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Sample records for brucella lipopolysaccharide genes

  1. Immunochemical characterization of rough Brucella lipopolysaccharides.

    OpenAIRE

    Moreno, E; Jones, L M; Berman, D T

    1984-01-01

    Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its re...

  2. Blastogenic response of bovine lymphocytes to Brucella abortus lipopolysaccharide.

    OpenAIRE

    Baldwin, C L; Winter, A J

    1985-01-01

    Brucella abortus lipopolysaccharide was tested in a blastogenesis assay with unfractionated and nylon wool-separated peripheral blood lymphocytes of Brucella-naive cattle and cattle immunized with B. abortus. Our results indicated that in cattle the lipopolysaccharide of B. abortus is not a B-cell mitogen. In immunized animals it stimulated predominantly nylon wool-adherent cells. The lipopolysaccharide of Escherichia coli O128:B12, in contrast, induced a substantially greater proliferative r...

  3. Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis Pathway

    Science.gov (United States)

    Wattam, Alice R.; Inzana, Thomas J.; Williams, Kelly P.; Mane, Shrinivasrao P.; Shukla, Maulik; Almeida, Nalvo F.; Dickerman, Allan W.; Mason, Steven; Moriyón, Ignacio; O’Callaghan, David; Whatmore, Adrian M.; Sobral, Bruno W.; Tiller, Rebekah V.; Hoffmaster, Alex R.; Frace, Michael A.; De Castro, Cristina; Molinaro, Antonio; Boyle, Stephen M.; De, Barun K.; Setubal, João C.

    2012-01-01

    ABSTRACT Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. PMID:22930339

  4. Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge.

    Science.gov (United States)

    Lalsiamthara, Jonathan; Lee, John Hwa

    2017-06-01

    Intracellular pathogen Salmonella exhibits natural infection broadly analogous to Brucella, this phenomenon makes Salmonella a pragmatic choice for an anti-Brucella vaccine delivery platform. In this study we developed and formulated a combination of four attenuated Salmonella Typhimurium live vector strains delivering heterologous Brucella antigens (rBs), namely lumazine synthase, proline racemase subunit A, lipoprotein outer membrane protein-19, and Cu-Zn superoxide dismutase. With an aim to develop a cross-protecting vaccine, Brucella pan-species conserved rBs were selected. The present study compared the efficacy of smooth and rough variants of Salmonella delivery vector and also evaluated the inclusion of purified Brucella lipopolysaccharide (LPS) in the formulation. Immunization of SPF-BALB/c mice with the vaccine combinations significantly (P≤0.05) reduced splenic wild-type Brucella abortus 544 colonization as compared to non-immunized mice as well as Salmonella only immunized mice. Increased induction of Brucella specific-IgG, sIgA production, and antigen-specific splenocyte proliferative responses were observed in the mice immunized with the formulations as compared to naïve or vector only immunized mice. Modulatory effects of rB and LPS on production of interleukin (IL)-4, IL-12, and interferon-γ were detected in splenocytes of mice immunized with the formulation. Rough Salmonella variant in combination with LPS could further enhance the efficacy of the delivery when applied intraperitoneally. Taken together, it is compelling that Brucella LPS-augmented Salmonella vector delivering immunogenic Brucella proteins may be more suitable than the current non-ideal live Brucella abortus vaccine. The vaccine system also provides a basis for the development of cross-protecting vaccine capable of preventing multispecies brucellosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Genomic Island 2 Is an Unstable Genetic Element Contributing to Brucella Lipopolysaccharide Spontaneous Smooth-to-Rough Dissociation ▿ †

    Science.gov (United States)

    Mancilla, Marcos; López-Goñi, Ignacio; Moriyón, Ignacio; Zárraga, Ana María

    2010-01-01

    Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages. PMID:20952568

  6. Importance of Lipopolysaccharide and Cyclic β-1,2-Glucans in Brucella-Mammalian Infections

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    Andreas F. Haag

    2010-01-01

    Full Text Available Brucella species are the causative agents of one of the most prevalent zoonotic diseases: brucellosis. Infections by Brucella species cause major economic losses in agriculture, leading to abortions in infected animals and resulting in a severe, although rarely lethal, debilitating disease in humans. Brucella species persist as intracellular pathogens that manage to effectively evade recognition by the host's immune system. Sugar-modified components in the Brucella cell envelope play an important role in their host interaction. Brucella lipopolysaccharide (LPS, unlike Escherichia coli LPS, does not trigger the host's innate immune system. Brucella produces cyclic β-1,2-glucans, which are important for targeting them to their replicative niche in the endoplasmic reticulum within the host cell. This paper will focus on the role of LPS and cyclic β-1,2-glucans in Brucella-mammalian infections and discuss the use of mutants, within the biosynthesis pathway of these cell envelope structures, in vaccine development.

  7. New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide

    Science.gov (United States)

    Casabuono, Adriana C.; Czibener, Cecilia; Del Giudice, Mariela G.; Valguarnera, Ezequiel; Ugalde, Juan E.; Couto, Alicia S.

    2017-12-01

    Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. [Figure not available: see fulltext.

  8. Differential Stimulatory Activities of Smooth and Rough Brucella abortus Lipopolysaccharide in Murine Macrophages

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    Raheela Akhtar1,2*, Yongqun O. He2, Charles B. Larson2, Zafar I. Chaudhary3 and Mansur ud-Din Ahmad4

    2012-06-01

    Full Text Available Brucella abortus lipopolysaccharide (LPS was isolated and purified from rough (RB51 and smooth (S2308 strains of Brucella. The LPS preparations were used to treat murine (RAW 264.7 macrophages in order to study their differential effects. Treated macrophages were tested by lysozyme release test (LRT, nitroblue tetrazolium test (NBT and nitric oxide (NO assay, respectively. Rough Brucella LPS induced significantly higher levels of lysozyme release, oxidative stress, and nitric oxide in murine macrophages than smooth Brucella LPS or combined LPS (rough + smooth LPS. These responses were dose-dependent. Macrophages treated with rough LPS were more Brucellacidal than those treated with smooth LPS. The minimal stimulation of murine macrophages by Brucella smooth LPS may provide basis for less active immune responses against smooth strains.

  9. The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

    Science.gov (United States)

    Iriarte, Maite; Manček-Keber, Mateja; Barquero-Calvo, Elías; Palacios-Chaves, Leyre; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Martirosyan, Anna; von Bargen, Kristine; Grilló, María-Jesús; Jerala, Roman; Brandenburg, Klaus; Llobet, Enrique; Bengoechea, José A.; Moreno, Edgardo

    2012-01-01

    Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines. PMID:22589715

  10. Study of Nitric Oxide production by murine peritoneal macrophages induced by Brucella Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Kavoosi G

    2001-07-01

    Full Text Available Brueclla is a gram negative bacteria that causes Brucellosis. Lipopolysaccharide (LPS ", the pathogenic agent of Brucella is composed of O-chain, core oligosaccharide and lipid A. in addition, the structural and biological properties of different LPS extracted from different strains are not identical. The first defense system against LPS is nonspecific immunity that causes macrophage activation. Activated macrophages produce oxygen and nitrogen radicals that enhance the protection against intracellular pathogens.In this experiment LPS was extracted by hot phenol- water procedure and the effect of various LPSs on nitric oxide prodution by peritoneal mouse macrophages was examined.Our results demonstrated that the effect of LPS on nitric oxide production is concentration-dependent we observed the maximum response in concentration of 10-20 microgram per milliliter. Also our results demonstrate that LPS extracted from vaccine Brucella abortus (S 19 had a highe effect on nitric oxide production than the LPS from other strains

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

    Science.gov (United States)

    Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G; Gorvel, Jean-Pierre; Moreno, Edgardo

    2015-05-01

    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.

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

    Science.gov (United States)

    Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L.; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G.; Gorvel, Jean-Pierre; Moreno, Edgardo

    2015-01-01

    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. PMID:25946018

  13. Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

    Science.gov (United States)

    Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

    2015-10-05

    Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural

  14. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  15. Diversity of virulence genes in Brucella melitensis and Brucella abortus detected from patients with rheumatoid arthritis.

    Science.gov (United States)

    Rahdar, Hossein Ali; Golmohammadi, Reza; Mirnejad, Reza; Ataee, Ramezan Ali; Alishiri, Gholam Hossein; Kazemian, Hossein

    2018-03-22

    The presence of Brucella melitensis and Brucella abortus genomes were investigated in the synovial fluid (SF) samples from 90 patients with rheumatoid arthritis (RA). DNA extraction and PCR assay were performed for simultaneous identification and discrimination of B. melitensis and B. abortus from the SF using three specific primers. After gel electrophoresis, the PCR products were confirmed by DNA sequencing. The cbg, omp31, manA, virB, and znuA virulence genes typing were performed by multiplex-PCR. Of the 90 samples, 14 were positive for B. melitensis (n = 9; 10%) and B. abortus (n = 5; 5.5%). The virulotyping of positive samples revealed the presence of all five virulence genes in B. melitensis. The virB, cbg, and om31 were detected in all five samples of B. abortus. In addition, zhuA and manA were detected in three (60%) and four (80%) samples, respectively, of the B. abortus-positive samples. Moreover, a total of 94.2% and 89.2% of the 14 positive samples were also found positive for manA and znuA, respectively. Our findings revealed that the Brucella spp. genomes can be detected in the SF of RA patients by the PCR-based method. We thus suggest that physicians should consider the Brucella spp. as indicators of potential RA for the timely diagnosis and treatment of RA. Copyright © 2018. Published by Elsevier Ltd.

  16. Brucella

    Science.gov (United States)

    The genus Brucella encompasses a group of gram negative bacteria that survive almost exclusively in infected hosts with preference for localization in intracellular compartments of cells. The genus has traditionally been divided into species based on microbe characteristics and host preference, bu...

  17. Comparison of Biological and Immunological Characterization of Lipopolysaccharides From Brucella abortus RB51 and S19.

    Science.gov (United States)

    Kianmehr, Zahra; Kaboudanian Ardestani, Sussan; Soleimanjahi, Hoorieh; Fotouhi, Fatemeh; Alamian, Saeed; Ahmadian, Shahin

    2015-11-01

    Brucella abortus RB51 is a rough stable mutant strain, which has been widely used as a live vaccine for prevention of brucellosis in cattle instead of B. abortus strain S19. B. abortus lipopolysaccharide (LPS) has unique properties in comparison to other bacterial LPS. In the current study, two types of LPS, smooth (S-LPS) and rough (R-LPS) were purified from B. abortus S19 and RB51, respectively. The aim of this study was to evaluate biological and immunological properties of purified LPS as an immunogenical determinant. Primarily, S19 and RB51 LPS were extracted and purified by two different modifications of the phenol water method. The final purity of LPS was determined by chemical analysis (2-keto-3-deoxyoctonate (KDO), glycan, phosphate and protein content) and different staining methods, following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). C57BL/6 mice were immunized subcutaneously three times at biweekly intervals with the same amount of purified LPSs. The humoral immunity was evaluated by measuring specific IgG levels and also different cytokine levels, such as IFN-γ, TNF-α, IL-4 and IL-10, were determined for assessing T-cell immune response. Biochemical analysis data and SDS-PAGE profile showed that the chemical nature of S19 LPS is different from RB51 LPS. Both S and R-LPS induce an immune response. T-cell immune response induced by both S and R-LPS had almost the same pattern whereas S19 LPS elicited humoral immunity, which was higher than RB51 LPS. Purified LPS can be considered as a safe adjuvant and can be used as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies.

  18. Analysis of pan-genome to identify the core genes and essential genes of Brucella spp.

    Science.gov (United States)

    Yang, Xiaowen; Li, Yajie; Zang, Juan; Li, Yexia; Bie, Pengfei; Lu, Yanli; Wu, Qingmin

    2016-04-01

    Brucella spp. are facultative intracellular pathogens, that cause a contagious zoonotic disease, that can result in such outcomes as abortion or sterility in susceptible animal hosts and grave, debilitating illness in humans. For deciphering the survival mechanism of Brucella spp. in vivo, 42 Brucella complete genomes from NCBI were analyzed for the pan-genome and core genome by identification of their composition and function of Brucella genomes. The results showed that the total 132,143 protein-coding genes in these genomes were divided into 5369 clusters. Among these, 1710 clusters were associated with the core genome, 1182 clusters with strain-specific genes and 2477 clusters with dispensable genomes. COG analysis indicated that 44 % of the core genes were devoted to metabolism, which were mainly responsible for energy production and conversion (COG category C), and amino acid transport and metabolism (COG category E). Meanwhile, approximately 35 % of the core genes were in positive selection. In addition, 1252 potential essential genes were predicted in the core genome by comparison with a prokaryote database of essential genes. The results suggested that the core genes in Brucella genomes are relatively conservation, and the energy and amino acid metabolism play a more important role in the process of growth and reproduction in Brucella spp. This study might help us to better understand the mechanisms of Brucella persistent infection and provide some clues for further exploring the gene modules of the intracellular survival in Brucella spp.

  19. Detection of virulence-associated genes in Brucella melitensis ...

    African Journals Online (AJOL)

    The current study involved detection of three virulence genes (bvfA, virB, ure) by PCR in 52 isolates of Brucella melitensis biovar 3, recovered from different animal species (28 sheep, 10 goats, 9 cattle and 5 buffaloes). Of the 52 B. melitensis strains; 48 (92.3%) isolates carried bvfA genes, 51 (98.1%) isolates had virB genes ...

  20. Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope

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    Raquel Conde-Álvarez

    2018-01-01

    Full Text Available The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP of the lipopolysaccharide (LPS, free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides, while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the

  1. Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope

    Science.gov (United States)

    Conde-Álvarez, Raquel; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Salvador-Bescós, Miriam; Bárcena-Varela, Marina; Aragón-Aranda, Beatriz; Martínez-Gómez, Estrella; Zúñiga-Ripa, Amaia; de Miguel, María J.; Bartholomew, Toby Leigh; Hanniffy, Sean; Grilló, María-Jesús; Vences-Guzmán, Miguel Ángel; Bengoechea, José A.; Arce-Gorvel, Vilma; Gorvel, Jean-Pierre; Moriyón, Ignacio; Iriarte, Maite

    2018-01-01

    The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the

  2. Construction of pTM series plasmids for gene expression in Brucella species.

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Ontology-based Brucella vaccine literature indexing and systematic analysis of gene-vaccine association network.

    Science.gov (United States)

    Hur, Junguk; Xiang, Zuoshuang; Feldman, Eva L; He, Yongqun

    2011-08-26

    Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals. The VO-based SciMiner (VO-SciMiner) was developed to incorporate a total of 67 Brucella vaccine terms. A set of rules for term expansion of VO terms were learned from training data, consisting of 90 biomedical articles related to Brucella vaccine terms. VO-SciMiner demonstrated high recall (91%) and precision (99%) from testing a separate set of 100 manually selected biomedical articles. VO-SciMiner indexing exhibited superior performance in retrieving Brucella vaccine-related papers over that obtained with MeSH-based PubMed literature search. For example, a VO-SciMiner search of "live attenuated Brucella vaccine" returned 922 hits as of April 20, 2011, while a PubMed search of the same query resulted in only 74 hits. Using the abstracts of 14,947 Brucella-related papers, VO-SciMiner identified 140 Brucella genes associated with Brucella vaccines. These genes included known protective antigens, virulence factors, and genes closely related to Brucella vaccines. These VO-interacting Brucella genes were significantly over-represented in biological functional categories, including metabolite transport and metabolism, replication and repair, cell wall biogenesis, intracellular trafficking and secretion, posttranslational modification, and chaperones. Furthermore, a comprehensive interaction network of Brucella vaccines and genes were identified. The asserted

  4. Ontology-based Brucella vaccine literature indexing and systematic analysis of gene-vaccine association network

    Science.gov (United States)

    2011-01-01

    Background Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals. Results The VO-based SciMiner (VO-SciMiner) was developed to incorporate a total of 67 Brucella vaccine terms. A set of rules for term expansion of VO terms were learned from training data, consisting of 90 biomedical articles related to Brucella vaccine terms. VO-SciMiner demonstrated high recall (91%) and precision (99%) from testing a separate set of 100 manually selected biomedical articles. VO-SciMiner indexing exhibited superior performance in retrieving Brucella vaccine-related papers over that obtained with MeSH-based PubMed literature search. For example, a VO-SciMiner search of "live attenuated Brucella vaccine" returned 922 hits as of April 20, 2011, while a PubMed search of the same query resulted in only 74 hits. Using the abstracts of 14,947 Brucella-related papers, VO-SciMiner identified 140 Brucella genes associated with Brucella vaccines. These genes included known protective antigens, virulence factors, and genes closely related to Brucella vaccines. These VO-interacting Brucella genes were significantly over-represented in biological functional categories, including metabolite transport and metabolism, replication and repair, cell wall biogenesis, intracellular trafficking and secretion, posttranslational modification, and chaperones. Furthermore, a comprehensive interaction network of Brucella vaccines and genes were

  5. Brucella melitensis global gene expression study provides novel information on growth phase-specific gene regulation with potential insights for understanding Brucella:host initial interactions

    Directory of Open Access Journals (Sweden)

    Garner Harold R

    2009-05-01

    Full Text Available Abstract Background Brucella spp. are the etiological agents of brucellosis, a zoonotic infectious disease that causes abortion in animals and chronic debilitating illness in humans. Natural Brucella infections occur primarily through an incompletely defined mechanism of adhesion to and penetration of mucosal epithelium. In this study, we characterized changes in genome-wide transcript abundance of the most and the least invasive growth phases of B. melitensis cultures to HeLa cells, as a preliminary approach for identifying candidate pathogen genes involved in invasion of epithelial cells. Results B. melitensis at the late logarithmic phase of growth are more invasive to HeLa cells than mid-logarithmic or stationary growth phases. Microarray analysis of B. melitensis gene expression identified 414 up- and 40 down-regulated genes in late-log growth phase (the most invasive culture compared to the stationary growth phase (the least invasive culture. As expected, the majority of up-regulated genes in late-log phase cultures were those associated with growth, including DNA replication, transcription, translation, intermediate metabolism, energy production and conversion, membrane transport, and biogenesis of the cell envelope and outer membrane; while the down-regulated genes were distributed among several functional categories. Conclusion This Brucella global expression profile study provides novel information on growth phase-specific gene expression. Further characterization of some genes found differentially expressed in the most invasive culture will likely bring new insights into the initial molecular interactions between Brucella and its host.

  6. Brucella abortus: pathogenicity and gene regulation of virulence

    Directory of Open Access Journals (Sweden)

    Olga Rivas-Solano

    2015-06-01

    Full Text Available Brucella abortus is a zoonotic intracellular facultative pathogen belonging to the subdivision α2 of class Proteobacteria. It causes a worldwide distributed zoonotic disease called brucellosis. The main symptoms are abortion and sterility in cattle, as well as an undulant febrile condition in humans. In endemic regions like Central America, brucellosis has a high socioeconomic impact. A basic research project was recently conducted at the ITCR with the purpose of studying gene regulation of virulence, structure and immunogenicity in B. abortus. The present review was written as part of this project. B. abortus virulence seems to be determined by its ability to invade, survive and replicate inside professional and non-professional phagocytes. It reaches its intracellular replicative niche without the activation of host antimicrobial mechanisms of innate immunity. It also has gene regulation mechanisms for a rapid adaptation to an intracellular environment such as the two-component signal transduction system BvrR/BvrS and the quorum sensing regulator called Vjbr, as well as other transcription factors. All of them integrate a complex gene regulation network.

  7. Detection of virulence-associated genes in Brucella melitensis ...

    African Journals Online (AJOL)

    Ibrahim Eldaghayes

    2018-03-20

    Mar 20, 2018 ... isolated from goats. This discrepancies may indicate that B. melitensis field strains prevailing in Egypt are more virulent than the strains of B. melitensis isolated from caprines in Iran. As, it was emphasized that the. T4SS of Brucella encoded by the virB operon is a major virulence factor (Delrue et al., 2005).

  8. Identification of Brucella ovis exclusive genes in field isolates from Argentina.

    Science.gov (United States)

    Alvarez, Lucía Paula; García-Effrón, Guillermo; Robles, Carlos Alejandro

    2016-03-01

    Brucellosis caused by Brucella ovis is one of the most important infectious diseases of sheep. The aim of this study was to determine the presence of genes both inside and outside the specific B. ovis pathogenicity island 1 (BOPI-1) in a large collection of field isolates of B. ovis and other Brucella spp. from Argentina. The BOV_A0500 gene from B. ovis BOPI-1 was identified in all 104 B. ovis isolates studied. The BOPI-1 complete sequence was found to be conserved in 10 B. ovis strains from the collection, for which whole genome sequencing was performed. The BOV_0198 gene, which is outside BOPI-1 and considered exclusive to B. ovis, showed 90-100% identity with genomic regions of B. ovis, B. melitensis, B. abortus, B. canis, B. suis, B. microti, B. ceti and B. pinnipedialis. The results demonstrate that BOPI-1 is the only exclusive genetic region of B. ovis and marine Brucella spp. and that it is highly conserved in B. ovis field isolates from Argentina. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Molecular prevalence of putative virulence-associated genes in Brucella melitensis and Brucella abortus isolates from human and livestock specimens in Iran.

    Science.gov (United States)

    Hashemifar, Iman; Yadegar, Abbas; Jazi, Faramarz Masjedian; Amirmozafari, Nour

    2017-04-01

    Molecular prevalence of nine putative virulence factors in two more prevalent Brucella species in Iranian patients and livestock was investigated. During five years (2010-2015), 120 human and animal specimens were collected from three geographical areas of Iran. All samples were cultured in blood culture media and subcultured into Brucella agar medium. Nine primer pairs were designed for detection of VirB2, VirB5, VceC, BtpA, BtpB, PrpA, BetB, BPE275 and BSPB virulence factors using PCR and sequence analysis. Totally, 68 Brucella isolates including 60 B. melitensis and 8 B. abortus were isolated from the human and animal specimens examined. Approximately, all B. melitensis and B. abortus strains were positive (100%) regarding btpA, btpB, virB5, vceC, bpe275, bspB, and virB2 genes except for prpA and betB that were detected in 86% and 97% of the strains, respectively. Significant relationships were found between the presence of prpA and human B. melitensis isolates (P = 0.04), and also between the presence of betB and human isolates of B. abortus (P = 0.03). In conclusion, our results revealed that Iranian Brucella strains, regardless of human or animal sources, are extremely virulent due to high prevalence of virulence attributes in almost all strains studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Microarray-based identification of differentially expressed genes in intracellular Brucella abortus within RAW264.7 cells.

    Directory of Open Access Journals (Sweden)

    Mingxing Tian

    Full Text Available Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella's virulence and potential for chronic infection include the ability to circumvent the host cell's internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334 of all Brucella abortus genes were up-regulated and 5.4% (180/3334 were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292 had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.

  11. Heterogeneity in Tandem Octanucleotides within Haemophilus influenzae Lipopolysaccharide Biosynthetic Gene losA Affects Serum Resistance

    OpenAIRE

    Erwin, Alice L.; Bonthuis, Paul J.; Geelhood, Jennifer L.; Nelson, Kevin L.; McCrea, Kirk W.; Gilsdorf, Janet R.; Smith, Arnold L.

