Immunogenicity and protective response induced by recombinant Brucella abortus proteins Adk, SecB and combination of these two recombinant proteins against a virulent strain B. abortus 544 infection in BALB/c mice.

In this study, two recombinant proteins encoded by Brucella abortus genes Adk and SecB were evaluated as single subunit vaccine (SSV) as well as combined subunit vaccine (CSV) against B. abortus infection in BALB/c mice. These genes were cloned into pcold-TF expression system and recombinant proteins were expressed in Escherichia coli DH5α. The immunoreactivity of purified rAdk and rSecB was analyzed by immunoblotting showing that purified rAdk and rSecB as well as pcold-TF vector strongly reacted with Brucella-positive serum. Mice were immunized intraperitoneally with SSVs, CSV, pcold-TF, RB51 and PBS. The analysis of cytokine revealed that SSVs and CSV can strongly induce production of proinflammatory cytokines TNF and IL-6, suggesting that these subunit vaccines elicited innate immune response, particularly, activated antimicrobial mechanism of macrophages to limit the initial infection. On the other hand, immunization with SSVs and CSV elicited strong IFN-γ production and decreased IL-10 production compared to PBS group. The secretion profiles of IFN-γ and IL-10 together with an enhancement of blood CD4+ population and significantly induced specific IgG1 and IgG2a antibodies indicated that SSVs and CSV induced not only humoral immunity but also T helper 1 T cell immunity. Finally, spleen proliferation and bacterial burden in the spleen of mice vaccinated with these subunit vaccines were significantly lower than those of PBS group, which conferred significant protection against B. abortus infection. Altogether, the potential of these antigens of B. abortus could be prospective candidates to develop subunit vaccines against brucellosis.

Interleukin 10 suppresses lysosome-mediated killing of Brucella abortus in cultured macrophages.

Brucella abortus is a Gram-negative zoonotic pathogen for which there is no 100% effective vaccine. Phagosomes in B. abortus-infected cells fail to mature, allowing the pathogen to survive and proliferate. Interleukin 10 (IL10) promotes B. abortus persistence in macrophages by mechanisms that are not fully understood. In this study, we investigated the regulatory role of IL10 in the immune response to B. abortus infection. B. abortus-infected macrophages were treated with either IL10 siRNA or recombinant IL10 (rIL10), and the expression of phagolysosome- or inflammation-related genes was evaluated by qRT-PCR and Western blotting. Phagolysosome fusion was monitored by fluorescence microscopy. We found that the synthesis of several membrane-trafficking regulators and lysosomal enzymes was suppressed by IL10 during infection, resulting in a significant increase in the recruitment of hydrolytic enzymes by Brucella-containing phagosomes (BCPs) when IL10 signaling was blocked. Moreover, blocking IL10 signaling also enhanced proinflammatory cytokine production. Finally, concomitant treatment with STAT3 siRNA significantly reduced the suppression of proinflammatory brucellacidal activity but not phagolysosome fusion by rIL10. Thus, our data provide the first evidence that clearly indicates the suppressive role of IL10 on phagolysosome fusion and inflammation in response to B. abortus infection through two distinct mechanisms, STAT3-independent and -dependent pathways, respectively, in murine macrophages.

Interleukin 6 Promotes Brucella abortus Clearance by Controlling Bactericidal Activity of Macrophages and CD8+ T Cell Differentiation.

To date, the implications of interleukin 6 (IL-6) for immune responses in the context of Brucella infection are still unknown. In the present study, we found that Brucella abortus infection induced marked production of IL-6 in mice that was important for sufficient differentiation of CD8+ T cells, a key factor in Brucella clearance. Blocking IL-6 signaling also significantly induced serum IL-4 and IL-10, together with a decreased gamma interferon (IFN-γ) level, suggesting that IL-6 is essential for priming the T-helper (Th) 1 cell immune response during Brucella infection. The IL-6 pathway also activated the bactericidal activity of primary and cultured macrophages. Bacterial killing was markedly abrogated when IL-6 signaling was suppressed, and this phenomenon was mainly associated with decreased activity of lysosome-mediated killing. Interestingly, suppressor of cytokine signaling 3 (SOCS3) was important for regulating the IL-6-dependent anti-Brucella activity through the JAK/STAT pathway. During early infection, in the absence of SOCS3, IL-6 exhibited anti-inflammatory effects and lysosome-mediated killing inhibition; however, the increase in SOCS3 successfully shifted functional IL-6 toward proinflammatory brucellacidal activity in the late stage. Our data clearly indicate that IL-6 contributes to host resistance against B. abortus infection by controlling brucellacidal activity in macrophages and priming cellular immune responses.

