Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

BACKGROUND: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. OBJECTIVES: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. METHODS: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. RESULTS: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1ß, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. CONCLUSIONS: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Quantification of Brucella abortus population structure in a natural host.

Cattle are natural hosts of the intracellular pathogen Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of uniquely barcoded B. abortus strains to temporally and spatially quantify population structure during colonization of cattle through a natural route of infection. Introducing 108 bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a 1-wk time point. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached a parotid lymph node. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present at 1 wk postinfection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

Inquérito sorológico de brucelose em machos e fêmeas bovinas em fazendas do Estado de Mato Grosso do Sul / Serological survey of brucellosis in males and females of cattle in the State of Mato Grosso do Sul

Objetivou-se avaliar a ocorrência de animais soro reagentes à brucelose bovina em fazendas localizadas no Estado de Mato Grosso do Sul, por meio de exame sorológico utilizando o Antígeno Acidificado Tamponado (AAT) e discutir as possíveis diferenças entre as soroprevalências de fêmeas e machos. Foram avaliados, a partir do teste de triagem com Antígeno Acidificado Tamponado (AAT), 724 bovinos da raça Nelore, sendo 274 machos e 450 fêmeas, provenientes de oito propriedades com histórico de problemas reprodutivos. O teste foi procedido conforme o protocolo determinado pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Os resultados demonstraram baixa soroprevalência da doença nos bovinos testados, sendo detectada prevalência para a doença de 1,10% nos machos e 2,88% nas fêmeas.Quando se considera o touro isoladamente nos rebanhos, pode-se perceber que a fertilidade é muito mais importante nos machos do que nas fêmeas individualmente, uma vez que os touros podem se acasalar com um número muito maior de fêmeas, seja na monta natural ou na inseminação artificial, demonstrando a importância do inquérito epidemiológico na população geral, principalmente nos machos. A maior frequência da doença foi encontrada nas fêmeas podendo estar relacionada à infecção por Brucella spp. no ambiente decorrente de parto ou aborto tornando as fêmeas transmissoras permanentes da doença.

The objective of this study was to evaluate the occurrence of seroreactive animals to bovine brucellosis in farms located in the State of Mato Grosso do Sul, by means of a serological examination using the Acidified Buffered Antigen (AAT) and to discuss the possible differences between the seroprevalence of females and males. A total of 724 Nellore cattle, 274 males and 450 females, from eight farms with a history of reproductive problems, were evaluated using the screening test with Acidified Buffered Antigen (AAT). The test was carried out according to the protocol determined by the Ministry of Agriculture, Livestock and Food Supply (MAPA).The results showed a low seroprevalence of the disease in the tested cattle, with a prevalence of 1.10% in males and 2.88% in females.When considered the bull alone in herds, it can be shown that fertility is much more important in males than in females individually, since bulls can mate with a much larger number of females, either in natural mating or in artificial insemination, demonstrating the importance of epidemiological survey in the general population, especially in males. The highest frequency of the disease was found in females and may be related to infection by Brucella spp. in the environment from childbirth or abortion making females permanent transmitters of the disease.

Brucella abortus and Pregnancy in Mice: Impact of Chronic Infection on Fertility and the Role of Regulatory T Cells in Tissue Colonization.

Stealthy intracellular bacterial pathogens are known to establish persistent and sometimes lifelong infections. Some of these pathogens also have a tropism for the reproductive system, thereby increasing the risk of reproductive disease and infertility. To date, the pathogenic mechanism involved remains poorly understood. Here, we demonstrate that Brucella abortus, a notorious reproductive pathogen, has the ability to infect the nonpregnant uterus, sustain infection, and induce inflammatory changes during both acute and chronic stages of infection. In addition, we demonstrated that chronically infected mice had a significantly reduced number of pregnancies compared to naive controls. To investigate the immunologic mechanism responsible for uterine tropism, we explored the role of regulatory T cells (Tregs) in the pathogenesis of Brucella abortus infection. We show that highly suppressive CD4+FOXP3+TNFR2+ Tregs contribute to the persistence of Brucella abortus infection and that inactivation of Tregs with tumor necrosis factor receptor II (TNFR2) antagonistic antibody protected mice by significantly reducing bacterial burden both systemically and within reproductive tissues. These findings support a critical role of Tregs in the pathogenesis of persistence induced by intracellular bacterial pathogens, including B. abortus Results from this study indicate that adverse reproductive outcomes can occur as sequelae of chronic infection in nonpregnant animals and that fine-tuning Treg activity may provide novel immunotherapeutic and prevention strategies against intracellular bacterial infections such as brucellosis.