    2006-01-01

    Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nonty...

  12. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus.

    Directory of Open Access Journals (Sweden)

    Fang Chen

    Full Text Available Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK. Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella

  13. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus.

    Science.gov (United States)

    Chen, Fang; He, Yongqun

    2009-08-28

    Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK). Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella pathogenesis and

  14. A new cis-encoded sRNA, BsrH, regulating the expression of hemH gene in Brucella abortus 2308.

    Science.gov (United States)

    Peng, Xiaowei; Dong, Hao; Wu, Qingmin

    2015-01-01

    A total of 129 sRNA candidates were identified in Brucella abortus 2308 in our previous work, and one candidate with potential to regulate expression of hemH gene was further analyzed in this study. We found that the novel sRNA can inhibit the expression of hemH and called it BsrH (Brucella sRNA regulating HemH). The expression level of BsrH was tested in four different stress conditions. A significant upregulation was detected during the growth in acidic and Brucella minimal media, as well as in the presence of hydroxyl peroxide, while iron deficiency caused the opposite effect. As expected, BsrH strongly affected the survival ratio of the Brucella cells under iron-limitation conditions, though overexpression of BsrH did not affect Brucella virulence. Thus, we conclude that BsrH plays a regulatory role in bacterial heme biosynthesis and can be considered as the first Brucella sRNA involved in stress responses. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Genome degradation in Brucella ovis corresponds with narrowing of its host range and tissue tropism.

    Directory of Open Access Journals (Sweden)

    Renee M Tsolis

    Full Text Available Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.

  16. Combinatorial control of adhesion of Brucella abortus 2308 to host cells by transcriptional rewiring of the trimeric autotransporter btaE gene.

    Science.gov (United States)

    Sieira, Rodrigo; Bialer, Magalí G; Roset, Mara S; Ruiz-Ranwez, Verónica; Langer, Tomás; Arocena, Gastón M; Mancini, Estefanía; Zorreguieta, Angeles

    2017-02-01

    Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host. © 2016 John Wiley & Sons Ltd.

  17. Brucella, nitrogen and virulence.

    Science.gov (United States)

    Ronneau, Severin; Moussa, Simon; Barbier, Thibault; Conde-Álvarez, Raquel; Zuniga-Ripa, Amaia; Moriyon, Ignacio; Letesson, Jean-Jacques

    2016-08-01

    The brucellae are α-Proteobacteria causing brucellosis, an important zoonosis. Although multiplying in endoplasmic reticulum-derived vacuoles, they cause no cell death, suggesting subtle but efficient use of host resources. Brucellae are amino-acid prototrophs able to grow with ammonium or use glutamate as the sole carbon-nitrogen source in vitro. They contain more than twice amino acid/peptide/polyamine uptake genes than the amino-acid auxotroph Legionella pneumophila, which multiplies in a similar vacuole, suggesting a different nutritional strategy. During these two last decades, many mutants of key actors in nitrogen metabolism (transporters, enzymes, regulators, etc.) have been described to be essential for full virulence of brucellae. Here, we review the genomic and experimental data on Brucella nitrogen metabolism and its connection with virulence. An analysis of various aspects of this metabolism (transport, assimilation, biosynthesis, catabolism, respiration and regulation) has highlighted differences and similarities in nitrogen metabolism with other α-Proteobacteria. Together, these data suggest that, during their intracellular life cycle, the brucellae use various nitrogen sources for biosynthesis, catabolism and respiration following a strategy that requires prototrophy and a tight regulation of nitrogen use.

  18. Defects in rhizobial cyclic glucan and lipopolysaccharide synthesis alter legume gene expression during nodule development

    DEFF Research Database (Denmark)

    D'Antuono, Alejandra L; Ott, Thomas; Krusell, Lene

    2008-01-01

    higher expression of phenylalanine ammonia lyase than wild-type nodules. Differences in expression pattern of genes involved in early recognition and signaling were observed in plants inoculated with the M. loti mutant strain affected in the synthesis of cyclic glucan. Udgivelsesdato: 2008-Jan......cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic beta(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsbeta2). Expression of genes associated...

  19. Identification of an IS711 Element Interrupting the wboA Gene of Brucella abortus Vaccine Strain RB51 and a PCR Assay To Distinguish Strain RB51 from Other Brucella Species and Strains

    Science.gov (United States)

    Vemulapalli, Ramesh; McQuiston, John R.; Schurig, Gerhardt G.; Sriranganathan, Nammalwar; Halling, Shirley M.; Boyle, Stephen M.

    1999-01-01

    Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested. PMID:10473532

  20. Vaccination of elk (Cervus canadensis) with Brucella abortus strain RB51 overexpressing superoxide dismutase and glycosyltransferase genes does not induce adequate protection against experimental brucella abortus challenge

    Science.gov (United States)

    In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area (GYA). In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the d...

  1. Lipopolysaccharide triggers nuclear import of Lpcat1 to regulate inducible gene expression in lung epithelia.

    Science.gov (United States)

    Ellis, Bryon; Kaercher, Leah; Snavely, Courtney; Zhao, Yutong; Zou, Chunbin

    2012-07-26

    To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene transcription. Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation. Lpcat1 translocates into the nucleus from the cytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli, two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overexpressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment. These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.

  2. Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization assay.

    Science.gov (United States)

    Nielsen, K; Smith, P; Conde, S; Draghi de Benitez, G; Gall, D; Halbert, G; Kenny, K; Massengill, C; Muenks, Q; Rojas, X; Perez, B; Samartino, L; Silva, P; Tollersrud, T; Jolley, M

    2004-01-01

    Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

  3. Complementation of Brucella abortus RB51 with a Functional wboA Gene Results in O-Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation

    OpenAIRE

    Vemulapalli, Ramesh; He, Yongqun; Buccolo, Larissa S.; Boyle, Stephen M.; Sriranganathan, Nammalwar; Schurig, Gerhardt G.

    2000-01-01

    Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760–764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to pr...

  4. Deletion of the small RNA chaperone protein Hfq down regulates genes related to virulence and confers protection against wild-type Brucella challenge in mice

    Directory of Open Access Journals (Sweden)

    Shuangshuang eLei

    2016-01-01

    Full Text Available Brucellosis is one of the most common zoonotic epidemics worldwide. Brucella, the etiological pathogen of brucellosis, has unique virulence characteristics, including the ability to survive within the host cell. Hfq is a bacterial chaperone protein that is involved in the survival of the pathogen under stress conditions. Moreover, hfq affects the expression of a large number of target genes. In the present study, we characterized the expression and regulatory patterns of the target genes of Hfq during brucellosis. The results revealed that hfq expression is highly induced in macrophages at the early infection stage and at the late stage of mouse infection. Several genes related to virulence, including omp25, omp31, vjbR, htrA, gntR, and dnaK, were found to be regulated by hfq during infection in BALB/c mice. Gene expression and cytokine secretion analysis revealed that an hfq-deletion mutant induced different cytokine profiles compared with that induced by 16M. Infection with the hfq-deletion mutant induced protective immune responses against 16M challenge. Together, these results suggest that hfq is induced during infection and its deletion results in significant attenuation which affects the host immune response caused by Brucella infection. By regulating genes related to virulence, hfq promotes the virulence of Brucella. The unique characteristics of the hfq-deletion mutant, including its decreased virulence and the ability to induce protective immune response upon infection, suggest that it represents an attractive candidate for the design of a live attenuated vaccine against Brucella.

  5. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Science.gov (United States)

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

    Directory of Open Access Journals (Sweden)

    Peng Li

    2017-09-01

    Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

  7. Brucella abortus

    Science.gov (United States)

    Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Roop Ii, R Martin; Comerci, Diego J; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Caswell, Clayton C; Baker, Kate S; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Letesson, Jean J; De Bolle, Xavier; Guzmán-Verri, Caterina

    2016-01-01

    Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella . As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain B. abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised.

  8. Vaccination of elk (Cervus canadensis with Brucella abortus strain RB51 overexpressing superoxide dismutase and glycosyltransferase genes does not induce adequate protection against experimental Brucella abortus challenge

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    Pauline eNol

    2016-02-01

    Full Text Available In recent years, elk (Cervus canadensis have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosytransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further work is needed for development of an effective brucellosis vaccine for use in elk

  9. Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge.

    Science.gov (United States)

    Nol, Pauline; Olsen, Steven C; Rhyan, Jack C; Sriranganathan, Nammalwar; McCollum, Matthew P; Hennager, Steven G; Pavuk, Alana A; Sprino, Phillip J; Boyle, Stephen M; Berrier, Randall J; Salman, Mo D

    2016-01-01

    In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

  10. Mutation of purD and purF genes further attenuates Brucella abortus strain RB51.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Watarai, Masahisa; Hahn, Tae-Wook

    2015-02-01

    In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Effects of lipopolysaccharide-induced inflammation on expression of growth-associated genes by corticospinal neurons

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    Lieberman AR

    2006-01-01

    Full Text Available Abstract Background Inflammation around cell bodies of primary sensory neurons and retinal ganglion cells enhances expression of neuronal growth-associated genes and stimulates axonal regeneration. We have asked if inflammation would have similar effects on corticospinal neurons, which normally show little response to spinal cord injury. Lipopolysaccharide (LPS was applied onto the pial surface of the motor cortex of adult rats with or without concomitant injury of the corticospinal tract at C4. Inflammation around corticospinal tract cell bodies in the motor cortex was assessed by immunohistochemistry for OX42 (a microglia and macrophage marker. Expression of growth-associated genes c-jun, ATF3, SCG10 and GAP-43 was investigated by immunohistochemistry or in situ hybridisation. Results Application of LPS induced a gradient of inflammation through the full depth of the motor cortex and promoted c-Jun and SCG10 expression for up to 2 weeks, and GAP-43 upregulation for 3 days by many corticospinal neurons, but had very limited effects on neuronal ATF3 expression. However, many glial cells in the subcortical white matter upregulated ATF3. LPS did not promote sprouting of anterogradely labelled corticospinal axons, which did not grow into or beyond a cervical lesion site. Conclusion Inflammation produced by topical application of LPS promoted increased expression of some growth-associated genes in the cell bodies of corticospinal neurons, but was insufficient to promote regeneration of the corticospinal tract.

  12. Heterogeneity in tandem octanucleotides within Haemophilus influenzae lipopolysaccharide biosynthetic gene losA affects serum resistance.

    Science.gov (United States)

    Erwin, Alice L; Bonthuis, Paul J; Geelhood, Jennifer L; Nelson, Kevin L; McCrea, Kirk W; Gilsdorf, Janet R; Smith, Arnold L

    2006-06-01

    Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA. Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies (losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA-on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA, a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.

  13. Identification of Secondary Mutations Which Enhance and Stabilize the Attenuation of Brucella HTRA Mutants: Improving Brucella HTRA-Based Strains as Vaccine Candidates

    National Research Council Canada - National Science Library

    Roop, R

    2000-01-01

    Results to date from the studies funded under this contract suggest that Brucella genes involved in maintaining efficient stationary phase physiology and allowing the brucellae to make effective use...

  14. Development and trial of vaccines against Brucella

    Science.gov (United States)

    Lalsiamthara, Jonathan

    2017-01-01

    The search for ideal brucellosis vaccines remains active today. Currently, no licensed human or canine anti-brucellosis vaccines are available. In bovines, the most successful vaccine (S19) is only used in calves, as adult vaccination results in orchitis in male, prolonged infection, and possible abortion complications in pregnant female cattle. Another widely deployed vaccine (RB51) has a low protective efficacy. An ideal vaccine should exhibit a safe profile as well as enhance protective efficacy. However, currently available vaccines exhibit one or more major drawbacks. Smooth live attenuated vaccines suffer shortcomings such as residual virulence and serodiagnostic interference. Inactivated vaccines, in general, confer relatively low levels of protection. Recent developments to improve brucellosis vaccines include generation of knockout mutants by targeting genes involved in metabolism, virulence, and the lipopolysaccharide synthesis pathway, as well as generation of DNA vaccines, mucosal vaccines, and live vectored vaccines, have all produced varying degrees of success. Herein, we briefly review the bacteriology, pathogenesis, immunological implications, candidate vaccines, vaccinations, and models related to Brucella. PMID:28859268

  15. Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein.

    Science.gov (United States)

    Oliveira, S C; Splitter, G A

    1994-11-01

    The Brucella abortus L7/L12 ribosomal gene was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5 alpha was transformed with the pMAL-L7/L12 construct, and gene expression was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The resulting fusion protein was purified by affinity chromatography and confirmed by Western blot (immunoblot) analysis using an anti-maltose-binding protein antibody. Additionally, purified recombinant L7/L12 protein induced T-lymphocyte proliferation of B. abortus-primed bovine peripheral blood mononuclear cells. Phenotypic analysis of the proliferating cell population demonstrated an increase in the percentage of CD4+ T lymphocytes when peripheral blood mononuclear cells were cultured with recombinant L7/L12 compared with cells cultured in medium alone. Subcloning and expression of a B. abortus gene encoding a previously demonstrated immunodominant protein for bovine lymphocytes are important steps in selecting Brucella proteins that have potential as a component of a genetically engineered candidate vaccine.

  16. Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation.

    Science.gov (United States)

    Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

    2015-01-01

    The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M.

  17. Characterization of ribonuclease III from Brucella.

    Science.gov (United States)

    Wu, Chang-Xian; Xu, Xian-Jin; Zheng, Ke; Liu, Fang; Yang, Xu-Dong; Chen, Chuang-Fu; Chen, Huan-Chun; Liu, Zheng-Fei

    2016-04-01

    Bacterial ribonuclease III (RNase III) is a highly conserved endonuclease, which plays pivotal roles in RNA maturation and decay pathways by cleaving double-stranded structure of RNAs. Here we cloned rncS gene from the genomic DNA of Brucella melitensis, and analyzed the cleavage properties of RNase III from Brucella. We identified Brucella-encoding small RNA (sRNA) by high-throughput sequencing and northern blot, and found that sRNA of Brucella and Homo miRNA precursor (pre-miRNA) can be bound and cleaved by B.melitensis ribonuclease III (Bm-RNase III). Cleavage activity of Bm-RNase III is bivalent metal cations- and alkaline buffer-dependent. We constructed several point mutations in Bm-RNase III, whose cleavage activity indicated that the 133th Glutamic acid residue was required for catalytic activity. Western blot revealed that Bm-RNase III was differently expressed in Brucella virulence strain 027 and vaccine strain M5-90. Collectively, our data suggest that Brucella RNase III can efficiently bind and cleave stem-loop structure of small RNA, and might participate in regulation of virulence in Brucella. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Type IV secretion and Brucella virulence.

    Science.gov (United States)

    Boschiroli, Maria Laura; Ouahrani-Bettache, Safia; Foulongne, Vincent; Michaux-Charachon, Sylvie; Bourg, Gisele; Allardet-Servent, Annick; Cazevieille, Chantal; Lavigne, Jean-Phillipe; Liautard, Jean Pierre; Ramuz, Michel; O'Callaghan, David

    2002-12-20

    The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate. Copyright 2002 Elsevier Science B.V.

  19. ChIP-seq analysis of the LuxR-type regulator VjbR reveals novel insights into the Brucella virulence gene expression network

    Science.gov (United States)

    Sycz, Gabriela; Bonomi, Hernán R.; Rodríguez, Romina M.; Zorreguieta, Angeles

    2017-01-01

    Abstract LuxR-type transcription factors control diverse physiological functions necessary for bacterial adaptation to environmental changes. In the intracellular pathogen Brucella, the LuxR homolog VjbR has been shown to regulate the expression of virulence factors acting at early stages of the intracellular infection and, directly or indirectly, hundreds of additional genes. However, the precise determination of VjbR direct targets has so far proved elusive. Here, we performed chromatin immunoprecipitation of VjbR followed by next-generation sequencing (ChIP-seq). We detected a large amount of VjbR-binding sites distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif, an unusual feature among LuxR-type regulators. RNA-seq analysis performed under conditions mimicking the eukaryotic intracellular environment revealed that, among all loci associated to VjbR-binding, this regulator directly modulated the expression of only a subset of genes encoding functions consistent with an intracellular adaptation strategy for survival during the initial stages of the host cell infection. Other VjbR-binding events, however, showed to be dissociated from transcription and may require different environmental signals to produce a transcriptional output. Taken together, our results bring new insights into the extent and functionality of LuxR-type-related transcriptional networks. PMID:28334833

  20. Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Goya Rodolfo G

    2011-03-01

    Full Text Available Abstract Background Insulin-like growth factor-I (IGF-I exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65 was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65 to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory

  1. Detection of virulence-associated genes in Brucella melitensis biovar 3, the prevalent field strain in different animal species in Egypt

    Directory of Open Access Journals (Sweden)

    Mahmoud E.R. Hamdy

    2018-03-01

    Full Text Available The current study involved detection of three virulence genes (bvfA, virB, ure by PCR in 52 isolates of Brucella melitensis biovar 3, recovered from different animal species (28 sheep, 10 goats, 9 cattle and 5 buffaloes. Of the 52 B. melitensis strains; 48 (92.3% isolates carried bvfA genes, 51 (98.1% isolates had virB genes and 50 (96.2% isolates were positive for ure genes. The distribution of the virulence genes is not affected by crossing the original host barriers of the animal species, as the three virulence factors (bvfA, virB and ure detected in 28 B. melitensis isolates obtained from ovine species in a ratio of 26/28 (92.9%, 27/28 (96.4% and 28/28 (100%, respectively. While 10 isolates originating from goats revealed a ratio of 10/10 (100%, 10/10 (100% and 9/10 (90% to the same order of virulence genes. Nearly, similar results of virulence genes detection were obtained in B. melitensis obtained from bovine (8/9, 9/9 and 8/9 and Buffalos (4/5, 5/5 and 5/5, respectively. The high prevalence of virulence-associated genes among the B. melitensis isolates detected from different animal species in Egypt indicates a potential virulence of this bacterium.

  2. A framework to identify gene expression profiles in a model of inflammation induced by lipopolysaccharide after treatment with thalidomide

    Directory of Open Access Journals (Sweden)

    Paiva Renata T

    2012-06-01

    Full Text Available Abstract Background Thalidomide is an anti-inflammatory and anti-angiogenic drug currently used for the treatment of several diseases, including erythema nodosum leprosum, which occurs in patients with lepromatous leprosy. In this research, we use DNA microarray analysis to identify the impact of thalidomide on gene expression responses in human cells after lipopolysaccharide (LPS stimulation. We employed a two-stage framework. Initially, we identified 1584 altered genes in response to LPS. Modulation of this set of genes was then analyzed in the LPS stimulated cells treated with thalidomide. Results We identified 64 genes with altered expression induced by thalidomide using the rank product method. In addition, the lists of up-regulated and down-regulated genes were investigated by means of bioinformatics functional analysis, which allowed for the identification of biological processes affected by thalidomide. Confirmatory analysis was done in five of the identified genes using real time PCR. Conclusions The results showed some genes that can further our understanding of the biological mechanisms in the action of thalidomide. Of the five genes evaluated with real time PCR, three were down regulated and two were up regulated confirming the initial results of the microarray analysis.

  3. A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

    Science.gov (United States)

    Wang, Jing-Yu; Wu, Ning; Liu, Wan-Hua; Ren, Juan-Juan; Tang, Pan; Qiu, Yuan-Hao; Wang, Chi-Young; Chang, Ching-Dong; Liu, Hung-Jen

    2014-01-01

    The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The Optimization of Molecular Detection of Clinical Isolates of Brucella in Blood Cultures by eryD Transcriptase Gene for Confirmation of Culture-Negative Samples.

    Science.gov (United States)

    Tabibnejad, Mahsa; Alikhani, Mohammad Yousef; Arjomandzadegan, Mohammad; Hashemi, Seyed Hamid; Naseri, Zahra

    2016-04-01

    Brucellosis is a zoonosis disease which is widespread across the world. The aim of the present study is the evaluation of culture-negative blood samples. A total of 100 patients with suspected brucellosis were included in this experimental study and given positive serological tests. Diagnosis was performed on patients with clinical symptoms of the disease, followed by the detection of a titer that was equal to or more than 1:160 (in endemic areas) by the standard tube agglutination method. Blood samples were cultured by a BACTEC 9050 system, and subsequently by Brucella agar. At the same time, DNA from all blood samples was extracted by Qiagen Kit Company (Qia Amp Mini Kit). A molecular assay of blood samples was carried out by detection of eryD transcriptase and bcsp 31 genes in specific double PCR reactions. The specificity of the primers was evaluated by DNA from pure and approved Brucella colonies found in the blood samples, by DNA from other bacteria, and by ordinary PCR. DNA extraction from the pure colonies was carried out by both Qiagen Kit and Chelex 100 methods; the two were compared. 39 cases (39%) had positive results when tested by the BACTEC system, and 61 cases (61%) became negative. 23 culture-positive blood samples were randomly selected for PCR reactions; all showed 491 bp for the eryD gene and 223 bp for the bcsp 31 gene. Interestingly, out of 14 culture-negative blood samples, 13 cases showed positive bonds in PCR. The specificity of the PCR method was equal to 100%. DNA extraction from pure cultures was done by both Chelex 100 and Qiagen Kit; these showed the same results for all samples. The results prove that the presented double PCR method could be used to detect positive cases from culture-negative blood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.

  5. Amplification, cloning and expression of Brucella melitensis bp26 gene (OMP28 isolated from Markazi province (Iran and purification of Bp26 Protein

    Directory of Open Access Journals (Sweden)

    Hosseini, S.D.

    2013-12-01

    Full Text Available Brucellosis is a debilitative disease that imposes costs on both economy and society. It is shown that although the vaccine can prevent abortion, it does not provide complete protection against infection. In Iran, Brucella melitensis is a common causative agent for brucellosis and BP26 protein of this bacterium having a good antigenesity and an important vaccine candidate. In this study B. melitensis bp26 gene was cloned first in to PTZ57R/T vector and accessed on the PET28a vector and sequenced. Recombinant vector transformed and expressed in to E. coli BL21 (DE3 and then recombinant protein was purified with Ni-NTA column of chromatography against His tag. Obtained rOmp28 could be used as a research experimental tool to find its potential as a detection kit and vaccine candidate.

  6. Brucella abortus Ornithine Lipids Are Dispensable Outer Membrane Components Devoid of a Marked Pathogen-Associated Molecular Pattern

    Science.gov (United States)

    Palacios-Chaves, Leyre; Conde-Álvarez, Raquel; Gil-Ramírez, Yolanda; Zúñiga-Ripa, Amaia; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Arce-Gorvel, Vilma; Gorvel, Jean-Pierre; Moreno, Edgardo; de Miguel, María-Jesús; Grilló, María-Jesús

    2011-01-01

    The brucellae are α-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-α release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity. PMID:21249206

  7. Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

    Directory of Open Access Journals (Sweden)

    Boermans Herman J

    2006-11-01

    Full Text Available Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR. Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

  8. Limited Genetic Diversity of Brucella spp.

    OpenAIRE

    Gándara, Benjamín; Merino, Ahidé López; Rogel, Marco Antonio; Martínez-Romero, Esperanza

    2001-01-01

    Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis. Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici....