Lipocalin 2 (Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages.

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.

Heat-stress-modulated induction of NF-κB leads to brucellacidal pro-inflammatory defense against Brucella abortus infection in murine macrophages and in a mouse model.

BACKGROUND: Brucella causes a chronic and debilitating infection that leads to great economic losses and a public health burden. In this study, we demonstrated the brucellacidal effect of heat shock mediated by the induction of pro-inflammatory cytokines, reactive oxygen species (ROS) accumulation and apoptosis in murine macrophages and in mice. RESULTS: RAW264.7 cells were incubated at 43 °C, and BALB/c mice were subjected to whole body hyperthermia. The data showed a reduction in bacterial survival in the mice after daily heat exposure. This was accompanied by increased levels of cytokines TNF, IL-6, IL-1ß and IFN-γ in the sera of the mice. Gene expression of NF-κB and inducible nitric oxide production were also induced in the mouse splenic cells. In parallel with the bacterial reduction in the mouse model, an increased bactericidal effect was observed in RAW264.7 cells after exposure to heat stress. In addition, the heat stress increased both the nuclear translocation of NF-κB and the expression of the heat shock proteins HSP70 and HSP90 in murine macrophages. Furthermore, heat exposure induced the increase of pro-inflammatory cytokines, ROS accumulation and apoptosis but did not affect the production of nitric oxide (NO) in macrophages. CONCLUSION: This study demonstrated the induction of innate immune responses by heat stress that significantly reduced the intracellular survival of B. abortus in vitro and in vivo. Transcriptional factor NF-κB, which is a master regulator, could be termed a key activator of heat-induced immunity against Brucella. The increase in the expression and activation of NF-κB in splenic cells and macrophages was followed by enhanced antimicrobial effectors, including cytokines, ROS and NO that may contribute to the reduction of bacterial survival.

Effects of gallic acid on signaling kinases in murine macrophages and immune modulation against Brucella abortus 544 infection in mice.

In this study, we investigated the effects of gallic acid (GA) in intracellular signaling within murine macrophages and its contribution to host immunity during Brucella infection. In vitro analysis revealed that GA treatment decreased F-actin content and suppressed p38α phosphorylation level. In vivo analysis showed that GA treatment reduced inflammation and proliferation of Brucella in spleens of mice in comparison to PBS treatment yielding a significant protection unit. For the analysis of immune response, the uninfected GA-treated mice showed increased production of IFN-γ and MCP-1, and the Brucella-infected GA-treated mice showed elevated levels of IL-12p70, TNF, IFN-γ, MCP-1, IL-10 and IL-6 in comparison to negative and positive control groups, respectively. These findings demonstrate the therapeutic effects of GA against Brucella infection through interference on intracellular signaling pathway, induction of cytokine production and protection from bacterial proliferation in spleens of mice.

Simultaneous RNA-seq based transcriptional profiling of intracellular Brucella abortus and B. abortus-infected murine macrophages.

Brucella is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living B. abortus. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during B. abortus infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (Tnf), interleukin-1α (Il1α), interleukin-1ß (Il1ß), interleukin-6 (Il6), interleukin-12 (Il12), chemokine C-X-C motif (CXCL) family, nuclear factor kappa B (Nf-κb), signal transducer and activator of transcription 1 (Stat1), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon B. abortus infection. In conclusion, these data suggest that Brucella modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.

The in vitro and in vivo protective effects of tannin derivatives against Salmonella enterica serovar Typhimurium infection.