ChIP-seq analysis of Brucella reveals transcriptional regulation of GntR.

Transcriptional regulator GntR controls diverse physiological functions necessary for Brucella survival. In the intracellular pathogen Brucella, GntR has been shown to regulate the expression of genes related to virulence. However, the precise determination of GntR direct targets has so far proved elusive. Therefore, we performed chromatin immunoprecipitation of GntR10 followed by next-generation sequencing (ChIP-seq). We selected target gene BAB1_1163 directly regulated by GntR10 and created the mutant (2308Δ1163) from virulent Brucella abortus 2308 (S2308). 2308Δ1163 strain survival capability in murine macrophages (RAW 264.7) was detected and the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were also measured. We detected 88 intergenic ChIP-seq peaks of GntR10 binding distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif with 12 bp length. 2308Δ1163 showed reduced survival capability in RAW 264.7. After the macrophages were infected with 2308Δ1163, the levels of TNF-α and IL-1ß were decreased and were significantly lower than that for the S2308-infected group, indicating that the 2308Δ1163 mutant could inhibit the secretion of inflammatory cytokines. Taken together, the research has recorded valuable data about GntR10 and provided new insights into the functionality of GntR10.

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.

Inmunopatología de la brucelosis osteoarticular / Immunopathology of osteoarticular brucelosis

La brucelosis es una de las enfermedades zoonóticas más importantes a nivel mundial capaz de producir enfermedad crónica en los seres humanos. La localización osteoarticular es la presentación más común de la enfermedad activa en el hombre. Sin embargo, algunos de los mecanismos moleculares implicados en la enfermedad osteoarticular han comenzado a dilucidarse recientemente. Brucella abortus induce daño óseo a través de diversos mecanismos en los cuales están implicados TNF-α y RANKL. En estos procesos participan células inflamatorias que incluyen monocitos/macrófagos, neutrófilos, linfocitos T del tipo Th17 y linfocitos B. Además, B. abortus puede afectar directamente las células osteoarticulares. La bacteria inhibe la deposición de la matriz ósea por los osteoblastos y modifica el fenotipo de estas células para producir metaloproteinasas de matriz (MMPs) y la secreción de citoquinas que contribuyen a la degradación del hueso. Por otro lado, la infección por B. abortus induce un aumento en la osteoclastogénesis, lo que aumenta la resorción de la matriz ósea orgánica y mineral y contribuye al daño óseo. Dado que la patología inducida por Brucella afecta el tejido articular, se estudió el efecto de la infección sobre los sinoviocitos. Estos estudios revelaron que, además de inducir la activación de estas células para secretar quemoquinas, citoquinas proinflamatorias y MMPs, la infección inhibe la muerte por apoptosis de los sinoviocitos. Brucella es una bacteria intracelular que se replica en el retículo endoplásmico de los macrófagos. El análisis de los sinoviocitos infectados con B. abortus indicó que las bacterias también se multiplican en el retículo endoplasmático, lo que sugiere que la bacteria podría usar este tipo celular para la multiplicación intracelular durante la localización osteoarticular de la enfermedad. Los hallazgos presentados en esta revisión intentan responder a preguntas sobre los mediadores inflamatorios implicados en el daño osteoarticular causado por Brucella. (AU)

Brucellosis is one of the most important zoonotic diseases that can produce chronic disease in humans worldwide. Osteoarticular involvement is the most common presentation of human active disease. The molecular mechanisms implicated in bone damage have started to be elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and RANKL are implicated. These processes are driven by inflammatory cells, including monocytes/macrophages, neutrophils, Th17 lymphocytes and B cells. Also, Brucella abortus (B. abortus) can directly affect osteoarticular cells. The bacterium inhibits bone matrix deposition by osteoblast and modifies the phenotype of these cells to produce matrix methalloproteinases (MMPs) and cytokine secretion that contribute to bone matrix degradation. B. abortus also affects osteoclast increasing mineral and organic bone matrix resorption and contributing to bone damage. Since the pathology induced by Brucella species involves joint tissue, experiments conducted in sinoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits sinoviocyte apoptosis. Brucella is an intracellular bacterium that replicate in the endoplasmic reticulum of macrophages. The analysis of B. abortus infected sinoviocytes indicated that bacteria also replicate in their reticulum suggesting that the bacterium could use this cell type for intracellular replication during the osteoarticular localization of the disease. The findings presented in this review try to answer key questions about the inflammatory mediators involved in osteoarticular damage caused by Brucella. (AU)