  9. Whole-genome analyses of speciation events in pathogenic Brucellae

    Energy Technology Data Exchange (ETDEWEB)

    Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Comerci, Diego J. [Universidad Nacional de General San Martin; Tolmasky, Marcelo E. [California State University; Larimer, Frank W [ORNL; Malfatti, Stephanie [Lawrence Livermore National Laboratory (LLNL); Vergez, Lisa [Lawrence Livermore National Laboratory (LLNL); Aguero, Fernan [Universidad Nacional de General San Martin; Land, Miriam L [ORNL; Ugalde, Rodolfo A. [Universidad Nacional de General San Martin; Garcia, Emilio [Lawrence Livermore National Laboratory (LLNL)

    2005-12-01

    Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

  10. Negative feedback regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by heme oxygenase-1 induction in macrophages.

    Science.gov (United States)

    Ashino, Takashi; Yamanaka, Rieko; Yamamoto, Masayuki; Shimokawa, Hiroaki; Sekikawa, Kenji; Iwakura, Yoichiro; Shioda, Seiji; Numazawa, Satoshi; Yoshida, Takemi

    2008-04-01

    Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.

  11. Brucella melitensis VjbR and C12-HSL regulons: contributions of the N-dodecanoyl homoserine lactone signaling molecule and LuxR homologue VjbR to gene expression

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    Garner Harold R

    2010-06-01

    Full Text Available Abstract Background Quorum sensing is a communication system that regulates gene expression in response to population density and often regulates virulence determinants. Deletion of the luxR homologue vjbR highly attenuates intracellular survival of Brucella melitensis and has been interpreted to be an indication of a role for QS in Brucella infection. Confirmation for such a role was suggested, but not confirmed, by the demonstrated in vitro synthesis of an auto-inducer (AI by Brucella cultures. In an effort to further delineate the role of VjbR to virulence and survival, gene expression under the control of VjbR and AI was characterized using microarray analysis. Results Analyses of wildtype B. melitensis and isogenic ΔvjbR transciptomes, grown in the presence and absence of exogenous N-dodecanoyl homoserine lactone (C12-HSL, revealed a temporal pattern of gene regulation with variances detected at exponential and stationary growth phases. Comparison of VjbR and C12-HSL transcriptomes indicated the shared regulation of 127 genes with all but 3 genes inversely regulated, suggesting that C12-HSL functions via VjbR in this case to reverse gene expression at these loci. Additional analysis using a ΔvjbR mutant revealed that AHL also altered gene expression in the absence of VjbR, up-regulating expression of 48 genes and a luxR homologue blxR 93-fold at stationary growth phase. Gene expression alterations include previously un-described adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, transporters, membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, energy production genes, and the previously reported fliF and virB operons. Conclusions VjbR and C12-HSL regulate expression of a large and diverse number of genes. Many genes identified as virulence factors in other bacterial pathogens were found to be differently expressed, suggesting an important contribution to intracellular

  12. The genome sequence of Brucella pinnipedialis B2/94 sheds light on the evolutionary history of the genus Brucella

    Science.gov (United States)

    2011-01-01

    Background Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups. PMID:21745361

  13. The genome sequence of Brucella pinnipedialis B2/94 sheds light on the evolutionary history of the genus Brucella

    Directory of Open Access Journals (Sweden)

    Claverie Jean-Michel

    2011-07-01

    Full Text Available Abstract Background Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a genome segments unshared between B. microti and B. pinnipedialis, b gene deletion/fusion events and c positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups.

  14. Activation of inflammatory immune gene cascades by lipopolysaccharide (LPS) in the porcine colonic tissue ex-vivo model.

    Science.gov (United States)

    Bahar, B; O'Doherty, J V; Vigors, S; Sweeney, T

    2016-11-01

    The technique of challenging postmortem tissue explants with inflammation inducer such as lipopolysaccharide (LPS) followed by gene expression analysis is used widely for evaluating the immune-suppressing effect of bioactives. Using porcine colonic tissue as an ex-vivo model of mammalian intestinal gut, this study evaluated the effect of incubation time on the integrity of gene transcripts and activation of inflammatory immune gene cascade by LPS treatment. Post-slaughter colon was removed surgically and explants were incubated for 0, 3, 6 and 12 h and the abundance of mRNA transcripts of a panel of 92 immune genes were evaluated using quantitative polymerase chain reaction (qPCR) arrays. The mRNA transcripts were highly intact after 0 and 3 h of incubation; however, after 6 h the degradation was clearly evident. Following 3 h incubation, 98·8% and 100% mRNA transcripts were detectable in the colonic tissue harvested from weaned and mature pigs, respectively. In the explants of weaned piglets, LPS treatment activated inflammatory signalling pathways [high mobility group B1 (HMGB1), dendritic cell maturation, interleukin (IL)-6, IL-8, IL-17F], while these pathways were inhibited by dexamethasone treatment. Activations of inflammatory genes were also evident in the explants collected from the mature pigs subjected to ex-vivo incubation for 3 h in the absence or presence of LPS. It is concluded that the colonic explant remains physiologically viable and responsive to immunological challenge for up to 3 h ex-vivo. © 2016 British Society for Immunology.

  15. Comparison of Gene Expression by Sheep and Human Blood Stimulated with the TLR4 Agonists Lipopolysaccharide and Monophosphoryl Lipid A.

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    Perenlei Enkhbaatar

    Full Text Available Animal models that mimic human biology are important for successful translation of basic science discoveries into the clinical practice. Recent studies in rodents have demonstrated the efficacy of TLR4 agonists as immunomodulators in models of infection. However, rodent models have been criticized for not mimicking important characteristics of the human immune response to microbial products. The goal of this study was to compare genomic responses of human and sheep blood to the TLR4 agonists lipopolysaccharide (LPS and monophosphoryl lipid A (MPLA.Venous blood, withdrawn from six healthy human adult volunteers (~ 28 years old and six healthy adult female sheep (~3 years old, was mixed with 30 μL of PBS, LPS (1μg/mL or MPLA (10μg/mL and incubated at room temperature for 90 minutes on a rolling rocker. After incubation, 2.5 mL of blood was transferred to Paxgene Blood RNA tubes. Gene expression analysis was performed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip. Agilent gene expression microarrays were scanned with a G2565 Microarray Scanner. Differentially expressed genes were identified.11,431 human and 4,992 sheep probes were detected above background. Among them 1,029 human and 175 sheep genes were differentially expressed at a stringency of 1.5-fold change (p 1.5-fold changes in human samples. Genes of major inflammatory mediators, such as IL-1, IL-6 and IL-8, TNF alpha, NF-kappaB, ETS2, PTGS2, PTX3, CXCL16, KYNU, and CLEC4E were similarly (>2-fold upregulated by LPS and MPLA in both species.The genomic responses of peripheral blood to LPS and MPLA in sheep are quite similar to those observed in humans, supporting the use of the ovine model for translational studies that mimic human inflammatory diseases and the study of TLR-based immunomodulators.

  16. Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics

    Directory of Open Access Journals (Sweden)

    Yongqun eHe

    2012-02-01

    Full Text Available Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of ten classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning.

  17. Analyses of Brucella Pathogenesis, Host Immunity, and Vaccine Targets using Systems Biology and Bioinformatics

    Science.gov (United States)

    He, Yongqun

    2011-01-01

    Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning. PMID:22919594

  18. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide

    DEFF Research Database (Denmark)

    Jiang, Li; Sørensen, Peter; Røntved, Christine

    2008-01-01

    Liver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver...... they are challenged with LPS. Our work presents the first insight into the dynamic changes in gene expression in the liver that influences the induction, kinetics and clinical outcome of the APR in dairy cows...... in turn stimulated or repressed the expression of genes encoding acute phase proteins (APP), collectins, complement components, chemokines, cell adhesion molecules and key metabolic enzymes during the APR. Hormones, anti-inflammatory and other hypothalamus-pituitary-adrenal axis (HPAA) linked mediators...

  19. Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice.

    Science.gov (United States)

    Hagiwara, Teruki; Yoshida, Shigeru; Hidaka, Yuji

    2017-04-01

    The concentration-sensitive sodium channel (Na C ) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that Na C contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether Na C is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between Na C gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. Na C protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of Na C and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the Na C protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of Na C decreased in the lung, as well as those for α-, β-, and γ-ENaC during ALI. Thus, Na C expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. Our results suggest that suppression of the gene expression of Na C is involved in the development of pulmonary edema in ALI.

  20. Activation of camalexin biosynthesis in Arabidopsis thaliana in response to perception of bacterial lipopolysaccharides: a gene-to-metabolite study.

    Science.gov (United States)

    Beets, Caryn Ann; Huang, Ju-Chi; Madala, Ntakadzeni Edwin; Dubery, Ian

    2012-07-01

    Lipopolysaccharides (LPS), as lipoglycan microbe-associated molecular pattern molecules, trigger activation of signal transduction pathways involved in defence that generate an enhanced defensive capacity in plants. The transcriptional regulation of the genes for tryptophan synthase B, TSB1, and the cytochrome P450 monooxygenases CYP79B2 and CYP71B15, involved in the camalexin biosynthetic pathway, were investigated in response to LPS treatment. GUS-reporter assays for CYP71B15 and CYP79B2 gene promoter activation were performed on transgenic plants and showed positive histochemical staining in response to LPS treatment, indicating activation of the promoters. Quantitative PCR revealed that transcripts of TSB1, CYP79B2 and CYP71B15 exhibited differential, transient up-regulation. TSB1 transcript levels were up-regulated between 6 and 9 h after LPS-induction, while CYP71B15 and CYP79B2 both exhibited maxima at 12 h. To obtain information on the gene-to-metabolite network, the effect of the transcriptome changes on the metabolome was correlated to camalexin production. Increases in camalexin concentration were quantified by ultra pressure liquid chromatography-mass spectrometry and both absorbance spectra and elemental composition confirmed its identity. The concentrations increased from 0.03 to 3.7 μg g(-1) fresh weight over a 24-h time period, thus indicating that the up-regulation of the biosynthetic pathway in response to LPS was accompanied by a time-dependent increase in camalexin concentration. Metabolomic analysis through principal component analysis-derived scores plots revealed clusters of sample replicates for 0, 6, 12, 18 and 24 h while loadings plots for LPS data identified camalexin as a biomarker that clearly demonstrated the variability between samples.

  1. Brucella papionis sp. nov., isolated from baboons (Papio spp.).

    Science.gov (United States)

    Whatmore, Adrian M; Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S; Brew, Simon D; Perrett, Lorraine L; Koylass, Mark S; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C; Dick, Edward J; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E

    2014-12-01

    Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP

  2. A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata Reveals Characteristics Departing from Classical Brucellae

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    Pedro Franscico Soler Llorens

    2016-09-01

    Full Text Available Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the ‘classical’ Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted ‘classical’ species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1 and BO2, which is remarkable for this bacterial genus.This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

  3. A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae

    Science.gov (United States)

    Soler-Lloréns, Pedro F.; Quance, Chris R.; Lawhon, Sara D.; Stuber, Tod P.; Edwards, John F.; Ficht, Thomas A.; Robbe-Austerman, Suelee; O'Callaghan, David; Keriel, Anne

    2016-01-01

    Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the “classical” Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted “classical” species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required. PMID:27734009

  4. A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae.

    Science.gov (United States)

    Soler-Lloréns, Pedro F; Quance, Chris R; Lawhon, Sara D; Stuber, Tod P; Edwards, John F; Ficht, Thomas A; Robbe-Austerman, Suelee; O'Callaghan, David; Keriel, Anne

    2016-01-01

    Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog ( Ceratophyrus ornate ) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi . We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata -like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella , but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

  5. Comparative effects of the herbal constituent parthenolide (Feverfew on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

    Directory of Open Access Journals (Sweden)

    Smolinski Alexa T

    2005-06-01

    Full Text Available Abstract Background Parthenolide, a major sesquiterpene lactone present in extracts of the herb Feverfew, has been investigated for its inhibitory effects on mediators of inflammation, including the proinflammatory cytokines. Although parthenolide's anti-inflammatory effects have been investigated in vitro, little in vivo data are available. Moreover, the molecular mechanisms for these inhibitory effects are not fully understood. The objective of this study was to test the hypothesis that parthenolide suppresses lipopolysaccharide (LPS-induced serum (interleukin IL-6, tumor necrosis factor (TNF-α, IL-1β and cyclooxygenase (COX-2 expression in mice as indicated by reduced splenic and liver mRNA levels. Methods Mice were co-treated i.p. with LPS (1 mg/kg bw and parthenolide (5 mg/kg bw and blood, spleen and liver collected. Serum was analyzed for IL-6, TNF-α and IL-1β by ELISA. Total RNA was extracted from spleen and liver, and real-time RT-PCR was used to determine relative mRNA expression of IL-1β, IL-6, TNF-α and COX-2. Results LPS induced increases in serum IL-6 and TNF-α concentrations with only IL-6 being suppressed in parthenolide-treated mice. Induction of IL-6 mRNA was reduced, TNF-α and COX-2 mRNAs unchanged, and IL-1β mRNA increased in spleens of parthenolide plus LPS co-treated animals compared to LPS-only. No significant differences were observed in inflammatory gene expression between these two groups in liver samples. Overall, mRNA expression of each proinflammatory gene was much higher in spleen when compared to liver. Conclusion In summary, only one gene, IL-6, was modestly suppressed by parthenolide co-exposure which contrasts with many in vitro studies suggesting anti-inflammatory effects of this compound. Also, LPS evoked greater effects in spleen than liver on expression of proinflammatory genes. Further study of the effects of parthenolide and other herbal constituents on inflammatory gene expression using model

  6. Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Jorge U. Carmona

    2017-01-01

    Full Text Available Platelet-rich plasma (PRP preparations are used in horses with osteoarthritis (OA. However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG and leukoreduced platelet-rich gel (Lr-PRG supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs challenged with lipopolysaccharide (LPS. SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB, matrix metalloproteinase 13 (MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4, collagen type I alpha 1 (COL1A1, collagen type II alpha 1 (COL2A1, and cartilage oligomeric matrix protein (COMP. The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

  7. Regulation of Intracellular Signaling Leading to Gene Expression in Lipopolysaccharide Stimulated Murine Macrophages

    Science.gov (United States)

    1995-09-20

    responsive Promoter Elements 9 Cytoplasmic Regulation of LPS-inducible Gene Expression 10 LPS-inducible Phospholipid Metabolism 11 Ion Mobilization in LPS...existence of NO·-dependent and NO·-independent mechanisms of LPS-induced hypotension depending on the experimental inducers of endotoxemia . 3 It is clear...Inducible Phospholipid Metabolism Phospholipid metabolism results in the activation of at least three second messengers. Phosphatidylinositol 4,5

  8. Evaluation of Immunogenicity of Divalent DNA Vaccine Encoding Brucella melitensis Omp31 and P39 Genes in Balb/c Mice

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    A Doosti

    2007-10-01

    Full Text Available Introduction & Objective: Brucella is a facultative intracellular pathogen and one of the etiologic agents of brucellosis that can infect humans and domestic animals. Attenuated strains such as B. melitensis Rve1 and B. abortus S19 and Rb51 are being used to control brucellosis in domestic animals. However, no safe and effective vaccine is available for human use. This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the B. melitensis Omp31 protein and P39 protein, designated pCDNA3 recombinant vector. Materials & Methods: This experimental study was performed in Biotechnology Research Center of Islamic Azad University, Shahrekord branch in summer, 1386. Construction of pCDNA3 recombinant vector containing Omp31 and P39 genes of B. melitensis was completed. Then, 12 Balb/c mice were immunized intramuscularly with 100 mg per 50 micro liters of this DNA vaccine. Control mice, 12 Balb/c mice, were simultaneously injected with PBS. During the 1st, 7th, 15th and 30th days the mice received the injections. Afterwards, the ELISA cytokine assay was performed and data were analyzed by SPSS software. Results: Intramuscular injection of the divalent DNA vaccine elicited cellular immune responses in Balb/c mice. The ELISA cytokine assay with serum of vaccinated mice showed high level of IFN-γ and low changes of IL-4 in compare with control mice. Conclusion: Use of divalent genetic vaccine based on the Omp31 and P39 genes can elicit a strong cellular immune response against Brucellosis.

  9. Draft Genome Sequences of Brucella suis Biovar 4 Strain NCTC 10385, Brucella ceti Strain NCTC 12891T, Brucella inopinata Strain CAMP 6436T, and Brucella neotomae Strain ATCC 23459T

    OpenAIRE

    Wahab, Tara; Ferrari, Sevinc; Lindberg, Martina; Bäckman, Stina; Kaden, Rene

    2014-01-01

    With the aim of developing quantitative PCR methods for the detection and differentiation of Brucella species, the genomes of Brucella ceti, Brucella inopinata, Brucella netotomae, and Brucella suis biovar 4 were sequenced and analyzed.

  10. The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages.

    Science.gov (United States)

    Wei, Pan; Lu, Qiang; Cui, Guimei; Guan, Zhenhong; Yang, Li; Sun, Changjiang; Sun, Wanchun; Peng, Qisheng

    2015-02-15

    Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 or RB51 overexpressing superoxide dismutase and glycosyltransferase genes.

    Science.gov (United States)

    Olsen, S C; Boyle, S M; Schurig, G G; Sriranganathan, N N

    2009-04-01

    Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 (RB51) or a recombinant RB51 strain overexpressing superoxide dismutase (sodC) and glycosyltransferase (wboA) genes (RB51+sodC,wboA). Bison were vaccinated with saline only or with 4.6 x 10(10) CFU of RB51 or 7.4 x 10(10) CFU of RB51+sodC,wboA (n = eight animals/treatment). Bison vaccinated with RB51 or RB51+sodC,wboA had greater (P RB51 after vaccination than did nonvaccinates. However, bison vaccinated with RB51+sodC,wboA cleared the vaccine strain from draining lymph nodes faster than bison vaccinated with the parental RB51 strain. Immunologic responses of bison vaccinated with RB51+sodC,wboA were similar to responses of bison vaccinated with RB51. Pregnant bison were intraconjunctivally challenged in midgestation with 10(7) CFU of B. abortus strain 2308. Bison vaccinated with RB51, but not RB51+sodC,wboA vaccinates, had greater protection from abortion, fetal/uterine, mammary, or maternal infection than nonvaccinates. Our data suggest that the RB51+sodC,wboA strain is less efficacious as a calfhood vaccine for bison than the parental RB51 strain. Our data also suggest that the RB51 vaccine is a currently available management tool that could be utilized to help reduce brucellosis in free-ranging bison.

  12. Desenvolvimento e avaliação de uma cepa knockout de Brucella abortus obtida pela deleção do gene virB10 Development and evaluation of a strain of Brucella abortus gotten by the knockout of the virB10 gene

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    Fabiane G. de Souza

    2009-11-01

    Full Text Available Brucella spp. são bactérias gram-negativas, intracelulares facultativas que são patogênicas para muitas espécies de mamíferos causando a brucelose, uma zoonose difundida mundialmente. Por isso a busca de alternativas de controle mais eficientes se faz necessário como o desenvolvimento de novas cepas que possam ser testadas como potenciais imunógenos. Neste estudo realizou-se a deleção do gene virB10 da cepa S2308 de Brucella abortus gerando uma cepa knockout provavelmente incapaz de produzir a proteína nativa correspondente. O gene virB10 faz parte de um operon que codifica para um sistema de secreção do tipo IV, essencial para a sobrevivência intracelular e multiplicação da bactéria em células hospedeiras. A deleção foi realizada pela construção do plasmídeo suicida pBlue:virB10:kan e eletroporação deste em células eletrocompetentes de B. abortus S2308, ocorrendo a troca do gene selvagem pelo gene interrompido, com o gene de resistência a canamicina, por recombinação homóloga dupla. Camundongos BALB/c foram inoculados com as cepas S19, RB-51, ΔvirB10 de B. abortus e B. abortus S2308 selvagem; os resultados demonstraram que camundongos BALB/c inoculados com S19 e camundongos BALB/c inoculados com S2308 apresentaram queda mais rápida de linha de tendência, quando comparadas aos demais grupos, para recuperação bacteriana (RB e peso esplênico (PE respectivamente. Os grupos que receberam ΔvirB10 S2308 de B. abortus e RB-51 demonstraram comportamento semelhante para ambas as características. Na sexta semana após a inoculação, os resultados para RB (log de UFC ± desvio padrão e PE (peso esplênico ± desvio padrão, respectivamente, mostraram: grupos inoculados com as cepas S2308 (4,44±1,97 e 0,44±0,11, S19 (1,83±2,54 e 0,31±0,04, RB-51 (0,00±0,00 e 0,20±0,01 e ΔvirB10 S2308 (1,43±1,25 e 0,19±0,03. Considerado o clearance bacteriano, todos os grupos diferiram estatisticamente do grupo que recebeu S

  13. Colistin-Resistant, Lipopolysaccharide-Deficient Acinetobacter baumannii Responds to Lipopolysaccharide Loss through Increased Expression of Genes Involved in the Synthesis and Transport of Lipoproteins, Phospholipids, and Poly-β-1,6-N-Acetylglucosamine

    Science.gov (United States)

    Henry, Rebekah; Vithanage, Nuwan; Harrison, Paul; Seemann, Torsten; Coutts, Scott; Moffatt, Jennifer H.; Nation, Roger L.; Li, Jian; Harper, Marina; Adler, Ben

    2012-01-01

    We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971–4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-β-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface. PMID:22024825

  14. Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-β-1,6-N-acetylglucosamine.

    Science.gov (United States)

    Henry, Rebekah; Vithanage, Nuwan; Harrison, Paul; Seemann, Torsten; Coutts, Scott; Moffatt, Jennifer H; Nation, Roger L; Li, Jian; Harper, Marina; Adler, Ben; Boyce, John D

    2012-01-01

    We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-β-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

  15. Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strains RB51 or RB51 overexpressing superoxide dismutase and Glycosyltransferase genes

    Science.gov (United States)

    Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus stra...

  16. Brucella microti: the genome sequence of an emerging pathogen

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    Scholz Holger C

    2009-08-01

    Full Text Available Abstract Background Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel Brucella species, Brucella microti, was determined. B. microti is a member of the genus Brucella within the Alphaproteobacteria, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other Brucella species, B. microti is a fast growing and biochemically very active microorganism with a phenotype more similar to that of Ochrobactrum, a facultative human pathogen. The atypical phenotype of B. microti prompted us to look for genomic differences compared to other Brucella species and to look for similarities with Ochrobactrum. Results The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of B. microti is almost identical to that of Brucella suis 1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in B. microti. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between B. microti and Ochrobactrum anthropi but impaired in other sequenced Brucella. The most noticeable difference between B. microti and other Brucella species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of B. microti. Conclusion Contrary to expectations from the phenotypic analysis, the genome sequence of B. microti is highly similar to that of known Brucella species, and is remotely related to the one of O. anthropi. How the few differences in gene content between B. microti and B. suis 1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will

  17. Brucella microti: the genome sequence of an emerging pathogen.

    Science.gov (United States)

    Audic, Stéphane; Lescot, Magali; Claverie, Jean-Michel; Scholz, Holger C

    2009-08-04

    Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel Brucella species, Brucella microti, was determined. B. microti is a member of the genus Brucella within the Alphaproteobacteria, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other Brucella species, B. microti is a fast growing and biochemically very active microorganism with a phenotype more similar to that of Ochrobactrum, a facultative human pathogen. The atypical phenotype of B. microti prompted us to look for genomic differences compared to other Brucella species and to look for similarities with Ochrobactrum. The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of B. microti is almost identical to that of Brucella suis 1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in B. microti. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between B. microti and Ochrobactrum anthropi but impaired in other sequenced Brucella. The most noticeable difference between B. microti and other Brucella species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of B. microti. Contrary to expectations from the phenotypic analysis, the genome sequence of B. microti is highly similar to that of known Brucella species, and is remotely related to the one of O. anthropi. How the few differences in gene content between B. microti and B. suis 1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the

  18. Comparison of the efficacy of Brucella abortus strain RB51 and Brucella melitensis Rev. 1 live vaccines against experimental infection with Brucella melitensis in pregnant ewes.

