Brucella melitensis Biovar 1 and Brucella abortus S19 Vaccine Strain Infections in Milkers Working at Cattle Farms in the Khartoum Area,Sudan

BackgroundHuman brucellosis is a preventable zoonoses that may become persistent, causing, if left untreated, severe localized disease. Occupational exposure to infected animals or animal products and consumption of fresh contaminated dairy are main risk factors.MethodsOne hundred farmworkers employed at two cattle farms one in Khartoum North and one in Omdurman were screened for the presence of specific antibodies and seropositive workers were invited to donate a blood sample for blood culture. Molecular typing was used to characterize Brucella isolates.ResultsTen percent of farmworkers tested seropositive and while Brucella melitensis biovar 1 was isolated from the blood of three individuals, an isolate identical to the B. abortus S19 vaccine strain was isolated from a fourth person. All four bacteremic individuals were employed as milkers and did not have obvious disease.ConclusionsThe isolation of the highly infectious pathogen B. melitensis from seropositive workers is consistent with the notion that the pathogen may persist in the blood without causing overt disease. While vaccination with strain S19 is essential for the control of bovine brucellosis the vaccine strain may be transmitted to the human population and protective measures remain important to prevent exposure also in view of the presence of B. melitensis. To create awareness for this potentially severe disease more information on the prevalence of the pathogen in different risk groups and in livestock in the Sudan is needed.     

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca⁺⁺ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.     

CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection

Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.     

Yip1A,a Novel Host Factor for the Activation of the IRE1 Pathway of the Unfolded Protein Response during Brucella Infection

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments. 　On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.     

Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.     

In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice

Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40^-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.     

Aconitate decarboxylase 1 participates in the control of pulmonary Brucella infection in mice

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1^-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1^-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.     

FixL-like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells

Replication of Brucella inside eukaryotic cells is essential for pathogenesis, and successful infection requires rapid adaptation to the intracellular milieu. Close relatives of Brucella use the two-component system FixLJ to survive inside the host. We aimed to identify a homologous sensor in Brucella abortus. A predicted protein with transmembrane and conserved histidine kinase domains was identified as the Fix-like Brucella sensor, FlbS. Although it lacks the PAS domain, recombinant FlbS binds haem in vitro. An internal in-frame deletion in flbS severely decreased B. abortus survival inside professional and non-professional phagocytes. This phenotype was reverted by genetic complementation. These results indicate the critical role of this haemoprotein in the intracellular lifestyle of Brucella.     

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.     

Link between Geographical Origin and Occurrence of Brucella abortus Biovars in Cow and Water Buffalo Herds

Sixty-three Brucella isolates from water buffaloes and cattle slaughtered within the Italian national plan for brucellosis control were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Genotyping indicated a strong influence of geographic origin on the Brucella abortus biovar distribution in areas where brucellosis is endemic and highlighted the importance of rigorous management procedures aimed at avoiding inter- and intraherd spreading of pathogens.     

The two‐component systems PrrBA and NtrYX co‐ordinately regulate the adaptation of Brucella abortus to an oxygen‐limited environment

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortus PrrBA is a functional two‐component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two‐component systems PrrBA and NtrYX co‐ordinately regulate the expression of denitrification and high‐affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild‐type B. abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.     

Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein

BackgroundBrucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable.Methodology/Principal findingsIn this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen.ConclusionsB cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.     

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.     

An Atypical Riboflavin Pathway Is Essential for Brucella abortus Virulence

Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis.     

Brucellosis as an Emerging Threat in Developing Economies: Lessons from Nigeria

Nigeria is the most populous country in Africa, has a large proportion of the world''s poor livestock keepers, and is a hotspot for neglected zoonoses. A review of the 127 accessible publications on brucellosis in Nigeria reveals only scant and fragmented evidence on its spatial and temporal distribution in different epidemiological contexts. The few bacteriological studies conducted demonstrate the existence of Brucella abortus in cattle and sheep, but evidence for B. melitensis in small ruminants is dated and unclear. The bulk of the evidence consists of seroprevalence studies, but test standardization and validation are not always adequately described, and misinterpretations exist with regard to sensitivity and/or specificity and ability to identify the infecting Brucella species. Despite this, early studies suggest that although brucellosis was endemic in extensive nomadic systems, seroprevalence was low, and brucellosis was not perceived as a real burden; recent studies, however, may reflect a changing trend. Concerning human brucellosis, no studies have identified the Brucella species and most reports provide only serological evidence of contact with Brucella in the classical risk groups; some suggest brucellosis misdiagnoses as malaria or other febrile conditions. The investigation of a severe outbreak that occurred in the late 1970s describes the emergence of animal and human disease caused by the settling of previously nomadic populations during the Sahelian drought. There appears to be an increasing risk of re-emergence of brucellosis in sub-Saharan Africa, as a result of the co-existence of pastoralist movements and the increase of intensive management resulting from growing urbanization and food demand. Highly contagious zoonoses like brucellosis pose a threat with far-reaching social and political consequences.     

Identification of Recombination and Positively Selected Genes in Brucella

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.