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

B. Abortus Modulates Osteoblast Function Through the Induction of Autophagy.

Osteoarticular brucellosis is the most common localization of human active disease. Osteoblasts are specialized mesenchymal-derived cells involved in bone formation and are considered as professional mineralizing cells. Autophagy has been involved in osteoblast metabolism. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in osteoblast cells. Autophagy was revealed by upregulation of LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. Induction of autophagy was also corroborated by using the pharmacological inhibitors wortmannin, a PI 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Autophagy induction create a microenvironment that modifies osteoblast metabolism by the inhibition of the deposition of organic and mineral matrix, the induction of matrix metalloproteinase (MMP)-2, osteopontin, and RANKL secretion leading to bone loss. Accordingly, autophagy is also involved in the down-modulation of the master transcription factor in bone formation osterix during B. abortus infection. Taking together our results indicate that B. abortus induces the activation of autophagy pathway in osteoblast cells and this activation is involved in the modulation of osteoblast function and bone formation.

IL-1R and Inflammasomes Mediate Early Pulmonary Protective Mechanisms in Respiratory Brucella Abortus Infection.

Brucella spp. infection is frequently acquired through contaminated aerosols. The role of interleukin-1 beta (IL-1ß) in the early pulmonary response to respiratory Brucella infection is unknown. As shown here, IL-1ß levels in lung homogenates and bronchoalveolar lavage fluid (BALF) of mice intratracheally inoculated with B. abortus were increased at 3 and 7 days p.i. At 7 days p.i., pulmonary CFU numbers were higher in IL-1 receptor (IL-1R) knockout (KO) mice than in wild type (WT) mice. At different times p.i. CFU in lungs and BALF were higher in mice lacking some inflammasome components (caspase-1, AIM2, NLRP3) than in WT mice. At 2 days p.i. pulmonary levels of IL-1ß and CXCL1 (neutrophils chemoattractant) were lower in caspase-1/11 KO mice. At day 3 p.i., neutrophils counts in BALF were lower in caspase-1/11 KO mice than in WT mice. During in vitro infections, IL-1ß secretion was lower in alveolar macrophages from caspase-1/11, NLRP3 or AIM2 KO mice than in WT controls. Similarly, IL-1ß production by B. abortus-infected alveolar epithelial cells was reduced by pretreatment with a specific caspase-1 inhibitor. This study shows that IL-1R, probably through IL-1ß action, and the NLRP3 and AIM2 inflammasomes are involved in pulmonary innate immune protective mechanisms against respiratory B. abortus infection.

Pigs, pooches and pasteurisation: The changing face of brucellosis in Australia

BACKGROUND: Brucellosis, also known as undulant, Mediterranean or Malta fever, is a systemic infection that causes fever, sweats, arthralgias and myalgias. A globally important disease, brucellosis is re-emerging in Australia in association with feral pig hunting activities. OBJECTIVE: This article aims to provide clinicians with an overview of brucellosis, covering epidemiology, clinical features, diagnosis, management and prevention. DISCUSSION: Brucellosis should be suspected in all patients with non-specific, flu-like illness who fall into one of the major risk groups (feral pig hunters, overseas travellers and migrants). Depression is common and often severe, relative to other symptoms. Early diagnosis and treatment are important for preventing complications, which include osteoarticular, genitourinary or, more rarely, neurological or cardiovascular diseases. Diagnosing acute infections is based on serology and blood cultures; imaging and biopsy may be required for diagnosis of focal infections. Dual therapy with doxycycline and gentamicin is the recommended treatment. Relapse occurs in up to 10% of patients. Prevention is achieved through the use of protective gear during hunting and avoidance of unpasteurised dairy products in countries where occur in animals.

Endoribonuclease YbeY Is Linked to Proper Cellular Morphology and Virulence in Brucella abortus.

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in Brucella abortus is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of ybeY in B. abortus led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the ΔybeY strain was significantly attenuated in both macrophage and mouse models of infection. The ΔybeY strain also showed increased sensitivities to several in vitro-applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the ΔybeY strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of Brucella; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria.IMPORTANCEBrucella spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat Brucella infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in B. abortus Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.

Mitochondrial fragmentation affects neither the sensitivity to TNFα-induced apoptosis of Brucella-infected cells nor the intracellular replication of the bacteria.