    Science.gov (United States)

    el Idrissi, A H; Benkirane, A; el Maadoudi, M; Bouslikhane, M; Berrada, J; Zerouali, A

    2001-12-01

    To test the efficacy of rough Brucella strain vaccines in sheep, a vaccine recently developed in cattle (Brucella abortus strain RB51) was assessed in comparison with the conventional Rev. 1 vaccine. Forty-five ewes from twelve to fourteen months of age, from brucellosis-free flocks, were allotted to three groups of fifteen ewes each. Group one was vaccinated by the conjunctival route with 1.73 x 10(8) colony forming units (CFU) of Rev. 1 vaccine. Group two was vaccinated subcutaneously with 11 x 10(9) CFU of RB51 vaccine and group three was considered as a control. All sheep were challenged at two to three months of gestation with 5 x 10(7) CFU of virulent B. melitensis H38. Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide, as measured by conventional serological tests (Rose Bengal plate test and complement fixation test). Protection of sheep against abortion and excretion of virulent Brucella strain in vaginal fluid, aborted foetuses and/or non viable lambs at parturition and abortion was significantly lower than that afforded by Rev. 1 vaccine. The difference compared to the control group was not significant. Data from this study suggest that the RB51 vaccine used for cattle vaccination does not provide effective protection of sheep against abortion induced by B. melitensis.

  19. Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation.

    Science.gov (United States)

    Vemulapalli, R; He, Y; Buccolo, L S; Boyle, S M; Sriranganathan, N; Schurig, G G

    2000-07-01

    Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760-764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functional wboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboA gene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible

  20. Molecular investigation of virulence factors of Brucella melitensis and Brucella abortus strains isolated from clinical and non-clinical samples.

    Science.gov (United States)

    Mirnejad, Reza; Jazi, Faramarz Masjedian; Mostafaei, Shayan; Sedighi, Mansour

    2017-08-01

    Brucella is zoonotic pathogen that induces abortion and sterility in domestic mammals and chronic infections in humans called Malta fever. It is a facultative intracellular potential pathogen with high infectivity. The virulence of Brucella is dependent upon its potential virulence factors such as enzymes and cell envelope associated virulence genes. The aim of this study was to investigate the Brucella virulence factors among strains isolated from humans and animals in different parts of Iran. Seventy eight strains of Brucella species isolated from suspected human and animal cases from several provinces of Iran during 2015-2016 and identified by phenotypic and molecular methods. The multiplex-PCR (M-PCR) assay was performed in order to detect the ure, wbkA, omp19, mviN, manA and perA genes by using gene specific primers. Out of 78 isolates of Brucella spp., 57 (73%) and 21 (27%) isolates were detected as B. melitensis and B. abortus, respectively, by molecular method. The relative frequency of virulence genes ure, wbkA, omp19, mviN, manA and perA were 74.4%, 89.7%, 93.6%, 94.9%, 100% and 92.3%, respectively. Our results indicate that the most of Brucella strains isolated from this region possess high percent of virulence factor genes (ure, wbkA, omp19, mviN, manA and perA) in their genome. So, each step of infection can be mediated by a number of virulence factors and each strain may have a unique combination of these factors that affected the rate of bacterial pathogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis

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    García-Lobo Juan M

    2008-01-01

    Full Text Available Abstract Background The Brucella genome contains an insertion sequence (IS element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose. Results In this study we obtained evidence of transposition of IS711 from the B. ovis and B. pinnipedialis chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR. TcR mutants due to IS711 transposition were only detected in B. ovis and B. pinnipedialis strains. Conclusion Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

  2. Brucella microti sp. nov., isolated from the common vole Microtus arvalis.

    Science.gov (United States)

    Scholz, Holger C; Hubalek, Zdenek; Sedlácek, Ivo; Vergnaud, Gilles; Tomaso, Herbert; Al Dahouk, Sascha; Melzer, Falk; Kämpfer, Peter; Neubauer, Heinrich; Cloeckaert, Axel; Maquart, Marianne; Zygmunt, Michel S; Whatmore, Adrian M; Falsen, Enevold; Bahn, Peter; Göllner, Cornelia; Pfeffer, Martin; Huber, Birgit; Busse, Hans-Jürgen; Nöckler, Karsten

    2008-02-01

    Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular

  3. Characterisation of the genetic diversity of Brucella by multilocus sequencing

    Directory of Open Access Journals (Sweden)

    MacMillan Alastair P

    2007-04-01

    Full Text Available Abstract Background Brucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus. Results Nine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs. Diversity at each locus ranged from 1.03–2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences. Conclusion The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in

  4. The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs.

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    Scholz, Holger C; Mühldorfer, Kristin; Shilton, Cathy; Benedict, Suresh; Whatmore, Adrian M; Blom, Jochen; Eisenberg, Tobias

    2016-01-01

    The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.

  5. The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs

    Science.gov (United States)

    Scholz, Holger C.; Mühldorfer, Kristin; Shilton, Cathy; Benedict, Suresh; Whatmore, Adrian M.; Blom, Jochen; Eisenberg, Tobias

    2016-01-01

    The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer. PMID:28036367

  6. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    Science.gov (United States)

    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  7. Isolation of Brucella inopinata-Like Bacteria from White's and Denny's Tree Frogs.

    Science.gov (United States)

    Kimura, Masanobu; Une, Yumi; Suzuki, Michio; Park, Eun-Sil; Imaoka, Koichi; Morikawa, Shigeru

    2017-05-01

    Brucella inopinata strain BO1 and B. sp. strain BO2 isolated from human patients, respectively, are genetically different from classical Brucella species. We isolated bacteria of the genus Brucella from two species of wild-caught tropical frogs kept in the facilities in Japan: White's tree frog, which inhabits Oceania, and Denny's tree frog, which inhabits Southeast Asia. Phylogenetic analyses based on 16S rRNA and recA gene sequences and multilocus sequence analysis showed that two isolates of Brucella spp. showed significant similarity to BO1, BO2, and the isolates from other wild-caught frogs. These results suggest that a variety of frog species are susceptible to a novel clade of Brucella bacteria, including B. inopinata.

  8. Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

    Science.gov (United States)

    de Figueiredo, Paul; Ficht, Thomas A; Rice-Ficht, Allison; Rossetti, Carlos A; Adams, L Garry

    2015-06-01

    This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Purification of nonlipopolysaccharide antigen from Brucella abortus during preparation of antigen used for indirect hemolysis test.

    OpenAIRE

    Hoffmann, E M; Houle, J J

    1986-01-01

    The indirect hemolysis test (IHLT) for the diagnosis of brucellosis uses a lipopolysaccharide (LPS) antigen obtained by dimethyl sulfoxide extraction of Brucella abortus. We showed that a non-LPS antigen can be obtained as a by-product of the IHLT antigen preparation. The antigen was purified to homogeneity by a combination of gel-filtration chromatography and ion-exchange chromatography. The substance contained 8% protein and about 65% carbohydrate. The molecular weight of the primary unit w...

  10. Serological Diagnosis of Brucella Infections in Odontocetes▿

    Science.gov (United States)

    Hernández-Mora, Gabriela; Manire, Charles A.; González-Barrientos, Rocío; Barquero-Calvo, Elías; Guzmán-Verri, Caterina; Staggs, Lydia; Thompson, Rachel; Chaves-Olarte, Esteban; Moreno, Edgardo

    2009-01-01

    Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (Ri/g2 = 0.65 and κi/g = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (Ri/c2 = 0.46, Rg/c2 = 0.37 and κi/c = 0.62, κg/c = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study. PMID:19386800

  11. The waaL gene is involved in lipopolysaccharide synthesis and plays a role on the bacterial pathogenesis of avian pathogenic Escherichia coli.

    Science.gov (United States)

    Han, Yue; Han, Xiangan; Wang, Shaohui; Meng, Qingmei; Zhang, Yuxi; Ding, Chan; Yu, Shengqing

    2014-08-27

    Avian pathogenic Escherichia coli (APEC) is a Gram-negative bacterium that causes avian colibacillosis, resulting in economically devastating to poultry industries worldwide. Lipopolysaccharide (LPS) has been identified as an important virulence factor of E. coli. The waaL gene encodes O-antigen ligase, which is responsible for attaching the O-antigen to lipid A-core oligosaccharide. In this study, a mutant strain ΔwaaL was constructed from APEC serotype 2 strain DE17. The mutant strain showed a decreased swimming motility and resistance to complement-mediated killing but a similar growth rate in the culture, compared with its parent strain. In addition, the mutant LPS demonstrated different patterns in SDS-PAGE followed by silver staining and western blotting. Besides, the mutant strain significantly decreased its adherence and invasion abilities to DF-1 cells, compared to its parent strain DE17. Deletion of the waaL gene in DE17 reduced the bacterial virulence by 42.2-fold in ducklings, based on measurement of the median lethal dose (LD50). Additional analysis indicated that deletion of the waaL gene increased the biofilm formation ability and reduced the resistance to environmental stress. These results suggest that the waaL gene functions on the APEC LPS synthesis and bacterial pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Neonatal manipulation of oxytocin prevents lipopolysaccharide-induced decrease in gene expression of growth factors in two developmental stages of the female rat.

    Science.gov (United States)

    Bakos, Jan; Lestanova, Zuzana; Strbak, Vladimir; Havranek, Tomas; Bacova, Zuzana

    2014-10-01

    Oxytocin production and secretion is important for early development of the brain. Long-term consequences of manipulation of oxytocin system might include changes in markers of brain plasticity - cytoskeletal proteins and neurotrophins. The aim of the present study was (1) to determine whether neonatal oxytocin administration affects gene expression of nestin, microtubule-associated protein-2 (MAP-2), brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of two developmental stages of rat and (2) to evaluate whether neonatal oxytocin administration protects against lipopolysaccharide (LPS) induced inflammation. Neonatal oxytocin did not prevent a decrease of body weight in the LPS treated animals. Oxytocin significantly increased gene expression of BDNF in the right hippocampus in 21-day and 2-month old rats of both sexes. Gene expression of NGF and MAP-2 significantly increased in males treated with oxytocin. Both, growth factors and intermediate filament-nestin mRNA levels, were reduced in females exposed to LPS. Oxytocin treatment prevented a decrease in the gene expression of only growth factors. In conclusion, neonatal manipulation of oxytocin has developmental and sex-dependent effect on markers of brain plasticity. These results also indicate, that oxytocin may be protective against inflammation particularly in females. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Activation of bovine neutrophils by Brucella spp.

    Science.gov (United States)

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. [Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

    Science.gov (United States)

    Wang, Yue; Chen, Hong; Liu, Ying; Zhou, Jingzhu; Li, Shijun; Hang, Yan; Tang, Guangpeng; Wang, Dingming; Chen, Guichun

    2015-09-01

    To identify and characterize the Brucella strains from Guizhou province in 2010-2013. A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

  15. ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

    Science.gov (United States)

    Miller, Melissa A; Burgess, Tristan L; Dodd, Erin M; Rhyan, Jack C; Jang, Spencer S; Byrne, Barbara A; Gulland, Frances M D; Murray, Michael J; Toy-Choutka, Sharon; Conrad, Patricia A; Field, Cara L; Sidor, Inga F; Smith, Woutrina A

    2017-04-01

    We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

  16. ATP-Binding Cassette Systems of Brucella

    Directory of Open Access Journals (Sweden)

    Dominic C. Jenner

    2009-01-01

    Full Text Available Brucellosis is a prevalent zoonotic disease and is endemic in the Middle East, South America, and other areas of the world. In this study, complete inventories of putative functional ABC systems of five Brucella species have been compiled and compared. ABC systems of Brucella melitensis 16M, Brucella abortus 9-941, Brucella canis RM6/66, Brucella suis 1330, and Brucella ovis 63/290 were identified and aligned. High numbers of ABC systems, particularly nutrient importers, were found in all Brucella species. However, differences in the total numbers of ABC systems were identified (B. melitensis, 79; B. suis, 72; B. abortus 64; B. canis, 74; B. ovis, 59 as well as specific differences in the functional ABC systems of the Brucella species. Since B. ovis is not known to cause human brucellosis, functional ABC systems absent in the B. ovis genome may represent virulence factors in human brucellosis.

  17. Hemokinin-1 Gene Expression Is Upregulated in Microglia Activated by Lipopolysaccharide through NF-κB and p38 MAPK Signaling Pathways

    Science.gov (United States)

    Sawada, Makoto; Suzuki, Hidenori

    2012-01-01

    The mammalian tachykinins, substance P (SP) and hemokinin-1 (HK-1), are widely distributed throughout the nervous system and/or peripheral organs, and function as neurotransmitters or chemical modulators by activating their cognate receptor NK1. The TAC1 gene encoding SP is highly expressed in the nervous system, while the TAC4 gene encoding HK-1 is uniformly expressed throughout the body, including a variety of peripheral immune cells. Since TAC4 mRNA is also expressed in microglia, the resident immune cells in the central nervous system, HK-1 may be involved in the inflammatory processes mediated by these cells. In the present study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in primary cultured microglia. TAC4 mRNA expression was upregulated in the microglia upon their activation by lipopolysaccharide, a well-characterized Toll-like receptor 4 agonist, while TAC1 mRNA expression was downregulated. Furthermore, both nuclear factor-κB and p38 mitogen-activated protein kinase intracellular signaling pathways were required for the upregulation of TAC4 mRNA expression, but not for the downregulation of TAC1 mRNA expression. These findings suggest that HK-1, rather than SP, plays dominant roles in the pathological conditions associated with microglial activation, such as neurodegenerative and neuroinflammatory disorders. PMID:22384199

  18. Hemokinin-1 gene expression is upregulated in microglia activated by lipopolysaccharide through NF-κB and p38 MAPK signaling pathways.

    Directory of Open Access Journals (Sweden)

    Atsushi Sakai

    Full Text Available The mammalian tachykinins, substance P (SP and hemokinin-1 (HK-1, are widely distributed throughout the nervous system and/or peripheral organs, and function as neurotransmitters or chemical modulators by activating their cognate receptor NK(1. The TAC1 gene encoding SP is highly expressed in the nervous system, while the TAC4 gene encoding HK-1 is uniformly expressed throughout the body, including a variety of peripheral immune cells. Since TAC4 mRNA is also expressed in microglia, the resident immune cells in the central nervous system, HK-1 may be involved in the inflammatory processes mediated by these cells. In the present study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in primary cultured microglia. TAC4 mRNA expression was upregulated in the microglia upon their activation by lipopolysaccharide, a well-characterized Toll-like receptor 4 agonist, while TAC1 mRNA expression was downregulated. Furthermore, both nuclear factor-κB and p38 mitogen-activated protein kinase intracellular signaling pathways were required for the upregulation of TAC4 mRNA expression, but not for the downregulation of TAC1 mRNA expression. These findings suggest that HK-1, rather than SP, plays dominant roles in the pathological conditions associated with microglial activation, such as neurodegenerative and neuroinflammatory disorders.

  19. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    Science.gov (United States)

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  20. Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide.

    Science.gov (United States)

    Alencar, Valquíria Campos; Jabes, Daniela Leite; Menegidio, Fabiano Bezerra; Sassaki, Guilherme Lanzi; de Souza, Lucas Rodrigo; Puzer, Luciano; Meneghetti, Maria Cecília Zorél; Lima, Marcelo Andrade; Tersariol, Ivarne Luis Dos Santos; de Oliveira, Regina Costa; Nunes, Luiz R

    2017-02-07

    Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.

  1. Attenuated Bioluminescent Brucella melitensis Mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) Confer Protection in Mice

    OpenAIRE

    Rajashekara, Gireesh; Glover, David A.; Banai, Menachem; O'Callaghan, David; Splitter, Gary A.

    2006-01-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B....

  2. Isolation of prawn ( Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

    Science.gov (United States)

    Ge, Qianqian; Li, Jian; Duan, Yafei; Li, Jitao; Sun, Ming; Zhao, Fazhen

    2016-04-01

    The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn ( Exopalaemon carinicauda) ( EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

  3. JMJD2A attenuation affects cell cycle and tumourigenic inflammatory gene regulation in lipopolysaccharide stimulated neuroectodermal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Das, Amitabh, E-mail: amitabhdas.kn@gmail.com [Department of Bionanotechnology, Hanyang University, Seoul 133-791 (Korea, Republic of); Chai, Jin Choul, E-mail: jincchai@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Jung, Kyoung Hwa, E-mail: khjung2@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Das, Nando Dulal, E-mail: nando.hu@gmail.com [Clinical Research Centre, Inha University School of Medicine, Incheon 400-711 (Korea, Republic of); Kang, Sung Chul, E-mail: gujiju11@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Lee, Young Seek, E-mail: yslee@hanyang.ac.kr [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Seo, Hyemyung, E-mail: hseo@hanyang.ac.kr [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Chai, Young Gyu, E-mail: ygchai@hanyang.ac.kr [Department of Bionanotechnology, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of)

    2014-11-01

    JMJD2A is a lysine trimethyl-specific histone demethylase that is highly expressed in a variety of tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study were to identify JMJD2A-regulated genes and understand the function of JMJD2A in p53-null neuroectodermal stem cells (p53{sup −/−} NE-4Cs). We determined the effect of LPS as a model of inflammation in p53{sup −/−} NE-4Cs and investigated whether the epigenetic modifier JMJD2A alter the expression of tumourigenic inflammatory genes. Global gene expression was measured in JMJD2A knockdown (kd) p53{sup −/−} NE-4Cs and in LPS-stimulated JMJD2A-kd p53{sup −/−} NE-4C cells. JMJD2A attenuation significantly down-regulated genes were Cdca2, Ccnd2, Ccnd1, Crebbp, IL6rα, and Stat3 related with cell cycle, proliferation, and inflammatory-disease responses. Importantly, some tumour-suppressor genes including Dapk3, Timp2 and TFPI were significantly up-regulated but were not affected by silencing of the JMJD2B. Furthermore, we confirmed the attenuation of JMJD2A also down-regulated Cdca2, Ccnd2, Crebbp, and Rest in primary NSCs isolated from the forebrains of E15 embryos of C57/BL6J mice with effective p53 inhibitor pifithrin-α (PFT-α). Transcription factor (TF) motif analysis revealed known binding patterns for CDC5, MYC, and CREB, as well as three novel motifs in JMJD2A-regulated genes. IPA established molecular networks. The molecular network signatures and functional gene-expression profiling data from this study warrants further investigation as an effective therapeutic target, and studies to elucidate the molecular mechanism of JMJD2A-kd-dependent effects in neuroectodermal stem cells should be performed. - Highlights: • Significant up-regulation of epigenetic modifier JMJD2A mRNA upon LPS treatment. • Inhibition of JMJD2A attenuated key inflammatory and tumourigenic genes. • Establishing IPA based functional genomics in JMJD2A-attenuated p53{sup

  4. Systems Biology Analysis of Brucella Infected Peyer's Patch Reveals Rapid Invasion with Modest Transient Perturbations of the Host Transcriptome

    Science.gov (United States)

    Rossetti, Carlos A.; Drake, Kenneth L.; Siddavatam, Prasad; Lawhon, Sara D.; Nunes, Jairo E. S.; Gull, Tamara; Khare, Sangeeta; Everts, Robin E.; Lewin, Harris A.; Adams, Leslie Garry

    2013-01-01

    Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer's patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-α and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is

  5. Systems biology analysis of Brucella infected Peyer's patch reveals rapid invasion with modest transient perturbations of the host transcriptome.

    Directory of Open Access Journals (Sweden)

    Carlos A Rossetti

    Full Text Available Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer's patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-α and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process

  6. Case Report: Acute brucella meningomyeloencephalo ...

    African Journals Online (AJOL)

    African Health Sciences ... There are 4 species of brucella pathogenic to humans and each of them has a specific types of animal reservoir: B.arbotus (cattle, buffallo),B.melitensis (goats, sheep, camels), B.suis (pigs), B.canis (Dogs). Humans are infected when they are exposed to body fluids from an infected animal.

  7. Complement Fixation Test To Assess Humoral Immunity in Cattle and Sheep Vaccinated with Brucella abortus RB51

    OpenAIRE

    Adone, Rosanna; Ciuchini, Franco

    1999-01-01

    The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B. abortus 2308. The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS. The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in...

  8. An evaluation of ELISA using recombinant Brucella abortus bacterioferritin (Bfr) for bovine brucellosis.

    Science.gov (United States)

    Hop, Huynh Tan; Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-04-01

    To date, detection of antibodies against the lipopolysaccharide portion is the backbone of most serodiagnostic methods for brucellosis screening. However this pose a risk for false positive reactions related to other pathogens especially that of Yersinia enterocolitica O:9 which has the most prominent cross reactivity with Brucella spp. In this study, cloning and expression of Brucella abortus bacterioferritin (Bfr) was accomplished by PCR amplification into an expression vector system, and purification of a recombinant B. abortus Bfr (rBfr). The immunogenicity of rBfr was confirmed by Western blot with Brucella-positive bovine serum. To determine whether rBfr has a potential benefit for use in the serodiagnosis of bovine brucellosis, rBfr-based ELISA was performed. Interestingly, rBfr was able to detect anti-Brucella antibodies in positive sera in a dependent manner of TAT values but did not show an immunoreaction with negative samples. Particularly, average OD492 values at the lowest, medium and highest TAT titer levels were 1.4, 2.2 and 2.6-fold increase compared with the cutoff value, respectively. The accuracy, specificity and sensitivity of rBfr showed 89.09%, 93.6% and 85.33%, respectively. These findings suggest that rBfr might be a good candidate for serological diagnosis development of bovine brucellosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Expression pattern of inflammatory response genes and their regulatory micrornas in bovine oviductal cells in response to lipopolysaccharide: implication for early embryonic development.