In this study, we investigated the protective effects of tannin-derived components, gallic acid (GA) and tannic acid (TA), in vitro and in vivo against Salmonella infection in mice. Both GA and TA showed antibacterial effects against Salmonella (S.) Typhimurium as well as inhibitory effects on the adherence, invasion, and intracellular growth of the pathogens in macrophages. Following a lethal dose of Salmonella infection in mice, reduced virulence in both GA- and TA-treated groups was observed based on reduced mortality rates. In the non-infected groups, the average weights of the spleens and livers of GA- or TA-treated mice were not significantly different with the control group. In addition, the average weights of these organs in all of the Salmonella-infected groups were not significantly different but the numbers of bacteria in the spleens and livers in both GA- and TA-treated mice were significantly reduced. The levels of cytokine production in non-infected mice revealed that GA-treated and TA-treated mice elicited an increased level of IFN-γ, and both IFN-γ and MCP-1, respectively, as compared with the PBS-treated group. These findings highlight the potential of GA and TA as alternatives for the treatment of salmonellosis and as supplements to conventional antimicrobial food additives.

Nocodazole treatment interrupted Brucella abortus invasion in RAW 264.7 cells, and successfully attenuated splenic proliferation with enhanced inflammatory response in mice.

Brucellosis is one of the most important and widespread zoonosis worldwide responsible for serious economic losses and considerable public health burden. In this study, we investigated the modulatory effect of a microtubule-inhibitor, nocodazole, on B. abortus infection in murine macrophages and in a mouse model. Nocodazole activated macrophages and directly inhibited the growth of Brucella in a dose-dependent manner. Nocodazole increased adhesion but reduced invasion and intracellular growth of Brucella in macrophages although it did not affect co-localization of Brucella with LAMP-1. In addition, nocodazole negatively affected actin polymerization, and weakly activated ERK and p38α but significantly activated JNK in non-infected cells. After subsequent infection, nocodazole weakly inhibited activation of ERK and p38α. For the in vivo tests, nocodazole -treated mice displayed elevated levels of IFN-γ, MCP-1 and IL-10 while Brucella-infected nocodazole -treated mice showed high levels of TNF, IFN-γ, MCP-1, IL-10 and IL-6 as compared to controls. Furthermore, nocodazole treatment reduced inflammation and Brucella proliferation in the spleens of mice. These findings highlight the potential use of nocodazole for the control of brucellosis although further investigations are encouraged to validate its therapeutic use in animal hosts.

Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

BACKGROUND: Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. RESULTS: First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. CONCLUSIONS: This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

Dextran sulfate sodium upregulates MAPK signaling for the uptake and subsequent intracellular survival of Brucella abortus in murine macrophages.

Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection.

Evaluation of the combined use of the recombinant Brucella abortus Omp10, Omp19 and Omp28 proteins for the clinical diagnosis of bovine brucellosis.

Currently, there are several serodiagnostic tools available for brucellosis, however, it is difficult to differentiate an active infection from vaccination. Hence, there is a great need to develop alternative means that can distinguish between these two conditions without utilizing lipopolysaccharide (LPS). This study was an attempt to determine the efficacy of combined recombinant Brucella (B.) abortus outer membrane proteins (rOmps) and individual rOmps in the serodiagnosis of brucellosis by enzyme linked immunosorbent assay (ELISA), utilizing both that standard tube agglutination test (TAT)-positive and -negative serum samples from Korean native cattle. The results are very interesting and promising because the combined rOmp antigens used in the study were highly reactive with the TAT-positive serum samples. The combined rOmps sensitivity, specificity and accuracy were 215/232 (92.67%), 294/298 (98.66%) and 509/530 (96.04%), respectively. While these results are preliminary, the tests performed have very high potential in the serodiagnosis of brucellosis and likewise, the combined rOmps can be used for future vaccine production.

ß-Sitosterol Contributes in the Resistance to Invasion and Survival of Brucella abortus 544 within RAW264.7 Cells, and Cytokine Production with Reduced Susceptibility to Infection in BALB/c Mice.

We previously identified ß-sitosterol (BS) as one of the most abundant compounds found in Korean red ginseng oil. BS is a widely prevalent vegetable-derived phytosterol with many known health benefits. Here, we investigated the efficacy of BS against Brucella (B.) abortus infection. BS showed no effect on bacterial growth but attenuated internalization, intracellular survival and MAPKs-linked intracellular signaling in RAW264.7 cells. BS treatment in cells is also associated with increased nitrite concentration during infection at 24 h. Slightly enhanced resistance to B. abortus infection was observed in mice orally given BS, which could be mediated by induced production of proinflammatory cytokines. Taken together, our study demonstrates the contribution of BS treatment against B. abortus infection although further investigation is encouraged to maximize its beneficial effects against intracellular infection.

