins was attenuated in DSS-treated cells upon intracellular trafficking of the pathogen compared with control cells. The results of this study demonstrated consistency with other pathogens. The uptake and intracellular replication of B. abortus is hypothesized to be stimulated by various dextran receptors such as C-type lectins that are involved in phagocytosis which can either be direct phagocytic receptors, modulators of the expression of other receptors or as opsonins leading to an enhanced internalization of B. abortus. The complexity of these interactions thus would warrant further investigation into the role of DSS in the pathogenesis of brucellosis. In summary, we conclude that DSS enhanced adhesion, phagocytosis and intracellular replication of B. abortus in epithelial cells which could lead to suppression of the innate immune system in chronic Brucella infection.

Fast, cheap and sufficient serodiagnostic tools needs to be developed for the early detectionof brucellosis. Currently the tools cannot differentiate an active infection from vaccinated, norcan it differentiate other bacterial infections with lipopolysaccharides, especially Yersiniainfections. In this study, we purified recombinant outer membrane protein 10 and 28(rOmp10,rOmp28), and a dipstick assay(indirect or sandwich) was constructed with single(rOmp10 orrOmp28) and combined rOmps(rOmp10 and rOmp28) from Brucella(B.) abortus 544 to evaluatebovine Brucella positive serum collected during the beginning of the Korean outbreak from2006 to 2015. In application with single rOmp, rOmp10(70%; indirect, 92.11%; sandwichdipstick) and rOmp28(72.5%; indirect, 86.84%; sandwich dipstick) had comparable results. Inaddition, results indicated that dipstick with combined rOmps(rOmps10 and rOmp28) weresuperior in detecting positive serum samples, at 85% indirect and 100% sandwich dipstick. Surprisingly, the results were the same in detecting negative results at 97.78% for both singleand combined indirect dipsticks. The dipstick tools with rOmp10 and rOmp28 would be usefulfor a rapid screen method for bovine brucellosis.

Brucella abortus is a well-known intracellular zoonotic pathogen. Despite significant research to understand the pathogenesis, underlying mechanisms of the bacterial infection are not yet understood. Thus, prevention and control of the B. abortus infection is problematic in animal and human. Therefore, several methods involving random mutation have been used to identify the mechanisms and provide a solution for control and prevention of Brucella infection. B. abortus mutants were generated by random insertion of a transposon, Ez-Tn5TM pMODTM-3 <R6Kγori/MCS> into a chromosome. Characteristics of these mutants were investigated using biochemical testing, growth features, determination of biovar, antibiotic resistance and detection of virulence factors, T4SS, PGK, and CGS. In biochemical testing, B. abortus mutants were categorized according to 7 groups with different condition of ILATk, SUTC, and ELLM. Different growth features were also observed between wild type and mutants. In addition, both B. abortus wild type and mutants were determined as biovar type 1 by biovar test. Three virulence factors, T4SS, PGK, and CGS were detected by PCR. Therefore, B. abortus mutants were characterized by analysis of phenotyping and it might be useful for further studies of known pathogenesis of B. abortus infection and for identification of diagnostic markers of brucellosis.

The bacterial lipopolysaccharide (LPS) mainly contributes to the structural integrity, survival and protection barrier against harsh environments. Therefore, the early stages in LPS or lipid A biosynthesis are attractive targets in the identification and development of inhibitors which would be effective against infections caused by Gram-negative bacteria. The bacterial outer membrane proteins (OMPs) meanwhile function as maintenance for structure, adhesion to other cells and substances, as well as development of resistance to antimicrobials. The LPS and LPS-related molecules, and OMPs are important immunogenic components of several important pathogens including Brucella, which have been extensively used in immunological studies and in the diagnosis of diseases. Here we review the importance, structure, functions and immunogenic aspects of LPS and OMPs particularly of Brucella which can be targeted for the prevention and diagnosis of brucellosis.

To date, most serodiagnostic methods for brucellosis screening are based on antibodies against lipopolysaccharides of Brucella spp. However, this approach has the drawback of yielding false-positive results due to cross-reactivity with lipopolysaccharides of other related pathogens, especially Yersinia enterocolitica O:9. In this study, Brucella abortus AspC was cloned and expressed by PCR amplification into a pCold TF expression system to obtain recombinant AspC (rAspC). The immunogenicity of rAspC was confirmed by western blotting of Brucella-positive bovine serum. rAspC-based ELISA was performed to determine whether rAspC could be used in the serodiagnosis of bovine brucellosis. rAspC reacted strongly with anti-Brucella antibodies in positive sera in the tube agglutination test (TAT), but did not show strong reaction with most negative samples. In particular, the average OD492 value at the highest TAT titer showed a 1.4-fold increase with respect to the cutoff value. The accuracy, specificity, and sensitivity of rAspC were 71.88%, 78.33%, and 68%, respectively. These findings suggest that rAspC might be valuable for the serological diagnosis of bovine brucellosis.