    Directory of Open Access Journals (Sweden)

    Sally Ibrahim

    Full Text Available In the present study, we used an in vitro model to investigate the response of the oviduct with respect to inflammatory mediators and their regulatory microRNAs in case of bacterial infection and subsequent association with embryo survival. For this, we conducted two experiments. In the first experiment, cultured primary bovine oviductal cells (BOEC were challenged with lipopolysaccharide (LPS for 24h and the temporal expression pattern of inflammatory mediators and their regulatory microRNAs were measured at 0, 3, 6, 12, 24 and 48h after LPS treatment. Intriguingly, the temporal patterns of all miRNAs except miR-21 were significantly up-regulated at 6h after LPS treatment. Whereas, we observed significant overexpression of pro-inflammatory mediators as tumor necrosis factor alpha (TNFα and interleukin-1 beta (IL1β after LPS challenge for 24h. On the other hand, the expression level of essential elements like oviductal glycoprotein 1 (OVGP1 and insulin-like growth factor 2 (IGF2 was significantly decreased in challenged groups compared with control. Moreover, miR-155, miR-146a, miR-223, miR-21, miR-16 and miR-215 have shown a clear suppression in challenged group after LPS treatment. In the 2nd experiment there were four groups of blastocysts produced, namely embryo+LPS free media, embryo+LPS, BOEC+embryo and BOEC+embryo+LPS. The suboptimal oviduct environment due to LPS challenge is found to have a significant influence on the expression of inflammatory response genes (TNFα and CSF1, stress response genes (SOD and CAT, mitochondrial activity, reactive oxygen species (ROS accumulation and apoptotic level either in cultured or co-cultured blastocysts. Collectively, LPS challenge led to aberrant changes in oviductal transcriptome profile, which could lead to a suboptimal environment for embryo development.

  10. Dynamic expression of leukocyte innate immune genes in whole blood from horses with lipopolysaccharide-induced acute systemic inflammation

    DEFF Research Database (Denmark)

    Vinther, Anne Mette L.; Skovgaard, Kerstin; Heegaard, Peter M. H.

    2015-01-01

    Background: In horses, insights into the innate immune processes in acute systemic inflammation are limited even though these processes may be highly important for future diagnostic and therapeutic advances in high-mortality disease conditions as the systemic inflammatory response syndrome (SIRS...... expressions in blood leukocytes during equine acute LPS-induced systemic inflammation thoroughly characterized a highly regulated and dynamic innate immune response. These results provide new insights into the molecular mechanisms of equine systemic inflammation.......) and sepsis. Therefore, the aim of this study was to investigate the expression of 31 selected blood leukocyte immune genes in an equine model of acute systemic inflammation to identify significantly regulated genes and to describe their expression dynamics during a 24-h experimental period. Systemic...

  11. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

    Science.gov (United States)

    Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

    2017-01-01

    Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

  12. Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

    Science.gov (United States)

    Scholz, Holger C; Revilla-Fernández, Sandra; Al Dahouk, Sascha; Hammerl, Jens A; Zygmunt, Michel S; Cloeckaert, Axel; Koylass, Mark; Whatmore, Adrian M; Blom, Jochen; Vergnaud, Gilles; Witte, Angela; Aistleitner, Karin; Hofer, Erwin

    2016-05-01

    Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

  13. Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver.

    Science.gov (United States)

    Shepherd, Brian S; Spear, Allyn R; Philip, Anju M; Leaman, Douglas W; Stepien, Carol A; Sepulveda-Villet, Osvaldo J; Palmquist, Debra E; Vijayan, Mathilakath M

    2018-03-01

    Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1β: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1β mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1β, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout. Copyright © 2018. Published by Elsevier Ltd.

  14. Glucocorticoid augments lipopolysaccharide-induced activation of the IκBζ-dependent genes encoding the anti-microbial glycoproteins lipocalin 2 and pentraxin 3.

    Science.gov (United States)

    Yamazaki, Soh; Akira, Shizuo; Sumimoto, Hideki

    2015-05-01

    Bacterial lipopolysaccharide (LPS), one of the most potent inducers of inflammation, activates the transcription factor NF-κB to induce expression of both proinflammatory mediators and anti-microbial glycoproteins such as lipocalin 2 (Lcn2) and pentraxin 3 (PTX3) in macrophages. Glucocorticoids are known to inhibit LPS-induced expression of proinflammatory cytokines via glucocorticoid receptor (GR)-mediated transrepression of NF-κB, whereas their effect on induction of anti-microbial effectors has remained to be elucidated. Here we show that the synthetic glucocorticoid dexamethasone (Dex) strongly enhances LPS-induced transcription of Lcn2 and Ptx3, although Dex by itself fails to trigger their transcription. In macrophages deficient in IκBζ (an inducible coactivator of NF-κB), Lcn2 and Ptx3 are not activated by LPS either alone or in combination with Dex. Association of GR as well as Brg1 (a subunit of the chromatin remodelling Swi/Snf complex) with a functional glucocorticoid response element in Lcn2 requires both the costimulation with LPS and the presence of IκBζ. Although Ptx3 does not contain the element, LPS induces recruitment of Dex-liganded GR to NF-κB-binding sites in regulatory regions of Ptx3, an event that does not occur in IκBζ-deficient macrophages. Thus glucocorticoids likely regulate infection-induced inflammation by increasing anti-microbial effectors in an IκBζ-dependent manner, while repressing proinflammatory genes. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. Advancement of knowledge of Brucella over the past 50 years.

    Science.gov (United States)

    Olsen, S C; Palmer, M V

    2014-11-01

    Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. At that time, the genus was considered to contain only 3 species: Brucella abortus, Brucella melitensis, and Brucella suis. Since the early 1960s, at least 7 new species have been identified as belonging to the Brucella genus (Brucella canis, Brucella ceti, Brucella inopinata, Brucella microti, Brucella neotomae, Brucella ovis, and Brucella pinnipedialis) with several additional new species under consideration for inclusion. Although molecular studies have found such high homology that some authors have proposed that all Brucella are actually 1 species, the epidemiologic and diagnostic benefits for separating the genus based on phenotypic characteristics are more compelling. Although pathogenic Brucella spp have preferred reservoir hosts, their ability to infect numerous mammalian hosts has been increasingly documented. The maintenance of infection in new reservoir hosts, such as wildlife, has become an issue for both public health and animal health regulatory personnel. Since the 1960s, new information on how Brucella enters host cells and modifies their intracellular environment has been gained. Although the pathogenesis and histologic lesions of B. abortus, B. melitensis, and B. suis in their preferred hosts have not changed, additional knowledge on the pathology of these brucellae in new hosts, or of new species of Brucella in their preferred hosts, has been obtained. To this day, brucellosis remains a significant human zoonosis that is emerging or reemerging in many parts of the world. © The Author(s) 2014.

  16. The Effect of Artemisia fragrans Willd: Essential Oil on Inducible Nitric Oxide Synthase Gene Expression and Nitric Oxide Production in Lipopolysaccharide-stimulated Murine Macrophage Cell Line.

    Science.gov (United States)

    Farghadan, Maryam; Ghafoori, Hosein; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan; Farzaneh, Parvaneh; Kokhaei, Parviz

    2016-12-01

    The genus Artemisia is estimated to comprise over 800 species with anti-cancer, anti-fungal, anti-oxidant and anti-inflammatory properties. Artemisia fragrans (A. fragrans), a species that belongs to genus Artemisia, is rich in monoterpenes and sesquiterpenes derivatives. Due to anti-inflammatory properties of monoterpenes and sesquiterpenes, we aimed to investigate the effect of A. fragrans essential oil on mRNA expression of inducible nitric oxide synthase (iNOS) gene and nitric oxide (NO) production in Lipopolysaccharide (LPS) -stimulated RAW264.7 cell line. NO, which is synthesized by iNOS, is the main macrophage-derived inflammatory mediator. The oil obtained from the A. fragrans was prepared from aerial parts of the plant. Chemical composition of essential oil was analyzed by gas chromatography-mass spectrometry (GC/MS).The cytotoxicity of various concentrations of essential oil was evaluated by mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test assay. The effect of different doses (1.75-7 mg/mL) of A. fragrans oil on mRNA expression of iNOS gene and NO production in LPS-stimulated RAW 264.7 cells was assessed by real-time PCR method and Griess reagent, respectively. In GC/MS analyses of A. fragrans oil, 32 compounds were identified. The main components of the oil were camphor and 1, 8-cineole. The results demonstrated that the essential oil of A. fragrans (1.75- 7 mg/mL), in a dose-dependent manner, inhibits mRNA expression of iNOS induced by LPS in the RAW264.7 cells without cytotoxic effect even at higher doses. The results of iNOS were consistent with the results of NO production. Our preliminary results suggest the possible anti-inflammatory effect of A. fragrans. Further studies are needed to determine the full pharmacokinetics of A. fragrans activity in vivo.

  17. Potential proliferative effect of lipopolysaccharide preconditioning on ...

    African Journals Online (AJOL)

    User

    2015-04-01

    Apr 1, 2015 ... concentrations (0.1, 0.5 and 1 µg/ml) to assess its effect on EPCs proliferation. The following genes; ... Key words: Human umbilical cord blood (hUCB), Endothelial progenitors cells (EPCs), lipopolysaccharide. (LPS), Toll-like .... detergent reagent to each well and vibration for 10 min. The absorbance (A) in ...

  18. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus organism...

  19. [Insertional mutation in the AZOBR_p60120 gene is accompanied by defects in the synthesis of lipopolysaccharide and calcofluor-binding polysaccharides in the bacterium Azospirillum brasilense Sp245].

    Science.gov (United States)

    Katsy, E I; Prilipov, A G

    2015-03-01

    In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR_p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR_p60120 gene in the A. brasilense Sp245 LPSI- Cal- KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.

  20. Whole-genome analyses of the speciation events in the pathogenic Brucellae

    Energy Technology Data Exchange (ETDEWEB)

    Chain, P; Comerci, D; Tolmasky, M; Larimer, F; Malfatti, S; Vergez, L; Aguero, F; Land, M; Ugalde, R; Garcia, E

    2005-07-14

    Despite their high DNA identity and a proposal to group classical Brucella species as biovars of B. melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters as well as by a marked host range (e.g. B. suis for swine, B. melitensis for sheep and goats, B. abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucellae species and to the B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions and insertions support previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complement of both B. abortus strains is identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggest that these inactivations may play an important role in the establishment of host-specificity and may have been a primary driver of speciation in the Brucellae. Despite being non-motile, the Brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures, and may contribute to differences in host-specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g. glycogen, biotin, NAD, and choline) are consistent with adaptation of Brucellae to an intracellular lifestyle.

  1. mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin

    DEFF Research Database (Denmark)

    Andreassen, Stine Mandrup; Berg, Lise Charlotte; Nielsen, Søren Saxmose

    2015-01-01

    to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 μ g/ml LPS, 5 mg/ml fibrinogen or 5 U...

  2. Early in vivo cytokine genes expression in chickens after challenge with Salmonella typhimurium lipopolysaccharide and modulation by dietary n-3 polyunsaturated fatty acids

    NARCIS (Netherlands)

    Sijben, J.W.C.; Klasing, K.C.; Schrama, J.W.; Parmentier, H.K.; Poel, van der J.J.; Savelkoul, H.F.J.; Kaiser, P.

    2003-01-01

    We studied the effects of Salmonella typhimurium lipopolysaccharide (LPS) on in vivo cytokine mRNA levels in chickens, and investigated whether these levels could be altered by different nutrients. Two hundred and forty chicks were assigned in a 2 X 4 factorial design of treatments. Factors were

  3. Final Report Grant No. DE-FG02-98ER20307 Lipopolysaccharide Structures and Genes Required for Root Nodule Development August 1, 2004 to July 31, 2008

    Energy Technology Data Exchange (ETDEWEB)

    Noel, K. Dale [Marquette Univ., Milwaukee,WI (United States)

    2008-12-07

    This project dealt with the plant-bacterial symbiosis that gives rise to root nodules on leguminous plants in which the bacteria carry out nitrogen fixation. Nitrogen fixation, like carbon dioxide fixation, is essential for life on planet earth, and this symbiosis is estimated to account for half of all nitrogen fixed on land. Aside from being important for the sustenance of global life, this ability allows legumes to grow without nitrogen fertilizers. Basic studies such as this project are aimed at understanding the symbiosis well enough that eventually it can be engineered into important crop species so that they no longer depend on nitrogen fertilizer for growth. The production and distribution of excessive fertilizer needed for optimal crop yields is responsible for a significant portion of the energy costs in agriculture. The specific aims of this work were to further the understanding of a bacterial factor that is essential for the symbiotic infection process. This factor is a bacterial surface molecule, lipopolysaccharide O antigen. In this project we showed that, not only the presence, but the specific structure of this molecule is crucial for infection. Although the success of bacterial infections in many pathogenic and mutualistic interactions have been shown to depend on intact O antigen, it has been very rare to establish that specific features of the structure are important. One of the features in this case is the presence of one additional methyl group on one sugar in the O antigen. It is very surprising that such a minor change should have an observable effect. This work sets the stage for biochemical studies of possible plant receptors that may be involved. During the course of this grant period, we developed a method of testing the importance of this bacterial component at stages of nodule development beyond the step that is blocked by null mutation. The method works adequately for this purpose and is being improved. It has implications for testing

  4. Laboratory exposure to Brucella melitensis in Denmark

    DEFF Research Database (Denmark)

    Knudsen, A; Kronborg, G; Knudsen, Inge Jenny Dahl

    2013-01-01

    Brucella species are a frequent cause of laboratory-acquired infections. This report describes the handling of a laboratory exposure of 17 laboratory staff members exposed to Brucella melitensis in a large microbiology laboratory in a brucella-non-endemic area. We followed the US Centers...... for Disease Control and Prevention guidelines, but, of 14 staff members classified as high-risk exposure, none accepted post-exposure prophylaxis. However, in a period of 6 months of follow-up, none of the exposed laboratory workers developed brucellosis and all obtained sera were negative for antibrucella...

  5. Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

    Science.gov (United States)

    Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

    2013-01-01

    The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

  6. Progress in Brucella vaccine development

    Science.gov (United States)

    YANG, Xinghong; SKYBERG, Jerod A.; CAO, Ling; CLAPP, Beata; THORNBURG, Theresa; PASCUAL, David W.

    2012-01-01

    Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines. PMID:23730309

  7. Subclinical Brucella infection in man.

    Science.gov (United States)

    Henderson, R J; Hill, D M

    1972-07-15

    Much subclinical infection with Brucella abortus affects the dairy farming community, slaughterhouse workers, and veterinary surgeons. High titres of antibody to B. abortus by the saline agglutination, antihuman globulin, or complement fixation test may be shown in subjects from the first two groups and generally there are no symptoms or signs of brucella infection whatever. Veterinary surgeons are prone to subclinical infection and young practitioners frequently display high titres against the usual tests at the same time apparently enjoying good health. Repeated reinfection of this group, however, may result in hypersensitivity to B. abortus and bouts of ill health at intervals may be due to this. Infection seems to come more often from contact with infected material than by drinking untreated milk, particularly in the herdsman, slaughterhouse worker, and veterinary surgeon. A doctor investigating illness of a patient from the dairy farming community and allied occupations should not accept a high titre against B. abortus as the only clue to diagnosis of the patient's condition.

  8. Genomic comparisons of Brucella spp. and closely related bacteria using base compositional and proteome based methods

    DEFF Research Database (Denmark)

    Bohlin, Jon; Snipen, Lars; Cloeckaert, Axel

    2010-01-01

    in marker genes on the other. The proteome based methods found greater similarity between Brucella species and Ochrobactrum species than between species within genus Agrobacterium compared to each other. In other words, proteome comparisons of species within genus Agrobacterium were found to be more diverse...

  9. Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

    OpenAIRE

    Köhler, Stephan; Ouahrani-Bettache, Safia; Layssac, Marion; Teyssier, Jacques; Liautard, Jean-Pierre

    1999-01-01

    A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

  10. Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan.

    Science.gov (United States)

    Lindahl-Rajala, Elisabeth; Hoffman, Tove; Fretin, David; Godfroid, Jacques; Sattorov, Nosirjon; Boqvist, Sofia; Lundkvist, Åke; Magnusson, Ulf

    2017-03-01

    Brucellosis is one of the most common zoonoses globally, and Central Asia remains a Brucella hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as Brucella. This study investigates the presence of Brucella DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. Brucella DNA was detected in 10.3% of 564 cow milk samples by IS711-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, Brucella DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of Brucella DNA. In addition, sequence analysis of the rpoB gene suggests that one cow was infected with B. abortus and another cow was most likely infected with B. melitensis. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in Brucella surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control Brucella infections in the livestock population but further research focusing on the isolation of Brucella is required to obtain a comprehensive understanding of the Brucella spp. circulating among the livestock in this region.

  11. Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan.

    Directory of Open Access Journals (Sweden)

    Elisabeth Lindahl-Rajala

    2017-03-01

    Full Text Available Brucellosis is one of the most common zoonoses globally, and Central Asia remains a Brucella hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as Brucella. This study investigates the presence of Brucella DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. Brucella DNA was detected in 10.3% of 564 cow milk samples by IS711-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, Brucella DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of Brucella DNA. In addition, sequence analysis of the rpoB gene suggests that one cow was infected with B. abortus and another cow was most likely infected with B. melitensis. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in Brucella surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control Brucella infections in the livestock population but further research focusing on the isolation of Brucella is required to obtain a comprehensive understanding of the Brucella spp. circulating among the livestock in this region.

  12. Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan

    Science.gov (United States)

    Lindahl-Rajala, Elisabeth; Hoffman, Tove; Fretin, David; Godfroid, Jacques; Sattorov, Nosirjon; Boqvist, Sofia; Lundkvist, Åke; Magnusson, Ulf

    2017-01-01

    Brucellosis is one of the most common zoonoses globally, and Central Asia remains a Brucella hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as Brucella. This study investigates the presence of Brucella DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. Brucella DNA was detected in 10.3% of 564 cow milk samples by IS711-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, Brucella DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of Brucella DNA. In addition, sequence analysis of the rpoB gene suggests that one cow was infected with B. abortus and another cow was most likely infected with B. melitensis. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in Brucella surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control Brucella infections in the livestock population but further research focusing on the isolation of Brucella is required to obtain a comprehensive understanding of the Brucella spp. circulating among the livestock in this region. PMID:28296882

  13. Brucella melitensis Invades Murine Erythrocytes during Infection

    Science.gov (United States)

    Vitry, Marie-Alice; Hanot Mambres, Delphine; Deghelt, Michaël; Hack, Katrin; Machelart, Arnaud; Lhomme, Frédéric; Vanderwinden, Jean-Marie; Vermeersch, Marjorie; De Trez, Carl; Pérez-Morga, David; Letesson, Jean-Jacques

    2014-01-01

    Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature. PMID:25001604

  14. The presence of Brucella ceti ST26 in a striped dolphin (Stenella coeruleoalba) with meningoencephalitis from the Mediterranean Sea.

    Science.gov (United States)

    Alba, Patricia; Terracciano, Giuliana; Franco, Alessia; Lorenzetti, Serena; Cocumelli, Cristiano; Fichi, Gianluca; Eleni, Claudia; Zygmunt, Michel S; Cloeckaert, Axel; Battisti, Antonio

    2013-05-31

    Brucella spp. was isolated from brain, lung and intestinal lymph nodes of a dead adult male striped dolphin (Stenella coeruleoalba) found stranded on the Tyrrhenian coast (Tuscany, Italy) of the Mediterranean Sea in February 2012. Brucella spp. was associated with moderate to severe lesions of meningoencephalitis. A co-infection by Toxoplasma gondii was also demonstrated at brain level by means of molecular and histopathologic methods. The Brucella isolate was further characterized based on a fragment-specific polymerase chain reaction (PCR) approach, consisting of a set of five specific PCRs, targeting specific chromosomal IS711 locations for marine mammal Brucellae, as described previously. The isolate was thus classified as Brucella ceti I; V fragment-positive (or B. ceti dolphin type), according to previous studies. Multi Locus Sequence Analysis demonstrated that the isolate belongs to Sequence Type 26, while omp2 (omp2a and omp2b genes) sequence analysis further confirmed the isolate belonged to this group of strains. This is the first report of Brucella spp. from marine mammals in the Mediterranean Sea, and represents a further observation that this strain group is associated with hosts of the Family Delphinidae, and particularly with the striped dolphins, also in the Mediterranean area, thus constituting a further biological hazard of concern for this vulnerable subpopulation. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes

    NARCIS (Netherlands)

    Muendo, Esther N.; Mbatha, Peter M.; Macharia, Joseph; Abdoel, Theresia H.; Janszen, Paul V.; Pastoor, Rob; Smits, Henk L.

    2012-01-01

    Brucella melitensis biovar 1 was isolated from bovine milk samples from a herd in central Kenya, and Brucella abortus biovar 3 was isolated from aborted fetus materials and vaginal discharge fluids from cattle in central and eastern provinces of Kenya. All infections including those with B.

  16. Multivalent Fusion DNA Vaccine against Brucella abortus

    Science.gov (United States)

    Gómez, Leonardo; Llanos, Javiera; Escalona, Emilia; Sáez, Darwin; Álvarez, Francisco; Molina, Raúl; Flores, Manuel

    2017-01-01

    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. PMID:29082252

  17. Multivalent Fusion DNA Vaccine against Brucella abortus

    Directory of Open Access Journals (Sweden)

    Leonardo Gómez

    2017-01-01

    Full Text Available 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.

  18. Brucella serology in abattoir workers

    International Nuclear Information System (INIS)

    Mukhtar, F.; Kokab, F.

    2008-01-01

    Brucellosis is an occupational hazard with those particularly at risk either living in close proximity with animals or handling them. It is a public health problem in developing countries with adverse health implications both for animals and human beings as well as economic implications for individuals and communities. The Objectives were to estimate the seroprevalence of brucellosis among abattoir workers of Lahore District and to determine the association of brucellosis with nature of job of the workers. Data was collected in April 2008. It was a cross-sectional study in which four main slaughterhouses in Lahore were included. The slaughterhouse workers were divided into seven strata based on their nature of job: meat sellers, slaughterers, animal keepers, drivers, cleaners, loaders and vets/paravets. A total of 360 such workers were selected using stratified random sampling technique. Sampling frames for different strata were prepared and from each frame, proportionate numbers, were selected through simple random method using random number tables. Data was obtained using a questionnaire. Additionally blood samples were collected and analyzed for anti-Brucella Immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA) technique. The seroprevalence of anti-Brucella IgG was found to be 21.7%. A statistically significant difference was observed between the immune status of the respondents and their nature of job (p=0.005), age groups (p=0.013), and duration of job (p=0.003). The disease is an important public health problem in Pakistan. The disease can be prevented in the slaughterhouse workers through the use of personal protective devices. Public health authorities should educate the general public regarding prevention of the disease with specific emphasis on people working in slaughterhouses. (author)

  19. Antimicrobial Susceptibility of Brucella melitensis Isolates in Peru

    Science.gov (United States)

    2011-03-01

    during susceptibility testing: Escherichia coli 25922, Staphylococcus au reus 25923, Brucella abortus 2308, Brucella melitensis rev- I, and Bmcel/a canis...2011, American Society for Microbiology. All Rights Reserved. Antin1icrobial Susceptibility of Brucella melitensis Isolates in Peru 9 Ryan C. Maves,1...48 human Brucella melitensis biotype 1 strains from Peru between 2000 and 2006. MICs of isolates to doxycycline, azithromycin, gentamicin, rifampin

  20. Identifikasi Brucella abortus Isolat Lokal dengan Brucella abortus Strain Specific-Polymerase Chain Reaction (IDENTIFICATION OF LOCAL ISOLATES OF BRUCELLA ABORTUS USING BRUCELLA ABORTUS STRAIN SPECIFIC-POLYMERASE CHAIN REACTION ASSAY)

    OpenAIRE

    Susan Maphilindawati Noor; Pratiwi Pujilestari Sudarmono; Asmarani Kusumawati; Anis Karuniawati

    2014-01-01

    Brucella abortus Strain Specific-Polymerase Chain Reaction (BaSS-PCR) is a single multiplex PCRtechnique which able to identify and differentiate between Brucella abortus field strains (biovar 1, 2, and4), B. abortus vaccine strains, Brucella species, and non-Brucella species. In this study, BaSS-PCR wasapplied to identify local isolates of B. abortus in order to investigate the B. abortus strains that infectedcattle in Indonesia. Fifty local strains of B.abortus isolated from infected cattle...