Chemokine receptor 4 (CXCR4) blockade enhances resistance to bacterial internalization in RAW264.7 cells and AMD3100, a CXCR4 antagonist, attenuates susceptibility to Brucella abortus 544 infection in a murine model.

We investigated the involvement of chemokine receptor type 4 (CXCR4) signaling on the outcome of Brucella (B.) abortus 544 infection in murine macrophages and in a mouse model. CXCR4 manipulation were first evaluated for Brucella invasion and intracellular survival efficiency, mitogen-activated protein kinases (ERK1/2, JNK, p38α) activation and generation of nitric oxide (NO), and then in the splenic bacterial proliferation and cytokine production in BALB/c mice. CXCR4 blockade is involved in the successful control of Brucella invasion, reduction of ERK1/2 phosphorylation and inhibition of nitric oxide release from macrophages. Furthermore, using a reported CXCR4-specific antagonist AMD3100 resulted in splenomegaly but attenuated Brucella proliferation in these organs with elevated serum levels of MCP-1, TNF and IL-12. These findings provide insights on the contribution of CXCR4 signaling in the phagocytic pathway and immune modulation during B. abortus infection.

Interleukin 1 alpha (IL-1α) restricts Brucella abortus 544 survival through promoting lysosomal-mediated killing and NO production in macrophages.

The interleukin-1 (IL-1) family of cytokines, particularly IL-1α and IL-1ß, are potent regulators of innate immunity that play key roles in host defense against infection, hence we evaluated the role of these cytokines in the control of brucellosis within RAW 264.7 cells. Marked expression and secretion of IL-1α and IL-1ß were observed during Brucella infection in macrophages. Blocking of IL-1α and IL-1ß reduced induction of IL-10, IL-1ß and TNF, and IL-6 and TNF, respectively. However, interference of IL-1α and not IL-1ß signaling notably augmented susceptibility of macrophages to Brucella infection which indicates that IL-1α is required for a downstream signaling cascade of innate immunity for efficient clearance of Brucella. This protection requires binding to interleukin-1 receptor (IL-1R) mediated by myeloid differentiation factor 88 (MyD88) signaling and associated with increased lysosomal-mediated killing and nitric oxide (NO) production. Expression of pro-inflammatory cytokines was observed to be mediated via NF-κB-p50, HIF-1α and CEBPA, but negatively controlled by CEBPB while transcription of some important phagolysosomal genes was regulated via CEBPA and c-Jun which indicates the important role of these transcription factors in the control of Brucella infection in macrophages via IL-1α signaling pathway.

Substantial Protective Immunity Conferred by a Combination of Brucella abortus Recombinant Proteins against Brucella abortus 544 Infection in BALB/c Mice.

Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and DH5α, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with 5 × 104 CFU of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-γ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.

The immunomodulatory effect of antimicrobial peptide HPA3P restricts Brucella abortus 544 infection in BALB/c mice.

The discovery of antimicrobial peptides (AMPs) in recent years has been promising for the treatment of multidrug resistant pathogenic microbes. Brucellosis is still considered one of the most common zoonoses in the world. In this study, we evaluated the effect HPA3P peptide in the bacterial uptake and intracellular growth of Brucella abortus (B. abortus) 544 in murine macrophages RAW 264.7. HPA3P was further utilized in a mouse model for infection and treatment. This peptide did not show cytotoxicity or bactericidal effect to B. abortus. However, it inhibited bacterial internalization at 0, 15 and 30 min incubation at two different doses at 12 and 24 µM as well as reduced intracellular growth after 2, 24 and 48 h incubation. Mice treated with HPA3P demonstrated a significant 1.01-log reduction (P < 0.0001) and spleen weight reduction compared to the nanocarrier control (P < 0.01). Significant increases in key cytokines Interferon-γ (IFN-γ) and Tumor necrosis factor (TNF) at 3, 7 and 14 days post-infection were observed in HPA3P treated mice similar to the antibiotic control group with both compared to the nanocarrier control. Monocyte chemoattractant protein-1 (MCP-1) was also heightened at 14 days post-infection. Histopathological analysis also suggests reduced bacterial granuloma in the liver and spleens of HPA3P treated group compared with the nanocarrier control group. In this study, the modulation of crucial cytokines IFN-γ and TNF might have led to a considerable reduction in the proliferation of B. abortus in a mouse model of brucellosis. Further investigation might be required to maximize the efficacy of HPA3P treatment in murine brucellosis.