Mitochondria are complex organelles that participate in many cellular functions, ranging from ATP production to immune responses against viruses and bacteria. This integration of a plethora of functions within a single organelle makes mitochondria a very attractive target to manipulate for intracellular pathogens. We characterised the crosstalk that exists between Brucella abortus, the causative agent of brucellosis, and the mitochondria of infected cells. Brucella replicates in a compartment derived from the endoplasmic reticulum (ER) and modulates ER functionality by activating the unfolded protein response. However, the impact of Brucella on the mitochondrial population of infected cells still requires a systematic study. We observed physical contacts between Brucella containing vacuoles and mitochondria. We also found that B. abortus replication is independent of mitochondrial oxidative phosphorylation and that mitochondrial reactive oxygen species do not participate to the control of B. abortus infection in vitro. We demonstrated that B. abortus and B. melitensis induce a drastic mitochondrial fragmentation at 48 hours post-infection in different cell types, including myeloid and non-myeloid cells. This fragmentation is DRP1-independent and might be caused by a deficit of mitochondrial fusion. However, mitochondrial fragmentation does not change neither Brucella replication efficiency, nor the susceptibility of infected cells to TNFα-induced apoptosis.

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.

Multivalent Fusion DNA Vaccine against Brucella abortus.

As an alternative brucellosis prevention method, we evaluated the immunogenicity induced by new multivalent DNA vaccines in BALB/c mice. We constructed the vaccines by fusion of BAB1_0273 and/or BAB1_0278 open reading frames (ORFs) from genomic island 3 (GI-3) and the Brucella abortus 2308 sodC gene with a link based on prolines and alanines (pV273-sod, pV278-sod, and pV273-278-sod, resp.). Results show that immunization with all tested multivalent DNA vaccines induced a specific humoral and cellular immune response. These novel multivalent vaccines significantly increased the production of IgM, IgG, and IgG2a antibodies as well as IFN-γ levels and the lymphoproliferative response of splenocytes. Although immunization with these multivalent vaccines induced a typical T-helper 1- (Th1-) dominated immune response, such immunogenicity conferred low protection levels in mice challenged with the B. abortus 2308 pathogenic strain. Our results demonstrated that the expression of BAB1_0273 and/or BABl_0278 antigens conjugated to SOD protein can polarize mice immunity to a Th1-type phenotype, conferring low levels of protection.

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 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.

Chronic Brucella Infection Induces Selective and Persistent Interferon Gamma-Dependent Alterations of Marginal Zone Macrophages in the Spleen.

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209+ MZ macrophages (MZMs) and the CD169+ marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection.

Occurrence of oxidative stress in dairy cows seropositives for Brucella abortus.

Bovine brucellosis is an important zoonotic disease caused by the bacterium Brucella abortus that leads to economic losses due to animal discard and commercial restrictions. Since positive animals for brucellosis are culled, little is known about the pathogenesis of this disease. Therefore, the aims of this study were to evaluate possible changes in the activity of deaminase adenosine (ADA) and the oxidative stress in cows seropositives for brucellosis (Experiment I), and to evaluate the seroprevalence of B. abortus in dairy cows from the Western state of Santa Catarina, Southern Brazil (Experiment II). The Experiment I evaluated 20 pregnant cows: ten seropositives for B. abortus and ten seronegatives that were used as controls. The ADA activity and markers of oxidative stress (TBARS, catalase (CAT) and superoxide dismutase (SOD)) were evaluated in these animals. A reduction in the activity of ADA and catalase enzymes in seropositive animals was observed (p < 0.001). Conversely, there was an increase in TBARS levels and superoxide dismutase activity in cows infected by B. abortus (p < 0.001). The presence of oxidative stress and a reduction of ADA might be related to the modulation of the inflammatory response. The experiment II was performed due to a high number of herds with restrictions imposed by cases of brucellosis in the state of Santa Catarina in the last two years, and thus, the seroprevalence for B. abortus was evaluated in 1242 serum samples of cows of 69 herds. The serodiagnosis was performed using two tests: buffered acidified antigen and 2-mercaptoethanol. However, none of the serum samples were positive for B. abortus. Although we did not find seropositive animals for brucellosis in our study, the disease still requires continued surveillance, due to its economic impact, and to the oxidative stress caused by it, which may have contributed to cases of abortion in three seropositive cows (Experiment I) in the final third of the gestation.