55 research outputs found
Brucellosis and one health: inherited and future challenges
One Health is the collaborative efforts of multiple disciplines to attain optimal health for people, animals and the environment, a concept that historically owes much to the study of brucellosis, including recent political and ethical considerations. Brucellosis One Health actors include Public Health and Veterinary Services, microbiologists, medical and veterinary practitioners and breeders. Brucellosis awareness, and the correct use of diagnostic, epidemiological and prophylactic tools is essential. In brucellosis, One Health implementation faces inherited and new challenges, some aggravated by global warming and the intensification of breeding to meet growing food demands. In endemic scenarios, disease awareness, stakeholder sensitization/engagement and the need to build breeder trust are unresolved issues, all made difficult by the protean characteristics of this zoonosis. Extended infrastructural weaknesses, often accentuated by geography and climate, are critically important. Capacity-building faces misconceptions derived from an uncritical adoption of control/eradication strategies applied in countries with suitable means, and requires additional reference laboratories in endemic areas. Challenges for One Health implementation include the lack of research in species other than cattle and small ruminants, the need for a safer small ruminant vaccine, the need to fill in the infrastructure gap, the need for realistic capacity-building, the creation of reference laboratories in critical areas, and the stepwise implementation of measures not directly transposed from the so-called developed countries
Examining ecological validity in social interaction: problems of visual fidelity, gaze, and social potential
Social interaction is an essential part of the human experience, and much work has been done to study it. However, several common approaches to examining social interactions in psychological research may inadvertently either unnaturally constrain the observed behaviour by causing it to deviate from naturalistic performance, or introduce unwanted sources of variance. In particular, these sources are the differences between naturalistic and experimental behaviour that occur from changes in visual fidelity (quality of the observed stimuli), gaze (whether it is controlled for in the stimuli), and social potential (potential for the stimuli to provide actual interaction). We expand on these possible sources of extraneous variance and why they may be important. We review the ways in which experimenters have developed novel designs to remove these sources of extraneous variance. New experimental designs using a ‘two-person’ approach are argued to be one of the most effective ways to develop more ecologically valid measures of social interaction, and we suggest that future work on social interaction should use these designs wherever possible
Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models
The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular
parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an
infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular
niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative
fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase
(ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ppdK and mae mutants was severely
impaired and growth of the double fbp- glpX mutant was reduced. In macrophages, only the ppdK and mae mutants
showed reduced multiplication, and studies with the ppdK mutant confirmed that it reached the replicative niche. Similarly,
only the ppdK and mae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer
than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich
medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that
B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly
other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt
In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice
Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis
Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen Brucella melitensis 16 M
Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A DeltavjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
Morphological analysis of the sheathed flagellum of Brucella melitensis
<p>Abstract</p> <p>Background</p> <p>It was recently shown that <it>B. melitensis </it>is flagellated. However, the flagellar structure remains poorly described.</p> <p>Findings</p> <p>We analyzed the structure of the polar sheathed flagellum of <it>B. melitensis </it>by TEM analysis and demonstrated that the Ryu staining is a good method to quickly visualize the flagellum by optical microscopy. The TEM analysis demonstrated that an extension of the outer membrane surrounds a filament ending by a club-like structure. The Δ<it>ftcR</it>, Δ<it>fliF</it>, Δ<it>flgE </it>and Δ<it>fliC </it>flagellar mutants still produce an empty sheath.</p> <p>Conclusions</p> <p>Our results demonstrate that the flagellum of <it>B. melitensis </it>has the characteristics of the sheathed flagella. Our results also suggest that the flagellar sheath production is not directly linked to the flagellar structure assembly and is not regulated by the FtcR master regulator.</p
Production and characterization of monoclonal antibodies anti fragment Fc of bovine IgG
Systems Biology Approach Predicts Antibody Signature Associated with Brucella melitensis Infection in Humans
A complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking. Here we illustrate a systems biology approach to identify the antibody signature associated with Brucella melitensis (Bm) infection in humans and predict proteomic features of serodiagnostic antigens. By taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 Bm genes, we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens. The reactive antigens were then classified according to annotated functional features (COGs), computationally predicted features (e.g., subcellular localization, physical properties), and protein expression estimated by mass spectrometry (MS). Enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens, as were proteins predicted to have a signal peptide, a single transmembrane domain, and outer membrane or periplasmic location. These features accounted for 67% of the serodiagnostic antigens. An overlay of the seroreactive antigen set with proteomic data sets generated by MS identified an additional 24%, suggesting that protein expression in bacteria is an additional determinant in the induction of Brucella-specific antibodies. This analysis indicates that one-third of the proteome contains enriching features that account for 91% of the antigens recognized, and after B. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features. This systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to B. melitensis and a new framework for comparing the humoral responses against other microorganisms
Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export
Background: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.This work was funded by the European Commission (Research Contract QLK2-CT-2002-00918) and the Ministerio de Ciencia y Tecnología of Spain
(Proyecto AGL2004-01162/GAN)
Generation of the Brucella melitensis ORFeome version 1.1.
The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets
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