Brucellosis is an important and re-emerging zoonotic disease worldwide. The prevention of human infection is achieved predominantly through the control of brucellosis in agricultural animals, which in turn depends on accurate diagnosis and vaccination. However, conventional serological diagnosis of brucellosis has several limitations, and currently available vaccines for animals have several drawbacks, including the ability to cause infection in humans. Phosphoglycerate kinase (Pgk) is one of the specific proteins reactive with mouse sera in the early stage of Brucella infection, and deletion of the pgk gene in B. abortus strain 2308 resulted in extreme attenuation of this strain in vitro and in vivo. Furthermore, the B. abortus pgk mutant has been used as a live vaccine, and in challenge experiments, it induced protection that was superior to that conferred by commercial strains. In this study, the pgk gene from Brucella abortus 544 was successfully amplified and cloned into a maltose binding protein fusion protein expression vector (pMAL). The recombinant protein was expressed in Escherichia coli DH5α and purified. The immunogenicity of purified recombinant B. abortus 544 Pgk (rPgk) was evaluated by western blot analysis using Brucella-positive mouse sera. rPgk could be used as an antigenic component for future serological tests and potential vaccine development.

본 연구는 오배자 에탄올 추출물 (GRE), 염소산나트륨 (SC) 그리고 오배자 에탄올 추출물과 염소산나트륨 합제(GS)의 B. abortus에 대한 항균효과를 확인하기 위해 수행 되었다. GRE, SC 그리고 GS를 B. abortus에 처리하여 배양한 후, B. abortus의 생존수를 확인하였으며, 마우스 탐식세포 내 감염된 B. abortus의 증식 억제효과를 경시별 (2, 24, 48시간)로 조사하였다. GRE, SC 그리고 GS는 각각 400 μg/mL 이하, 15 mM 그리고 0.6GS (GS 1, GRE 1,000 μg/mL + SC 30 mM) 이하의 농도에서 세포독성을 나타나지 않았다. 모든 처리구에서 B. abortus의 생존율은 용량- 의존적으로 현저하게 감소하는 결과를 나타내었다. 또한, GRE (400 μg/mL), SC (15 mM) 그리고 0.5GS (GRE 500 μg/mL + SC 15 mM)를 처리한 세포에서 배양 48시간 후에, B. abortus의 증식이 통계적으로 유의성 있게 감소하였으며 (GRE, p < 0.01; SC and 0.5GS, p < 0.001), 특히, GS를 처리한 경우, B. abortus의 세포내 증식이 GRE와 SC의 상승작용에 의한 강력한 항균효과를 나타내었다. 결론적으로, GS는 B. abortus에 대한 항균물질로서 유용할 뿐만 아니라, 식육과 우유 위생 분야에 적용할 수 있을 것으로 생각된다.

This study investigated the antibacterial effects of GR ethanol extracts (GRE), sodium chlorate (SC) and a combination of GRE and SC (GS) on Brucella abortus (B. abortus). The antibacterial activities of GRE, SC and GS towards B. abortus were evaluated by incubating B. abortus with GRE, SC and GS. Following treatment with GRE, SC and GS, B. abortus survival and intracellular proliferation in macrophages were monitored. In the cellular cytotoxicity assay, GRE, SC and GS are not cytotoxic at concentrations less than 400 μg/ml, 15 mM and 0.6GS (1 of GS, GRE 1,000 μg/ml + SC 30 mM), respectively. The viability of B. abortus was markedly decreased in a dosedependent manner in all treatment groups. In addition, B. abortus intracellular proliferation within macrophages was significantly reduced in cells treated with GRE (400 μg/mL), SC (15 mM) and 0.5GS (GRE 500 μg/mL + SC 15 mM) after 48 hr-incubation (GRE, p < 0.01; SC and 0.5GS, p < 0.001). Especially, in the treatment of GS, the synergistic effect of GRE and SC treatment on B. abortus in macrophage was observed. In conclusion, GS is useful as an antibacterial candidate against B. abortus, and can be applied in the field of meat and milk hygiene.

This study investigated the therapeutic effects of Galla rhois (GR) ethanol extract (GRE), sodium chlorate (SC), and a combination of GRE and SC on mice infected with Brucella abortus (B. abortus). Mice were infected intraperitoneally with B. abortus and then treated with GRE, SC, and a combination GRE and SC in drinking water for 14 days. Then, serum antibodies were used in a tube agglutination test (TAT), after which the weight and CFUs from each spleen were measured. In addition, histopathological changes in each liver were examined at 14 days post-infection. At 14 days post-infection, negative reactions of serum antibodies in PC (positive control), SCT (SC 1.6 g/L drinking water), GRT (GRE 200 mg/L drinking water), and GST (GRE 200 mg + SC 1.6 g/L drinking water) were 0, 40, 60, and 80%, respectively. The average spleen weight was not significantly different between the groups. At 14 days post-infection, bacterial numbers in all treated groups were significantly lower compared to to that of the PC (GRT and SCT, P<0.05; GST, P<0.001). In terms of histopathological changes in the livers, there were numerous multifocal microgranulomas in the PC, whereas this number successively decreased in the SCT, GRT, and GST groups. Conclusively, a combination of GRE and SC exhibits therapeutic effects on mice infected with B. abortus. These results suggest the potential efficacy of a mixture of GRE and SC in the treatment of brucellosis.