  1. Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice.

    Science.gov (United States)

    Lalsiamthara, Jonathan; Gogia, Neha; Goswami, Tapas K; Singh, R K; Chaudhuri, Pallab

    2015-05-21

    Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. 9 CFR 113.32 - Detection of Brucella contamination.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of Brucella contamination... REQUIREMENTS Standard Procedures § 113.32 Detection of Brucella contamination. The test for detection of Brucella contamination provided in this section shall be conducted when such a test is prescribed in an...

  3. Prevalence of Brucella antibodies in sheep and springbok ...

    African Journals Online (AJOL)

    It was concluded that sheep and springbok on the eleven farms had not been exposed to Brucella melitensis and B. abortus infections and that on previously positive farms the infection had been eliminated in sheep and had not spread to springbok. Key words: springbok, sheep, Brucella melitensis, Brucella abortus, ...

  4. Pseudomonas aeruginosa B-band lipopolysaccharide genes wbpA and wbpI and their Escherichia coli homologues wecC and wecB are not functionally interchangeable.

    Science.gov (United States)

    Burrows, L L; Pigeon, K E; Lam, J S

    2000-08-15

    The O antigen unit of Pseudomonas aeruginosa serotype O5 is a complex trisaccharide containing 2-acetamido-3-acetiminido-2, 3-dideoxy-beta-D-mannuronic acid, 2-acetimido-3-acetimido-2, 3-dideoxy-beta-D-mannuronic acid, and 2-acetimido-2, 6-deoxy-beta-D-galactosamine. Specific knockout mutations in the putative UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) epimerase gene, wbpI, or the putative UDP-D-N-acetylmannosamine dehydrogenase gene, wbpA, resulted in strains that no longer produced B-band lipopolysaccharide, confirming the essential roles of these genes in B-band O antigen synthesis. Despite approximately 50% similarity of wbpI and wbpA to the Escherichia coli genes wecB (rffE) and wecC (rffD) involved in enterobacterial common antigen synthesis, cross-complementation experiments were not successful. These results imply that the P. aeruginosa UDP-D-GlcNAc precursor may be di-N-acetylated prior to further modification, preventing the E. coli enzymes from recognizing it as a substrate.

  5. Bison PRNP genotyping and potential association with Brucella spp. seroprevalence

    Science.gov (United States)

    Seabury, C.M.; Halbert, N.D.; Gogan, P.J.P.; Templeton, J.W.; Derr, J.N.

    2005-01-01

    The implication that host cellular prion protein (PrPC) may function as a cell surface receptor and/or portal protein for Brucella abortus in mice prompted an evaluation of nucleotide and amino acid variation within exon 3 of the prion protein gene (PRNP) for six US bison populations. A non-synonymous single nucleotide polymorphism (T50C), resulting in the predicted amino acid replacement M17T (Met ??? Thr), was identified in each population. To date, no variation (T50: Met) has been detected at the corresponding exon 3 nucleotide and/or amino acid position for domestic cattle. Notably, 80% (20 of 25) of the Yellowstone National Park bison possessing the C/C genotype were Brucella spp. seropositive, representing a significant (P = 0.021) association between seropositivity and the C/C genotypic class. Moreover, significant differences in the distribution of PRNP exon 3 alleles and genotypes were detected between Yellowstone National Park bison and three bison populations that were either founded from seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis. Unlike domestic cattle, no indel polymorphisms were detected within the corresponding regions of the putative bison PRNP promoter, intron 1, octapeptide repeat region or 3???-untranslated region for any population examined. This study provides the first evidence of a potential association between nucleotide variation within PRNP exon 3 and the presence of Brucella spp. antibodies in bison, implicating PrPC in the natural resistance of bison to brucellosis infection. ?? 2005 International Society for Animal Genetics.

  6. Brucella Infection in HIV Infected Patients

    Directory of Open Access Journals (Sweden)

    SeyedAhmad SeyedAlinaghi

    2011-12-01

    Full Text Available The purpose of this study was to assess the possible correlation between Brucella and HIV infections. Iran is a country where HIV infection is expanding and Brucellosis is prevalent. In the present study, 184 HIV infected patients were assigned and for all of them HIV infection was confirmed by western blot test. In order to identify the prevalence rate of Brucella infection and systemic brucellosis in these subjects, sera samples were obtained and Brucella specific serological tests were performed to reveal antibody titers. Detailed history was taken and physical examination was carried out for all of patients. 11 (6% subjects had high titers but only 3 of them were symptomatic. Most of these subjects were injection drug user (IDU men and one was a rural woman. Considering both prevalence rates of Brucella infection (3% and symptomatic brucellosis (0.1% in Iran, our HIV positive patients show higher rates of Brucella infection and systemic brucellosis. Preserved cellular immunity of participants and retention of granulocytes activity may explain this poor association; whereas other explanations such as immunological state difference and non-overlapping geographical distribution of the 2 pathogens have been mentioned by various authors.

  7. Brucella epididymo-orchitis: a consideration in endemic area

    Directory of Open Access Journals (Sweden)

    Jaffar A. Al-Tawfiq

    2006-06-01

    Full Text Available Brucellosis is a zoonotic disease caused by Brucella sp. and may affect many parts of the body. Brucella epididymo-orchitis had been reported in up to 20% of patients with brucellosis. This is a case report of Brucella epididymo-orchitis in a Saudi male patient. He presented with a unilateral swelling of the left testicle. He had fever, arthralgia and night sweats. Ultrasound examination revealed enlarged left epididymis and testicle. Brucella serology was positive and the patient responded to treatment with doxycycline and gentamicin. Thus, brucella infection should be considered in the differential diagnosis of patients presenting with epididymo-orchitis from an endemic area.

  8. Using real-time polymerase chain reaction as an alternative rapid method for enumeration of colony count in live Brucella vaccines

    Directory of Open Access Journals (Sweden)

    Waleed S. Shell

    2017-06-01

    Full Text Available Aim: Brucellosis is a major bacterial zoonosis of global importance affecting a range of animal species and man worldwide. It has economic, public health, and bio-risk importance. Control and prevention of animal brucellosis mainly depend on accurate diagnostic tools and implementation of effective and safe animal vaccination program. There are three types of animal Brucella live vaccines - Brucella melitensis Rev-1 vaccine, Brucella abortus S19, and B. abortus RB51. Evaluation of these vaccines depends mainly on enumeration of Brucella viable count. At present, used colony count method is time consuming, costly and requires especial skills. Hence, the aim of this study is to use and standardize real-time polymerase chain reaction (RT-PCR as an alternative, quantitative, sensitive, and rapid method to detect the colony count of Brucella in live Brucella vaccine. Materials and Methods: Four batches of different live Brucella vaccines were evaluated using of conventional bacterial count and RT-quantitative PCR (RT-qPCR using BSCP31 gene specific primers and probe. Standard curve was generated from DNA template extracted from 10-fold serial dilution of living B. abortus RB51 vaccine to evaluate the sensitivity of RT-qPCR. Results: Results revealed that three batches of living Brucella vaccines were acceptable for Brucella colony count when traditional bacterial enumeration method was used. Results of RT-qPCR were identical to that of conventional bacterial count. Conclusion: Results concluded that RT-qPCR was relatively sensitive compared to traditional bacterial colony count of these vaccines.

  9. Prevalence of Brucella spp in humans

    Directory of Open Access Journals (Sweden)

    Catharina de Paula Oliveira Cavalcanti Soares

    2015-10-01

    Full Text Available Objective: to determine the seroprevalence of Brucella spp in humans.Method: this is an observational study, developed with 455 individuals between 18 and 64 years old, who use the Estratégia de Saúde da Família (Brazil's family health strategy. The serum samples of volunteers underwent buffered acid antigen tests, such as screening, agar gel immunodiffusion and slow seroagglutination test in tubes and 2-Mercaptoethanol.Results: among the samples, 1.98% has responded to buffered-acid antigen, 2.85% to agar gel immunodiffusion test and 1.54% to the slow seroagglutination tests on tubes/2-Mercaptoethanol. The prevalence of Brucella spp was 4.4%, represented by the last two tests.Conclusion: the results of this research suggest that the studied population is exposed to Brucella spp infection.

  10. Metal acquisition and virulence in Brucella

    Science.gov (United States)

    Roop, R. Martin

    2013-01-01

    Similar to other bacteria, Brucella strains require several biologically essential metals for their survival in vitro and in vivo. Acquiring sufficient levels of some of these metals, particularly iron, manganese and zinc, is especially challenging in the mammalian host, where sequestration of these micronutrients is a well-documented component of both the innate and acquired immune responses. This review describes the Brucella metal transporters that have been shown to play critical roles in the virulence of these bacteria in experimental and natural hosts. PMID:22632611

  11. Brucella HTRA Protein and Pathogenesis: Brucella Delta HTA Strains as Vaccines

    National Research Council Canada - National Science Library

    Roop, Martin

    1998-01-01

    .... The results of studies funded by contract DAMD17-94-C-4054 confirmed that the Brucella HtrA contributes to the resistance of these intracellular pathogens to killing by host neutrophils and macrophages...

  12. Brucella HTRA Protein and Pathogenesis: Brucella Delta HTRA Strains as Vaccines

    National Research Council Canada - National Science Library

    Roop

    1997-01-01

    .... The results of studies described in previous reports confirmed that the Brucella HtrA contributes to the resistance of these intracellular pathogens to killing by host neutrophils and macrophages...

  13. Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

    Science.gov (United States)

    Gourley, Christopher R.

    The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

  14. Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

    Energy Technology Data Exchange (ETDEWEB)

    Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

    2015-12-01

    Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted asmonolayers at the air-water interface, and their properties, aswell as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region,butwas prevented fromthis penetration intothemodified lipopolysaccharides.Results correlatewith behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.

  15. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    Science.gov (United States)

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  16. Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

    Science.gov (United States)

    Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2016-01-01

    Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

  17. Establishment of Chronic Infection: Brucella's Stealth Strategy

    Science.gov (United States)

    Ahmed, Waqas; Zheng, Ke; Liu, Zheng-Fei

    2016-01-01

    Brucella is a facultative intracellular pathogen that causes zoonotic infection known as brucellosis which results in abortion and infertility in natural host. Humans, especially in low income countries, can acquire infection by direct contact with infected animal or by consumption of animal products and show high morbidity, severe economic losses and public health problems. However for survival, host cells develop complex immune mechanisms to defeat and battle against attacking pathogens and maintain a balance between host resistance and Brucella virulence. On the other hand as a successful intracellular pathogen, Brucella has evolved multiple strategies to evade immune response mechanisms to establish persistent infection and replication within host. In this review, we mainly summarize the “Stealth” strategies employed by Brucella to modulate innate and the adaptive immune systems, autophagy, apoptosis and possible role of small noncoding RNA in the establishment of chronic infection. The purpose of this review is to give an overview for recent understanding how this pathogen evades immune response mechanisms of host, which will facilitate to understanding the pathogenesis of brucellosis and the development of novel, more effective therapeutic approaches to treat brucellosis. PMID:27014640

  18. Identification and characterization of Brucella effector proteins

    NARCIS (Netherlands)

    de Jong, Maarten Frederik

    2012-01-01

    Brucella-bacteriën gebruiken de eiwitten VceB en VceC om het immuunsysteem van humane gastheercellen te omzeilen, blijkt uit het promotieonderzoek van Maarten de Jong. Dit biedt nieuwe aanknopingspunten voor de bestrijding van deze gevaarlijke bacterie. Brucellose is een wereldwijd voorkomende

  19. Brucella neotomae Infection in Humans, Costa Rica.

    Science.gov (United States)

    Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Jiménez-Rojas, César; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Víquez-Ruiz, Eunice; Rojas-Campos, Norman; Baker, Kate S; Oviedo-Sánchez, Gerardo; Amuy, Ernesto; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Guzmán-Verri, Caterina

    2017-06-01

    Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed.

  20. Isolation of Brucella microti from soil

    Czech Academy of Sciences Publication Activity Database

    Scholz, H. C.; Hubálek, Zdeněk; Nesvadbová, Jiřina; Tomaso, H.; Vergnaud, G.; Le Fleche, P.; Whatmore, A. M.; Al Dahouk, S.; Krüger, M.; Lodri, C.; Pfeffer, M.

    2008-01-01

    Roč. 14, č. 8 (2008), s. 1316-1317 ISSN 1080-6040 Institutional research plan: CEZ:AV0Z60930519 Keywords : Brucella microti * Microtus arvalis Subject RIV: EE - Microbiology, Virology Impact factor: 6.449, year: 2008

  1. Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution.

    Science.gov (United States)

    Suárez-Esquivel, Marcela; Baker, Kate S; Ruiz-Villalobos, Nazareth; Hernández-Mora, Gabriela; Barquero-Calvo, Elías; González-Barrientos, Rocío; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Chacón-Díaz, Carlos; Cloeckaert, Axel; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Guzmán-Verri, Caterina

    2017-07-01

    Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97-99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Brucella abortus vaccine strain RB51 produces low levels of M-like O-antigen.

    Science.gov (United States)

    Cloeckaert, Axel; Zygmunt, Michel S; Guilloteau, Laurence A

    2002-03-15

    Brucella abortus RB51 is a rough (R) stable vaccine strain used in cattle and is believed to be devoid of O-side chain. We analyzed by use of a panel of monoclonal antibodies (MAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes the O-chain expression in strain RB51. Two MAbs specific for the C/Y (A=M) and C (M>A) epitopes showed low bindings in ELISA to strain RB51. O-chain expression was further confirmed by Western blot after SDS-PAGE of strain RB51. In particular, the MAb of C (M>A) specificity, showing preferential binding to M-dominant smooth (S) Brucella strains, revealed in strain RB51 a typical smooth-lipopolysaccharide (S-LPS) pattern which resembled that of M-dominant S-LPS. Thus, the results clearly show that strain RB51 produces low levels of M-like O-antigen.

  3. Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence.

    Science.gov (United States)

    Mancilla, Marcos

    2015-01-01

    Dissociation encompasses changes in a series of phenotypes: colony and cell morphology, inmunological and biochemical reactions and virulence. The concept is generally associated to the in vitro transition between smooth (S) and rough (R) colonies, a phenotypic observation in Gram-negative bacteria commonly made since the beginning of microbiology as a science. It is also well known that the loss of the O-polysaccharide, the most external lipopolysaccharide (LPS) moiety, triggers the change in the colony phenotype. Although dissociation is related to one of the most basic features used to distinguish between species, i.e., colony morphology, and, in the case of pathogens, predict their virulence behavior, it has been considered a laboratory artifact and thus did not gain further attention. However, recent insights into genetics and pathogenesis of members of Brucella, causative agents of brucellosis, have brought a new outlook on this experimental fact, suggesting that it plays a role beyond the laboratory observations. In this perspective article, the current knowledge on Brucella LPS genetics and its connection with dissociation in the frame of evolution is discussed. Latest reports support the notion that, by means of a better understanding of genetic pathways linked to R phenotype and the biological impact of this intriguing "old" phenomenon, unexpected applications can be achieved.

  4. Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

    Science.gov (United States)

    Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

    1999-01-01

    The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

  5. Smooth to rough dissociation in Brucella: the missing link to virulence

    Directory of Open Access Journals (Sweden)

    Marcos eMancilla

    2016-01-01

    Full Text Available Dissociation encompasses changes in a series of phenotypes: colony and cell morphology, inmunological and biochemical reactions and virulence. The concept is generally associated to the in vitro transition between smooth (S and rough (R colonies, a phenotypic observation in Gram-negative bacteria commonly made since the beginning of microbiology as a science. It is also well known that the loss of the O-polysaccharide, the most external lipopolysaccharide (LPS moiety, triggers the change in the colony phenotype. Although dissociation is related to one of the most basic features used to distinguish between species, i.e. colony morphology, and, in the case of pathogens, predict their virulence behaviour, it has been considered a laboratory artifact and thus did not gain further attention. However, recent insights into genetics and pathogenesis of members of Brucella, causative agents of brucellosis, have brought a new outlook on this experimental fact, suggesting that it plays a role beyond the laboratory observations.In this perspective article, the current knowledge on Brucella LPS genetics and its connection with dissociation in the frame of evolution is discussed. Latest reports support the notion that, by means of a better understanding of genetic pathways linked to R phenotype and the biological impact of this intriguing old phenomenon, unexpected applications can be achieved.

  6. Brucella abortus strain RB51 leucine auxotroph as an environmentally safe vaccine for plasmid maintenance and antigen overexpression.

    Science.gov (United States)

    Rajasekaran, Parthiban; Seleem, Mohamed N; Contreras, Andrea; Purwantini, Endang; Schurig, Gerhardt G; Sriranganathan, Nammalwar; Boyle, Stephen M

    2008-11-01

    To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

  7. Validation of the Abbreviated Brucella AMOS PCR as a Rapid Screening Method for Differentiation of Brucella abortus Field Strain Isolates and the Vaccine Strains, 19 and RB51

    OpenAIRE

    Ewalt, Darla R.; Bricker, Betsy J.

    2000-01-01

    The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isola...

  8. Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella sp and Yersinia enterocolitica O : 9 in cattle and pigs

    DEFF Research Database (Denmark)

    Nielsen, K.; Smith, P.; Yu, W.

    2006-01-01

    A rapid, inexpensive and rugged serological test that distinguishes cattle and swine infected with Brucella sp. or Yersinia enterocolitica O:9 is described. The test protocol, which is an indirect enzyme immunoassay uses a high concentration of divalent cation chelating agents to minimize binding...... of Y. enterocolitica O:9 antibody to rough lipopolysaccharide antigen derived from B. abortus RB51. No false positive reactions were observed when testing 100 Canadian cattle and swine without any evidence of brucellosis. The assay detected 91.6% of cattle (n = 155) and 93.5% (n = 31) of swine infected...... with Brucella sp. Sera from 58 cattle and 38 swine exposed to Y. enterocolitica O:9 were negative while only 20 sera from 121 'false positive' reactors of unspecified origin gave low level positive reactions, eliminating 84% of the false positive reactions. Crown...

  9. Case report 469: Spondylitis (lumbar spine) due to Brucella abortus

    Energy Technology Data Exchange (ETDEWEB)

    Manaster, B.J.

    1988-03-01

    The current case is interesting in that, although the plain radiographs were diagnostic of infection and the patient's work history suggested brucellosis, both the negative serum antibody titers to brucella and the CT appearance of large calcified psoas abscesses made the diagnosis of tuberculous spondylitis most probable. Open biopsy with tissue culture proved brucella. From this experience it appears that the presence of large calcified psoas abscesses should not eliminate the diagnosis of brucella spondylitis in the proper clinical setting.

  10. Case report 469: Spondylitis (lumbar spine) due to Brucella abortus

    International Nuclear Information System (INIS)

    Manaster, B.J.

    1988-01-01

    The current case is interesting in that, although the plain radiographs were diagnostic of infection and the patient's work history suggested brucellosis, both the negative serum antibody titers to brucella and the CT appearance of large calcified psoas abscesses made the diagnosis of tuberculous spondylitis most probable. Open biopsy with tissue culture proved brucella. From this experience it appears that the presence of large calcified psoas abscesses should not eliminate the diagnosis of brucella spondylitis in the proper clinical setting. (orig.)

  11. Host response to Brucella infection: review and future perspective.

    Science.gov (United States)

    Elfaki, Mohamed G; Alaidan, Alwaleed Abdullah; Al-Hokail, Abdullah Abdulrahman

    2015-07-30

    Brucellosis is a zoonotic and contagious infectious disease caused by infection with Brucella species. The infecting brucellae are capable of causing a devastating multi-organ disease in humans with serious health complications. The pathogenesis of Brucella infection is influenced largely by host factors, Brucella species/strain, and the ability of invading brucellae to survive and replicate within mononuclear phagocytic cells, preferentially macrophages (Mf). Consequently, the course of human infection may appear as an acute fatal or progress into chronic debilitating infection with periodical episodes that leads to bacteremia and death. The existence of brucellae inside Mf represents one of the strategies used by Brucella to evade the host immune response and is responsible for treatment failure in certain human populations treated with anti-Brucella drugs. Moreover, the persistence of brucellae inside Mf complicates the diagnosis and may affect the host cell signaling pathways with consequent alterations in both innate and adaptive immune responses. Therefore, there is an urgent need to pursue the development of novel drugs and/or vaccine targets against human brucellosis using high throughput technologies in genomics, proteomics, and immunology.

  12. Successful Management of Prosthetic Valve Brucella Endocarditis with Antibiotherapy Alone

    Directory of Open Access Journals (Sweden)

    José Pedro Fonseca

    2018-01-01

    Full Text Available Objectives: To report a case of mechanical aortic prosthesis Brucella endocarditis successfully treated with antibiotics alone. Materials and methods: We describe a clinical case and present a review of the literature. Results: A 60-year-old female farmer with a mechanical aortic prosthetic valve presented with low back pain and fever. She was diagnosed with prosthetic valve Brucella mellitensis endocarditis and was cured with antibiotic therapy alone. Few cases of successfully treated prosthetic valve Brucella endocarditis without surgery have been reported. Conclusion: Prosthetic valve Brucella endocarditis usually requires surgical valve replacement. However, selected patients may be successfully treated with antibiotic therapy alone.

  13. Transcriptome analysis of the Brucella abortus BvrR/BvrS two-component regulatory system.

    Directory of Open Access Journals (Sweden)

    Cristina Viadas

    Full Text Available BACKGROUND: The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. METHODOLOGY/PRINCIPAL FINDINGS: A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d, lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. CONCLUSIONS/SIGNIFICANCE: All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche.

  14. Coordinated zinc homeostasis is essential for the wild-type virulence of Brucella abortus.

    Science.gov (United States)

    Sheehan, Lauren M; Budnick, James A; Roop, R Martin; Caswell, Clayton C

    2015-05-01

    Metal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence of Brucella abortus; however, the capacity of Brucella strains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region of znuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion of zur or zntR alone did not result in increased zinc toxicity in the corresponding mutants; however, deletion of zntA led to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion of zntR resulted in significant attenuation of B. abortus in a mouse model of chronic infection, and subsequent experiments revealed that overexpression of zntA in the zntR mutant is the molecular basis for its decreased virulence. The importance of zinc uptake for Brucella pathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export in Brucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation of Brucella virulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria

  15. MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identifies 7 major groups within Brucella ceti and Brucella pinnipedialis

    Directory of Open Access Journals (Sweden)

    Jacques Isabelle

    2009-07-01

    Full Text Available Abstract Background Since 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats Analysis (MLVA approach. A previously published assay comprising 16 loci (MLVA-16 that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used. Results 294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata and the two others comprising other seal species isolates. Conclusion The clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two

  16. Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro

    Directory of Open Access Journals (Sweden)

    Xiangguo eWang

    2016-02-01

    Full Text Available Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER, and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2 in goat trophoblast cells (GTCs and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm, a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA, a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

  17. Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

    Science.gov (United States)

    Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

    2017-04-04

    Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10 5 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

  18. Brucella dissociation is essential for macrophage egress and bacterial dissemination.

    Science.gov (United States)

    Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A

    2014-01-01

    It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.