Emodin Successfully Inhibited Invasion of Brucella abortus Via Modulting Adherence, Microtubule Dynamics and ERK Signaling Pathway in RAW 264.7 Cells.

The aim of this work is to investigate the protective efficacy of emodin, an active, naturally-occurring anthraquinone derivative of several traditional Chinese herbs, against Brucella abortus infection in macrophages. Brucella were incubated with different concentrations of emodin and showed that bacterial survival rates were markedly reduced in a dose-dependent manner at increasing incubation time points. Through bacterial infection assay, the highest non-cytotoxic concentration of emodin demonstrated attenuated invasion of Brucella into macrophages, however it did not inhibit the growth of these pathogens within the host cells. On the other hand, emodin effectively decreased the number of bacteria that adhered to host cells, which indicated its potential as an anti-adhesin agent. Furthermore, using immunoblotting and FACS assay for detecting MAPK signaling proteins and F-actin polymerization, respectively, the results showed that the emodin-incubated cells displayed modest reduction in the phosphorylation levels of ERK1/2 and inhibition of F-actin polymerization as compared to control cells. These findings indicate the potential use of emodin as a naturally-occurring alternative method for the prevention of animal brucellosis although this requires confirmation of safe clinical doses.

Immunization of BALB/c mice with a combination of four recombinant Brucella abortus proteins, AspC, Dps, InpB and Ndk, confers a marked protection against a virulent strain of Brucella abortus.

In this study, we assessed the protective efficacy of single subunit vaccines, encoded by the B. abortus 544 genes aspC, dps, yaeC and inpB, against B. abortus infection in mice. First, immunization with these antigens, with the exception of the YaeC protein, was found to elicit both humoral and cellular immune responses with IgG2a being dominant over IgG1. In addition, a massive production of IFN-γ but lower degree of IL-10 was observed, suggesting that all three antigens were able to induce predominantly cell-mediated immunity in response to B. abortus infection. Further investigation of a combined subunit vaccine (CSV) consisting of purified AspC, Dps, InpB and Ndk proteins showed a superior protective effect in mice against brucellosis. The intraperitoneal injection of this combination was shown to induce a remarkable production of IFN-γ and IL-2, which occurred in conjunction with an increase of blood CD4+ and CD8+ T cell proportions. In addition, the higher titer of IgG2a compared to IgG1 elicited by this CSV was obtained, suggesting that this CSV induced a typical T-helper-1-dominated immune response in vivo. Furthermore, the protection level induced by this combination was significantly higher than that induced by single antigens and was not significantly different compared to a group immunized with a live attenuated vaccine (RB51). Altogether, our findings suggest that the combination of different immunogenic antigens could be a useful approach for the development of a new, effective and safe brucellosis vaccine that can replace current vaccine strains.

The Bactericidal Effect of High Temperature Is an Essential Resistance Mechanism of Chicken Macrophage against Brucella abortus Infection.

Knowledge of avian host responses to brucellosis is critical to understanding how birds resist this infection; however, this mechanism is not well established. On the other hand, temperature has a major involvement in the physiology of living organisms, and cell death induced by heat is attributed to protein denaturation. This study demonstrates the direct bactericidal effect of a high temperature (41ºC) on Brucella abortus that resulted in the gradual reduction of intracellular bacteria and inhibited bacterial growth within avian macrophage HD11 in an increasing period of time. On the other hand, this study also revealed that high temperature does not affect the rate of bacterial uptake, as confirmed by the bacterial adherence assay. No significant difference was observed in the expression of target genes between infected and uninfected cells for both temperatures. This study suggests the susceptibility of B. abortus to bacterial death under a high temperature with an increased period of incubation, leading to suppression of bacterial growth.