  19. Brucella Dissociation Is Essential for Macrophage Egress and Bacterial Dissemination

    Directory of Open Access Journals (Sweden)

    Thomas A Ficht

    2014-03-01

    Full Text Available It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic de-tails of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA. Vis-ible plaques were detected at 4-5 days post infection (p.i. with cytotoxic Brucella 16M∆manBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16M∆manBA∆virB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci with replicating Brucella in the monolayers infected with 16M∆manBA∆virB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addi-tion of gentamicin to the culture medium inhibited plaque formation, suggesting that the cell-to-cell spreading occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella induced cytotoxicity is critical for Brucella egress and dissemination.

  20. A Novel PCR Assay for Detecting Brucella abortus and Brucella melitensis

    Science.gov (United States)

    Alamian, Saeed; Esmaelizad, Majid; Zahraei, Taghi; Etemadi, Afshar; Mohammadi, Mohsen; Afshar, Davoud; Ghaderi, Soheila

    2017-01-01

    Objectives Brucellosis is a major zoonotic disease that poses a significant public health threat worldwide. The classical bacteriological detection process used to identify Brucella spp. is difficult and time-consuming. This study aimed to develop a novel molecular assay for detecting brucellosis. Methods All complete sequences of chromosome 1 with 2.1-Mbp lengths were compared among all available Brucella sequences. A unique repeat sequence (URS) locus on chromosome 1 could differentiate Brucella abortus from Brucella melitensis. A primer set was designed to flank the unique locus. A total of 136 lymph nodes and blood samples were evaluated and classified by the URS-polymerase chain reaction (PCR) method in 2013–2014. Results Biochemical tests and bacteriophage typing as the golden standard indicated that all Brucella spp. isolates were B. melitensis biovar 1 and B. abortus biovar 3. The PCR results were the same as the bacteriological method for detecting Brucella spp. The sensitivity and specificity of the URS-PCR method make it suitable for detecting B. abortus and B. melitensis. Conclusion Quick detection of B. abortus and B. melitensis can provide the most effective strategies for control of these bacteria. The advantage of this method over other presented methods is that both B. abortus and B. melitensis are detectable in a single test tube. Furthermore, this method covered 100% of all B. melitensis and B. abortus biotypes. The development of this URS-PCR method is the first step toward the development of a novel kit for the molecular identification of B. abortus and B. melitensis. PMID:28443226

  1. A Novel PCR Assay for Detecting Brucella abortus and Brucella melitensis.

    Science.gov (United States)

    Alamian, Saeed; Esmaelizad, Majid; Zahraei, Taghi; Etemadi, Afshar; Mohammadi, Mohsen; Afshar, Davoud; Ghaderi, Soheila

    2017-02-01

    Brucellosis is a major zoonotic disease that poses a significant public health threat worldwide. The classical bacteriological detection process used to identify Brucella spp. is difficult and time-consuming. This study aimed to develop a novel molecular assay for detecting brucellosis. All complete sequences of chromosome 1 with 2.1-Mbp lengths were compared among all available Brucella sequences. A unique repeat sequence (URS) locus on chromosome 1 could differentiate Brucella abortus from Brucella melitensis . A primer set was designed to flank the unique locus. A total of 136 lymph nodes and blood samples were evaluated and classified by the URS-polymerase chain reaction (PCR) method in 2013-2014. Biochemical tests and bacteriophage typing as the golden standard indicated that all Brucella spp. isolates were B. melitensis biovar 1 and B. abortus biovar 3. The PCR results were the same as the bacteriological method for detecting Brucella spp. The sensitivity and specificity of the URS-PCR method make it suitable for detecting B. abortus and B. melitensis . Quick detection of B. abortus and B. melitensis can provide the most effective strategies for control of these bacteria. The advantage of this method over other presented methods is that both B. abortus and B. melitensis are detectable in a single test tube. Furthermore, this method covered 100% of all B. melitensis and B. abortus biotypes. The development of this URS-PCR method is the first step toward the development of a novel kit for the molecular identification of B. abortus and B. melitensis .

  2. An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

    Science.gov (United States)

    Liu, Qiu-Ning; Chai, Xin-Yue; Tu, Jie; Xin, Zhao-Zhe; Li, Chao-Feng; Jiang, Sen-Hao; Zhou, Chun-Lin; Tang, Bo-Ping

    2016-02-01

    The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. The Glutaminase-Dependent System Confers Extreme Acid Resistance to New Species and Atypical Strains of Brucella

    Directory of Open Access Journals (Sweden)

    Luca Freddi

    2017-11-01

    Full Text Available Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB and of the glutamate (Glu/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the “classical” species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q, relying on the deamination of glutamine (Gln into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH ≤2.5, in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC–glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis.

  4. Cloning and Expression of 31kDa Outer Membrane Protein of Brucella melitansis in E.coli

    Directory of Open Access Journals (Sweden)

    Sayeneh Khodadadi

    2012-04-01

    Full Text Available Background & Objectives: The identification of Brucella spp. antigens with the capacity to elicit a protective immune response is of the great interest for the researchers. So, characterization and assessment of diverse antigens of Brucella need to be evaluated. In this study, we report the cloning and expression of the gene coding for 31 KDa OMP (OMP31 of Brucella melitensis 16M.   Methods: Brucella melitensis Omp31 gene was amplified with specific primers, cloned into pJET1/2 and subsequently subcloned in pET28a (+ vector. Both these recombinant plasmids were sequenced and then after, expression of recombinant protein was induced by 1mM IPTG. Western blot analysis was also performed by polyclonal rabbit antiserum.   Results: Omp31 successfully was cloned in both plasmid vectors. The recombinant Omp31 was expressed in E.coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant omp31 and retaining of its partial epitopes.   Conclusion: Our results show that, an expression host such as E. coli is suitable for omp31 production.

  5. Lymphocyte proliferation in response to Brucella abortus 2308 or RB51 antigens in mice infected with strain 2308, RB51, or 19.

    OpenAIRE

    Stevens, M G; Olsen, S C; Pugh, G W

    1994-01-01

    Lymphocyte proliferation to 22 protein fractions (106 to 18 kDa) of Brucella abortus 2308 or the lipopolysaccharide O-antigen-deficient mutant of 2308, strain RB51, was measured for 20 weeks after infection of mice with strain 2308, RB51, or 19. Throughout the 20-week study, the 22 protein fractions of 2308 and RB51 induced a similar pattern of proliferation when they were incubated with lymphocytes from the infected mice. In addition, during the 20 weeks, lymphocytes from all groups of infec...

  6. The new strains Brucella inopinata BO1 and Brucella species 83-210 behave biologically like classic infectious Brucella species and cause death in murine models of infection.

    Science.gov (United States)

    Jiménez de Bagüés, María P; Iturralde, María; Arias, Maykel A; Pardo, Julián; Cloeckaert, Axel; Zygmunt, Michel S

    2014-08-01

    Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. [Development of immunochromatographic test system for the rapid detection of lipopolysaccharide antigen and cells of causative agent of bovine brucellosis].

    Science.gov (United States)

    Byzova, N A; Zherdev, A V; Eskendirova, S Z; Baltin, K K; Unysheva, G B; Mukanov, K K; Ramankulov, E M; Dzantiev, B B

    2012-01-01

    A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.

  8. 21 CFR 866.3085 - Brucella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...) caused by bacteria belonging to the genus Brucella and provides epidemiological information on diseases...

  9. Knowledge of Brucella as a food-borne pathogen

    Science.gov (United States)

    Although Brucella spp. are known for causing reproductive losses in domestic livestock, they are also capable of infecting humans and causing clinical disease. Human infection with Brucella is almost exclusively a result of direct contact with infected animals or consumption of products made from un...

  10. Mean platelet volume in brucellosis: correlation between brucella ...

    African Journals Online (AJOL)

    Background: Brucellosis, a zoonotic infection, was most widely diagnosed by the Brucella standard serum agglutination test (SAT). No previous publication has demonstrated a correlation between the degree of Brucella SAT agglutination positivity and the severity of brucellosis infection. Objective: To contribute to the ...

  11. Bioinformatics analysis of Brucella vaccines and vaccine targets using VIOLIN.

    Science.gov (United States)

    He, Yongqun; Xiang, Zuoshuang

    2010-09-27

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis, one of the commonest zoonotic diseases found worldwide in humans and a variety of animal species. While several animal vaccines are available, there is no effective and safe vaccine for prevention of brucellosis in humans. VIOLIN (http://www.violinet.org) is a web-based vaccine database and analysis system that curates, stores, and analyzes published data of commercialized vaccines, and vaccines in clinical trials or in research. VIOLIN contains information for 454 vaccines or vaccine candidates for 73 pathogens. VIOLIN also contains many bioinformatics tools for vaccine data analysis, data integration, and vaccine target prediction. To demonstrate the applicability of VIOLIN for vaccine research, VIOLIN was used for bioinformatics analysis of existing Brucella vaccines and prediction of new Brucella vaccine targets. VIOLIN contains many literature mining programs (e.g., Vaxmesh) that provide in-depth analysis of Brucella vaccine literature. As a result of manual literature curation, VIOLIN contains information for 38 Brucella vaccines or vaccine candidates, 14 protective Brucella antigens, and 68 host response studies to Brucella vaccines from 97 peer-reviewed articles. These Brucella vaccines are classified in the Vaccine Ontology (VO) system and used for different ontological applications. The web-based VIOLIN vaccine target prediction program Vaxign was used to predict new Brucella vaccine targets. Vaxign identified 14 outer membrane proteins that are conserved in six virulent strains from B. abortus, B. melitensis, and B. suis that are pathogenic in humans. Of the 14 membrane proteins, two proteins (Omp2b and Omp31-1) are not present in B. ovis, a Brucella species that is not pathogenic in humans. Brucella vaccine data stored in VIOLIN were compared and analyzed using the VIOLIN query system. Bioinformatics curation and ontological representation of Brucella vaccines

  12. Bioinformatics analysis of Brucella vaccines and vaccine targets using VIOLIN

    Science.gov (United States)

    2010-01-01

    Background Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis, one of the commonest zoonotic diseases found worldwide in humans and a variety of animal species. While several animal vaccines are available, there is no effective and safe vaccine for prevention of brucellosis in humans. VIOLIN (http://www.violinet.org) is a web-based vaccine database and analysis system that curates, stores, and analyzes published data of commercialized vaccines, and vaccines in clinical trials or in research. VIOLIN contains information for 454 vaccines or vaccine candidates for 73 pathogens. VIOLIN also contains many bioinformatics tools for vaccine data analysis, data integration, and vaccine target prediction. To demonstrate the applicability of VIOLIN for vaccine research, VIOLIN was used for bioinformatics analysis of existing Brucella vaccines and prediction of new Brucella vaccine targets. Results VIOLIN contains many literature mining programs (e.g., Vaxmesh) that provide in-depth analysis of Brucella vaccine literature. As a result of manual literature curation, VIOLIN contains information for 38 Brucella vaccines or vaccine candidates, 14 protective Brucella antigens, and 68 host response studies to Brucella vaccines from 97 peer-reviewed articles. These Brucella vaccines are classified in the Vaccine Ontology (VO) system and used for different ontological applications. The web-based VIOLIN vaccine target prediction program Vaxign was used to predict new Brucella vaccine targets. Vaxign identified 14 outer membrane proteins that are conserved in six virulent strains from B. abortus, B. melitensis, and B. suis that are pathogenic in humans. Of the 14 membrane proteins, two proteins (Omp2b and Omp31-1) are not present in B. ovis, a Brucella species that is not pathogenic in humans. Brucella vaccine data stored in VIOLIN were compared and analyzed using the VIOLIN query system. Conclusions Bioinformatics curation and ontological

  13. Nucleotide sequences specific to Brucella and methods for the detection of Brucella

    Energy Technology Data Exchange (ETDEWEB)

    McCready, Paula M [Tracy, CA; Radnedge, Lyndsay [San Mateo, CA; Andersen, Gary L [Berkeley, CA; Ott, Linda L [Livermore, CA; Slezak, Thomas R [Livermore, CA; Kuczmarski, Thomas A [Livermore, CA

    2009-02-24

    Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  14. Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy.

    Science.gov (United States)

    Hamer, Isabelle; Goffin, Emeline; De Bolle, Xavier; Letesson, Jean-Jacques; Jadot, Michel

    2014-09-02

    Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway. Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild

  15. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

    Science.gov (United States)

    Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

    2006-05-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

  16. Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    International Nuclear Information System (INIS)

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-01-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 μM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 μM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-α and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-α and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 μM) significantly inhibited LPS-induced TNF-α and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-α and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-α and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated

  17. Characterisation of North American Brucella isolates from marine mammals.

    Science.gov (United States)

    Whatmore, Adrian M; Dawson, Claire; Muchowski, Jakub; Perrett, Lorraine L; Stubberfield, Emma; Koylass, Mark; Foster, Geoffrey; Davison, Nicholas J; Quance, Christine; Sidor, Inga F; Field, Cara L; St Leger, Judy

    2017-01-01

    Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

  18. Characterisation of North American Brucella isolates from marine mammals.

    Directory of Open Access Journals (Sweden)

    Adrian M Whatmore

    Full Text Available Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

  19. Infecção em cão por Brucella abortus: relato de caso Brucella abortus infection in dog: case report

    Directory of Open Access Journals (Sweden)

    J. Megid

    2007-12-01

    Full Text Available Brucella abortus infection is reported in a dog from a rural area that presented at clinical evaluation left testicular enlargement and right testicular decrease. Serum resulted negative to rapid agglutination test and agar gel immunodifusion with Brucella ovis antigen but positive to buffered plate agglutination test, tube agglutination test and 2- Mercapthoetanol with B. abortus antigen. Brucella isolation was negative in blood, testicular material, semen and urine. Brucella DNA was detected in PCR from urine and blood.

  20. Brucella contamination in raw milk by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Mohammad Khalili

    2016-10-01

    Full Text Available Background: Human brucellosis is a significant public health problem in many middle east countries including Iran. Brucella organisms, which are small aerobic, facultative intracellular coccobacilli, localize in the reproductive organs of host animals, causing abortions and sterility. They are shed in large numbers in the animal’s urine, milk, placental fluid, and other fluids. Dairy product from raw milk are a potential threat to public health in endemic developing countries. The gold standard for the diagnosis of brucellosis is isolation of Brucella species. However, isolation Brucella species is time consuming and needed to level 3 biocontainment facilities and highly skilled technical personnel to handle samples and live bacteria for eventual identification. Handling Brucella species increase risk of laboratory infection. Polymerase chain reaction (PCR with high sensitivity and specifity overcomed to these disadvantages. The aim of this study was to detect Brucella species in milk from dairy cattle farms in Kerman province, Iran by PCR technique. Methods: Forty and eight bulk tank milk (BTM were collected from October 2015 to March 2016 from 48 dairy cattle farm including 4200 cows. DNA of milk samples extracted by lysis buffer and proteinase K method. All milk samples were examined by PCR to detect Brucella-specific DNA targeting IS 711. Positive samples must be showed 317 bp amplified, corresponding to the expected size of the IS 711 genome region in all Brucella species. Results: Using IS711 primer were detected in 4 samples (8.3% Brucella spp. from 48 BTM samples in this area. Conclusion: The results indicate that brucellosis by Brucella species is endemic in the Kerman province dairy farms. Consumption of raw milk dairy products by individual farmers operating under poor hygienic conditions represents an high risk to public health. The need for implementing control measures and raising public awareness on zoonotic transmission of

  1. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    Science.gov (United States)

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis

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    Jacqueline Ribeiro de Castro

    2014-01-01

    Full Text Available ABSTRACT. Castro J.R., Silva C.B., Souza M.A., Salaberry S.R.S., Guimarães E.C., Mundim A.V. & Lima-Ribeiro A.M.C. [Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis.] Altera- ções hematológicas em cães naturalmente infectados por Leptospira spp., Brucella abortus e Brucella canis. Revista Brasileira de Medicina Veterinária, 36(1:49-54, 2014. Laboratório de Doenças Infectocontagiosas, Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Av. Ceará s/n, Bloco 2D, Sala 33, Campus Umuarama, Uberlândia, MG 38400-902, Brasil. E-mail: jack_ufu@yahoo.com.br The investigations of leptospirosis and brucellosis canine act as sanitary control in public health and zoonoses because they were established by close contact between dog and human. The aim was to determine the main hematological reagents in asymptomatic dogs against Leptospira spp. Brucella abortus and Brucella canis naturally infected, living in urban areas in the city of Uberlandia, Minas Gerais. We examined 140 blood samples from clinically healthy dogs, males and females and different ages. Leptospirosis was diagnosed by microscopic agglutination test (MAT, with a collection of twelve serovars, whereas, brucellosis was identified through the tests of Agar Gel Immunodiffusion (AGID for B. canis and buffered acidified antigen (TAA confirmed 2-Mercaptoethanol (2-ME for B. abortus. The results were analyzed using descriptive statistics with the calculation of simple percentages, mean and standard deviation. He applied and short sample t test for two independent samples to assess whether there were significant differences (p<0.05 between hematological parameters obtained. Dogs evaluated, 15% (21/140 and 2.85% (4/140 were reactive to Leptospira spp. and B. abortus, respectively. There was no sample reagent against B. canis. It was concluded that although no specific thrombocytopenia may be a significant finding in dogs

  3. MyD88 and STING Signaling Pathways Are Required for IRF3-Mediated IFN-β Induction in Response to Brucella abortus Infection

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    de Almeida, Leonardo A.; Carvalho, Natalia B.; Oliveira, Fernanda S.; Lacerda, Thais L. S.; Vasconcelos, Anilton C.; Nogueira, Lucas; Bafica, Andre; Silva, Aristóbolo M.; Oliveira, Sergio C.

    2011-01-01

    Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis. PMID:21829705

  4. MyD88 and STING signaling pathways are required for IRF3-mediated IFN-β induction in response to Brucella abortus infection.

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    Leonardo A de Almeida

    Full Text Available Type I interferons (IFNs are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis.

  5. Clinical Problems: Subclinical Brucella Infection in Man

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    Henderson, R. J.; Hill, D. M.

    1972-01-01

    Much subclinical infection with Brucella abortus affects the dairy farming community, slaughterhouse workers, and veterinary surgeons. High titres of antibody to B. abortus by the saline agglutination, antihuman globulin, or complement fixation test may be shown in subjects from the first two groups and generally there are no symptoms or signs of brucella infection whatever. Veterinary surgeons are prone to subclinical infection and young practitioners frequently display high titres against the usual tests at the same time apparently enjoying good health. Repeated reinfection of this group, however, may result in hypersensitivity to B. abortus and bouts of ill health at intervals may be due to this. Infection seems to come more often from contact with infected material than by drinking untreated milk, particularly in the herdsman, slaughterhouse worker, and veterinary surgeon. A doctor investigating illness of a patient from the dairy farming community and allied occupations should not accept a high titre against B. abortus as the only clue to diagnosis of the patient's condition. PMID:4625113

  6. Brucella abortus S19 vaccine protects dairy cattle against natural infection with Brucella melitensis.

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    van Straten, Michael; Bardenstein, Svetlana; Keningswald, Gaby; Banai, Menachem

    2016-11-21

    Brucellosis is a zoonotic disease that can cause severe illness in humans and considerable economic loss in the livestock industry. Although small ruminants are the preferential host for Brucella melitensis, this pathogen has emerged as a cause for Brucella outbreaks in cattle. S19 vaccination is implemented in many countries where B. abortus is endemic but its effectiveness against B. melitensis has not been validated. Here we show that vaccine effectiveness in preventing disease transmission between vaccinated and unvaccinated cohorts, as determined by seroconversion, was 87.2% (95% CI 69.5-94.6%). Furthermore, vaccination was associated with a reduced risk for abortion. Together, our data emphasize the role S19 vaccination could play in preventing B. melitensis outbreaks in areas where this pathogen is prevalent in small ruminant populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Detection of Brucella melitensis and Brucella abortus strains using a single-stage PCR method

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    Alamian, S.

    2015-04-01

    Full Text Available Brucella melitensis and Brucella abortus are of the most important causes of brucellosis, an infectious disease which is transmitted either directly or indirectly including consuming unpasteurized dairy products. Both strains are considered endemic in Iran. Common diagnostic methods such as bacteriologic cultures are difficult and time consuming regarding the bacteria. The aim of this study was to suggest a single-stage PCR method using a pair of primers to detect both B. melitensis and B. abortus. The primers were named UF1 and UR1 and the results showed that the final size of PCR products were 84 bp and 99 bp for B. melitensis and B. abortus, respectively. Therefore the method could be useful for rapid detection of B. melitensis and B. abortus simultaneously.

  8. Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes.

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    Muendo, Esther N; Mbatha, Peter M; Macharia, Joseph; Abdoel, Theresia H; Janszen, Paul V; Pastoor, Rob; Smits, Henk L

    2012-01-01

    Brucella melitensis biovar 1 was isolated from bovine milk samples from a herd in central Kenya, and Brucella abortus biovar 3 was isolated from aborted fetus materials and vaginal discharge fluids from cattle in central and eastern provinces of Kenya. All infections including those with B. melitensis were in cattle with reproductive problems kept in mixed herds indicating that cross infection occurs from small ruminants. Multiple-locus variable-number tandem repeat analysis genotyping revealed a close molecular homology of the B. melitensis isolates with an isolate from Israel and a close homology of the B. abortus isolates with an isolate from Uganda indicating that these genotypes have a wide geographic distribution. Infection of cattle with B. melitensis may complicate the control of brucellosis in this country.

  9. Genetic diversity of Brucella abortus and Brucella melitensis in Kazakhstan using MLVA-16.

    Science.gov (United States)

    Shevtsov, Alexandr; Ramanculov, Erlan; Shevtsova, Elena; Kairzhanova, Alma; Tarlykov, Pavel; Filipenko, Maxim; Dymova, Maya; Abisheva, Gulzada; Jailbekova, Aygul; Kamalova, Dinara; Chsherbakov, Andrei; Tulegenov, Samat; Akhmetova, Assel; Sytnik, Igor; Karibaev, Talgat; Mukanov, Kasim

    2015-08-01

    Brucellosis is an endemic disease in Central Asia characterized by high infection rates in humans and animals. Currently, little is known about the genetic diversity of Brucella spp. circulating in the region, despite the high prevalence of brucellosis. This study aimed to analyze the genetic diversity of Brucella melitensis and Brucella abortus strains circulating in the Republic of Kazakhstan. We genotyped 128 B. melitensis and 124 B. abortus strains collected in regions with the highest prevalence of brucellosis. Genotyping was performed using multi-locus variable-number tandem-repeat analysis (MLVA). Analysis of a subset of 8 loci (MLVA-8) of 128 B. melitensis strains identified genotypes 42 (n=108), 43 (n=2), and 63 (n=19) related to the 'East Mediterranean' group. An MLVA-16 assay sorted 128 B. melitensis strains into 25 different genotypes. Excluding one variable locus, MLVA-15 of B. melitensis was distinct from strains originating in the Mediterranean region; however, 77% of them were identical to strains isolated in China. A minimum spanning tree for B. melitensis using MLVA-15 analysis clustered the local strains together with strains previously collected in China. MLVA-8 analysis of 124 B. abortus strains identified them as genotype 36, suggesting Eurasian distribution of this lineage. Complete MLVA-16 assay analysis clustered the strains into five genotypes, revealing little diversity of B. abortus when compared on the global scale. A minimum spanning tree for B. abortus obtained using MLVA-15 analysis clustered the 2 most prevalent genotypes (n=117) together with strains previously collected in China. Thus, MLVA analysis was used to characterize 252 strains of Brucella collected in Kazakhstan. The analysis revealed genetic homogeneity among the strains. Interestingly, identical MLVA-15 profiles were found in seemingly unrelated outbreaks in China, Turkey, and Kazakhstan. Further analysis is needed for better understanding of the epidemiology of

  10. Persistence Testing of Brucella suis on Outdoor Materials ...

    Science.gov (United States)

    Report This report presents the results of an investigation to evaluate Brucella suis persistence on five materials (typically found in the outdoor environment) under various environmental conditions and exposure durations.

  11. The changing nature of the Brucella-containing vacuole.

    Science.gov (United States)

    Celli, Jean

    2015-07-01

    Bacteria of the genus Brucella are intracellular vacuolar pathogens of mammals that cause the worldwide zoonosis brucellosis, and reside within phagocytes of infected hosts to promote their survival, persistence and proliferation. These traits are essential to the bacterium's ability to cause disease and have been the subject of much investigation to gain an understanding of Brucella pathogenic mechanisms. Although the endoplasmic reticulum-derived nature of the Brucella replicative niche has been long known, major strides have recently been made in deciphering the molecular mechanisms of its biogenesis, including the identification of bacterial determinants and host cellular pathways involved in this process. Here I will review and discuss the most recent advances in our knowledge of Brucella intracellular pathogenesis, with an emphasis on bacterial exploitation of the host endoplasmic reticulum-associated functions, and how autophagy-related processes contribute to the bacterium's intracellular cycle. © 2015 John Wiley & Sons Ltd.

  12. Systems Biology Analysis of Temporal In vivo Brucella melitensis and Bovine Transcriptomes Predicts host:Pathogen Protein–Protein Interactions

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    Carlos A. Rossetti

    2017-07-01

    Full Text Available To date, fewer than 200 gene-products have been identified as Brucella virulence factors, and most were characterized individually without considering how they are temporally and coordinately expressed or secreted during the infection process. Here, we describe and analyze the in vivo temporal transcriptional profile of Brucella melitensis during the initial 4 h interaction with cattle. Pathway analysis revealed an activation of the “Two component system” providing evidence that the in vivo Brucella sense and actively regulate their metabolism through the transition to an intracellular lifestyle. Contrarily, other Brucella pathways involved in virulence such as “ABC transporters” and “T4SS system” were repressed suggesting a silencing strategy to avoid stimulation of the host innate immune response very early in the infection process. Also, three flagellum-encoded loci (BMEII0150-0168, BMEII1080-1089, and BMEII1105-1114, the “flagellar assembly” pathway and the cell components “bacterial-type flagellum hook” and “bacterial-type flagellum” were repressed in the tissue-associated B. melitensis, while RopE1 sigma factor, a flagellar repressor, was activated throughout the experiment. These results support the idea that Brucella employ a stealthy strategy at the onset of the infection of susceptible hosts. Further, through systems-level in silico host:pathogen protein–protein interactions simulation and correlation of pathogen gene expression with the host gene perturbations, we identified unanticipated interactions such as VirB11::MAPK8IP1; BtaE::NFKBIA, and 22 kDa OMP precursor::BAD and MAP2K3. These findings are suggestive of new virulence factors and mechanisms responsible for Brucella evasion of the host's protective immune response and the capability to maintain a dormant state. The predicted protein–protein interactions and the points of disruption provide novel insights that will stimulate advanced hypothesis

  13. Analyzing the molecular mechanism of lipoprotein localization in Brucella

    Science.gov (United States)

    Goolab, Shivani; Roth, Robyn L.; van Heerden, Henriette; Crampton, Michael C.

    2015-01-01

    Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria

  14. Analyzing the molecular mechanism of lipoprotein localization in Brucella.

    Science.gov (United States)

    Goolab, Shivani; Roth, Robyn L; van Heerden, Henriette; Crampton, Michael C

    2015-01-01

    Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria

  15. The ferrous iron transporter FtrABCD is required for the virulence of Brucella abortus 2308 in mice.

    Science.gov (United States)

    Elhassanny, Ahmed E M; Anderson, Eric S; Menscher, Evan A; Roop, R Martin

    2013-06-01

    Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host. © 2013 John Wiley & Sons Ltd.

  16. Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

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    García-Lobo Juan M

    2010-04-01

    Full Text Available Abstract Background Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown. Results Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium. Conclusions Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

  17. Brucella species survey in polar bears (ursus maritimus) of northern Alaska.

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    O'Hara, Todd M; Holcomb, Darce; Elzer, Philip; Estepp, Jessica; Perry, Quinesha; Hagius, Sue; Kirk, Cassandra

    2010-07-01

    We report on the presence of specific antibodies to Brucella spp. and Yersinia enterocolitica in polar bears (Ursus maritimus) from northern Alaska (southern Beaufort Sea) during 2003-2006. Based on numerous known stressors (e.g., climate change and loss of sea ice habitat, contaminants), there is increased concern regarding the status of polar bears. Considering these changes, it is important to assess exposure to potentially pathogenic organisms and to improve understanding of transmission pathways. Brucella or specific antibodies to Brucella spp. has been reported in marine mammals. Various assays were used to elucidate the pathway or source of exposure (e.g., "marine" vs. "terrestrial" Brucella spp.) of northern Alaska polar bears to Brucella spp. The standard plate test (SPT) and the buffered Brucella antigen card test (BBA) were used for initial screening for antibodies specific to Brucella. We then evaluated positive reactors (presence of serum antibody specific for Brucella spp.) using immunoblots and competitive enzyme-linked immunosorbent assay (cELISA; based on pinniped-derived Brucella spp. antigen). Annual prevalence of antibody (BBA and SPT) for Brucella spp. ranged from 6.8% to 18.5% over 2003-2006, with an overall prevalence of 10.2%. Prevalence of Brucella spp. antibody did vary by age class. Western blot analyses indicated 17 samples were positive for Brucella spp. antibody; of these, 13 were negative by marine (pinniped) derived Brucella antigen cELISA and four were positive by marine cELISA. Of the four samples positive for Brucella antibody by marine cELISA, three cross-reacted with Y. enterocolitica and Brucella spp. (one sample was Brucella negative and Y. enterocolitica positive). It appears the polar bear antibody does not react with the antigens used on the marine cELISA assay, potentially indicating a terrestrial (nonpinniped) source of Brucella spp.

  18. Type IV secretion system of Brucella spp. and its effectors.

    Science.gov (United States)

    Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

    2015-01-01

    Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

  19. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. Contamination of bovine, sheep and goat meat with Brucella spp.

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    Francesco Casalinuovo

    2016-06-01

    Full Text Available A study was conducted in order to evaluate the contamination by Brucella spp. of meat from animals slaughtered because they had resulted positive for brucellosis at some time during their life. After slaughter and before delivery to market outlets, swab samples were taken from 307 carcasses of infected animals: 40 cattle, 60 sheep and 207 goats. The swabs were subsequently analysed by means of polymerase chain reaction (PCR tests. In addition, bacteriological tests were carried out on the lymph nodes and internal organs of the same animals. Brucella spp. was detected by means of PCR in 25/307 carcasses (8%: 1 bovine (2.5%, 9 sheep (15% and 15 goats (7.2% and was isolated by means of a cultural method in 136/307 carcasses (44%. Moreover, additional analysis, performed on lymph nodes from the same carcasses that had proved positive by PCR, allowed highlighting type 3 Brucella abortus in the bovine carcass and type 3 Brucella melitensis in the sheep and goat carcasses. The study shows that cattle, sheep and goats meat of animals slaughtered because they had tested positive for brucellosis may be contaminated by Brucella spp. As this could constitute a real risk of transmission to both butchery personnel and consumers, the meat of animals infected by Brucella spp. should be analysed before being marketed. In this respect, PCR technique performed on swabs proved to be more useful, practical and faster than the traditional bacteriological method.

  1. Detection of Brucella abortus in Chiredzi district in Zimbabwe

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    Calvin Gomo

    2012-02-01

    Full Text Available Brucellosis is an endemic disease in Zimbabwe caused by the genus Brucella. Brucella seroprevalence was recently reported to be high in the wildlife-livestock interface in the Chiredzi district and the neighbouring Gonarezhou National Park (GNP in Zimbabwe, and higher amongst communal cattle with an abortion history and access to grazing in GNP than amongst communal cattle with no abortion history or access to grazing in GNP. The aim of this study was to investigate Brucella species in brucellosis seropositive cattle in the Chiredzi district with access to GNP using isolation and identification. Isolation of Brucella species from whole blood (n = 18 and milk samples (n = 10 from seropositive animals with an abortion history was based on the rose Bengal test (RBT and enzyme-linked immunoassays (enzyme- linked immunosorbent assay [ELISA]; indirect ELISA and complement ELISA, using microbiology and polymerase chain reaction (PCR methods. Brucella abortus was cultured and identified from blood and milk collected from seropositive cows in both communal areas. The Brucella-specific 16-23S intergenic spacer (ITS PCR and multiplex AMOS-PCR assays verified the identification of the cultures. Our results confirmed that B. abortus is present in cattle on communal farms in the Chiredzi district in Zimbabwe and might cause cattle abortions. The need for implementing control measures and raising public awareness on zoonotic transmission of brucellosis are recommended.

  2. Estudo do envolvimento da enzima formil-transferase na biossíntese do LPS da Brucella abortus e avaliação da persistência e proteção de uma linhagem mutante para este gene de camundongos C57BL/6 e defifientes para IRF-1

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    Thais Lourdes Santos Lacerda

    2010-01-01

    Os membros do gênero Brucella são bactérias Gram-negativas, intracelulares facultativas que podem causar infecções crônicas em mamíferos. Várias tentativas têm sido feitas com o objetivo de se obter uma vacina eficaz para esta doença. No entanto, apesar de conferir proteção, as vacinas comerciais atualmente disponíveis apresentam desvantagens, tais como a interferência no diagnóstico de animais infectados e resistência a antibióticos. Considerando LPS Brucella de um dos seus principais fatore...

  3. Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

    Science.gov (United States)

    Cassataro, Juliana; Pasquevich, Karina; Bruno, Laura; Wallach, Jorge C.; Fossati, Carlos A.; Baldi, Pablo C.

    2004-01-01

    Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.   PMID:14715555

  4. Endogenous Brucella endophthalmitis: A case report

    Directory of Open Access Journals (Sweden)

    Merih Oray

    2017-04-01

    Full Text Available Brucellosis may be associated with a wide range of ophthalmic manifestations including endophthalmitis, which is a sight-threatening condition that needs to be rapidly recognized and treated to avoid permanent visual loss. A 26-year-old female with a 6-month history of vision loss in the left eye was treated with high dose systemic corticosteroids and azathioprine with an initial misdiagnosis elsewhere. A dense vitreous haze with opacities at the posterior hyaloid and a wide area of retinochoroiditis led to the diagnosis of endogenous endophthalmitis at presentation to us. The vitreous sample and blood cultures demonstrated growth of Brucella melitensis. She received 6 months of systemic antibiotherapy, which resulted in resolution of inflammation; however, visual acuity remained poor due to irreversible damage. Infectious etiology, including brucellosis in endemic countries, has to be considered in the differential diagnosis before administering immunomodulatory therapy in patients with panuveitis of unknown origin.

  5. Identification of new IS711 insertion sites in Brucella abortus field isolates

    Directory of Open Access Journals (Sweden)

    Moriyón Ignacio

    2011-08-01

    Full Text Available Abstract Background Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated. Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. Results We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Conclusions Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  6. Identification of new IS711 insertion sites in Brucella abortus field isolates.

    Science.gov (United States)

    Mancilla, Marcos; Ulloa, Marcos; López-Goñi, Ignacio; Moriyón, Ignacio; María Zárraga, Ana

    2011-08-03

    Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated). Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  7. Species identification and molecular typing of human Brucella isolates from Kuwait.

    Science.gov (United States)

    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  8. A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata)

    Science.gov (United States)

    2011-01-01

    Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population. PMID:21819589

  9. Brucella abortus RB51 and Hot Saline Extract from Brucella ovis as Antigens in a Complement Fixation Test Used To Detect Sheep Vaccinated with Brucella abortus RB51

    OpenAIRE

    Adone, Rosanna; Ciuchini, Franco

    2001-01-01

    The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 × 109 CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with R...

  10. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  11. Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

    Science.gov (United States)

    Rajasekaran, Parthiban; Surendran, Naveen; Seleem, Mohamed N; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2011-04-12

    Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (PBrucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Improved immunogenicity of tetanus toxoid by Brucella abortus S19 LPS adjuvant.

    Science.gov (United States)

    Mohammadi, Mohsen; Kianmehr, Zahra; Kaboudanian Ardestani, Sussan; Gharegozlou, Behnaz

    2014-09-01

    Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund's adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.

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

    Science.gov (United States)

    Hop, Huynh Tan; Arayan, Lauren Togonon; Reyes, Alisha Wehdnesday Bernardo; Huy, Tran Xuan Ngoc; Min, WonGi; Lee, Hu Jang; Son, Jee Soo; Kim, Suk

    2017-12-01

    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-1β (Il1β), 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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection.

    Science.gov (United States)

    Tabynov, Kaissar; Sansyzbay, Abylai; Kydyrbayev, Zhailaubay; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Assanzhanova, Nurika; Sultankulova, Kulaisan; Sandybayev, Nurlan; Khairullin, Berik; Kuznetsova, Irina; Ferko, Boris; Egorov, Andrej

    2014-04-10

    We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by

  15. In vitro susceptibilities of Brucella melitensis isolates to eleven antibiotics

    Directory of Open Access Journals (Sweden)

    Loukaides Feidias

    2006-10-01

    Full Text Available Abstract Background Brucellosis is an endemic disease present in many countries worldwide, but it is rare in Europe and North America. Nevertheless brucella is included in the bacteria potentially used for bioterrorism. The aim of this study was the investigation of the antibiotic susceptibility profile of brucella isolates from areas of the eastern Mediterranean where it has been endemic. Methods The susceptibilities of 74 Brucella melitensis isolates derived from clinical samples (57 and animal products (17 were tested in vitro. The strains originate from Crete (59, Cyprus (10, and Syria (5. MICs of tetracycline, rifampicin, streptomycin, gentamicin, norfloxacin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, ampicillin, amoxicillin/clavulanic acid, and erythromycin were detected by E-test method. The NCCLS criteria for slow growing bacteria were considered to interpret the results. Results All the isolates were susceptible to tetracycline, streptomycin, gentamicin, ciprofloxacin, norfloxacin, and levofloxacin. Two isolates presented reduced susceptibility to rifampicin (MIC value: 1.5 mg/l and eight to SXT (MIC values: 0.75–1.5 mg/l. Erythromycin had the highest (4 mg/l MIC90value and both norfloxacin and erythromycin the highest (1.5 mg/l MIC50 value. Conclusion Brucella isolates remain susceptible in vitro to most antibiotics used for treatment of brucellosis. The establishment of a standardized antibiotic susceptibility method for Brucella spp would be useful for resistance determination in these bacteria and possible evaluation of bioterorism risks.

  16. Desenvolvimento de uma Reação em Cadeia pela Polimerase (PCR) para detecção de Brucella spp. em amostras de sangue de cães naturalmente infectados

    OpenAIRE

    Nanci do Rosário Vieira

    2004-01-01

    Foram desenhados três pares de primers, sendo um deles dirigido ao gene que codifica uma proteína periplasmática da Brucella (BP26) e dois outros dirigidos para a região interespaçadora entre os gene 16S e 23S do RNA ribossômico (ITS). Com os primers acima, foram padronizadas três PCRs, potencialmente capazes de detectar qualquer espécie de Brucella e denominadas PCR-ITS1, PCR-ITS2 e PCR-BP26. Soluções de DNA de sangue de cão livre de Brucella foram contaminadas com quantidades decrescentes d...

  17. Isolation of a novel 'atypical' Brucella strain from a bluespotted ribbontail ray (Taeniura lymma).

    Science.gov (United States)

    Eisenberg, Tobias; Riße, Karin; Schauerte, Nicole; Geiger, Christina; Blom, Jochen; Scholz, Holger C

    2017-02-01

    A pleomorphic Gram-negative, motile coccobacillus was isolated from the gills of a wild-caught bluespotted ribbontail ray after its sudden death during quarantine. Strain 141012304 was observed to grow aerobically, to be clearly positive for cytochrome oxidase, catalase, urease and was initially identified as "Brucella melitensis" or "Ochrobactrum anthropi" by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and VITEK2-compact ® , respectively. Affiliation to the genus Brucella was confirmed by bcsp31 and IS711 PCR as well as by Brucella species-specific multiplex PCR, therein displaying a characteristic banding pattern recently described for Brucella strains obtained from amphibian hosts. Likewise, based on recA sequencing, strain 141012304 was found to form a separate lineage, within the so called 'atypical' Brucella, consisting of genetically more distantly related strains. The closest similarity was detected to brucellae, which have recently been isolated from edible bull frogs. Subsequent next generation genome sequencing and phylogenetic analysis confirmed that the ray strain represents a novel Brucella lineage within the atypical group of Brucella and in vicinity to Brucella inopinata and Brucella strain BO2, both isolated from human patients. This is the first report of a natural Brucella infection in a saltwater fish extending the host range of this medically important genus.

  18. Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

    Science.gov (United States)

    Maio, Elisa; Begeman, Lineke; Bisselink, Yvette; van Tulden, Peter; Wiersma, Lidewij; Hiemstra, Sjoukje; Ruuls, Robin; Gröne, Andrea; Roest, Hendrik-Ido-Jan; Willemsen, Peter; van der Giessen, Joke

    2014-09-17

    The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

    Science.gov (United States)

    Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

    2014-11-07

    The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Isolation of Brucella pinnipedialis from Grey Seals (Halichoerus grypus) in the Baltic Sea.

    Science.gov (United States)

    Hirvelä-Koski, Varpu; Nylund, Minna; Skrzypczak, Teresa; Heikkinen, Petra; Kauhala, Kaarina; Jay, Maryne; Isomursu, Marja

    2017-10-01

    Brucella infection in seals was reported for the first time in 1994 around the coast of Scotland. Since then, marine mammal Brucella infections were found to be widely distributed in the northern hemisphere. Two Brucella species affect marine mammals: Brucella pinnipedialis in pinnipeds and Brucella ceti in cetaceans. We examined the livers of Baltic grey seals (Halichoerus grypus) from the Finnish coast (n=122) hunted, found dead, or killed as by-catch in fishing gear in 2013-15 as part of population health monitoring. We detected B. pinnipedialis in the livers of three grey seals. The bacterium was isolated from livers displaying parasitic cholangitis. We also detected Brucella DNA in liver flukes (Pseudamphistomum truncatum) obtained from a Brucella-infected grey seal, suggesting that flukes might be possible vectors of this pathogen in the marine environment.

  1. Brucella ceti and Brucellosis in Cetaceans

    Science.gov (United States)

    Guzmán-Verri, Caterina; González-Barrientos, Rocío; Hernández-Mora, Gabriela; Morales, Juan-Alberto; Baquero-Calvo, Elías; Chaves-Olarte, Esteban; Moreno, Edgardo

    2012-01-01

    Since the first case of brucellosis detected in a dolphin aborted fetus, an increasing number of Brucella ceti isolates has been reported in members of the two suborders of cetaceans: Mysticeti and Odontoceti. Serological surveys have shown that cetacean brucellosis may be distributed worldwide in the oceans. Although all B. ceti isolates have been included within the same species, three different groups have been recognized according to their preferred host, bacteriological properties, and distinct genetic traits: B. ceti dolphin type, B. ceti porpoise type, and B. ceti human type. It seems that B. ceti porpoise type is more closely related to B. ceti human isolates and B. pinnipedialis group, while B. ceti dolphin type seems ancestral to them. Based on comparative phylogenetic analysis, it is feasible that the B. ceti ancestor radiated in a terrestrial artiodactyl host close to the Raoellidae family about 58 million years ago. The more likely mode of transmission of B. ceti seems to be through sexual intercourse, maternal feeding, aborted fetuses, placental tissues, vertical transmission from mother to the fetus or through fish or helminth reservoirs. The B. ceti dolphin and porpoise types seem to display variable virulence in land animal models and low infectivity for humans. However, brucellosis in some dolphins and porpoises has been demonstrated to be a severe chronic disease, displaying significant clinical and pathological signs related to abortions, male infertility, neurobrucellosis, cardiopathies, bone and skin lesions, strandings, and death. PMID:22919595

  2. A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Brucella abortus Mediates Host-Pathogen Interactions

    Science.gov (United States)

    Sheehan, Lauren M.

    2017-01-01

    ABSTRACT In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. PMID:28588127

  3. Brucella abortus ΔrpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis.

    Science.gov (United States)

    Willett, Jonathan W; Herrou, Julien; Czyz, Daniel M; Cheng, Jason X; Crosson, Sean

    2016-09-30

    The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Improved serodiagnosis of bovine brucellosis by novel synthetic oligosaccharide antigens representing the capping m epitope elements of Brucella O-polysaccharide.

    Science.gov (United States)

    McGiven, John; Howells, Laurence; Duncombe, Lucy; Stack, Judy; Ganesh, N Vijaya; Guiard, Julie; Bundle, David R

    2015-04-01

    Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-polysaccharide (OPS). Serology is the most cost-effective means of detecting brucellosis, as infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyl residues (D-Rha4NFo) that are variably α(1→2)- and α(1→3)-linked. Six d-Rha4NFo homo-oligosaccharides were synthesized, each containing a single α(1→3) link but with a varied number of α(1→2) links. After conjugation to bovine serum albumin (BSA), glycoconjugates 1 to 6 were used to develop individual indirect enzyme-linked immunosorbent assays (iELISAs). The diagnostic capabilities of these antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth lipopolysaccharide (sLPS) iELISA, and competitive enzyme-linked immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1→3)-linked disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic trisaccharide antigens 2 and 3 and tetrasaccharide antigen 4 identified an OPS epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic oligosaccharide conjugates function as effective surrogates for naturally derived antigens. The creation of discrete OPS epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds

  5. Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp.

    Science.gov (United States)

    Nan, Wenlong; Tan, Pengfei; Wang, Yong; Xu, Zouliang; Mao, Kairong; Peng, Daxin; Chen, Yiping

    2014-09-01

    Immunisation with attenuated Brucella spp. vaccines prevents brucellosis, but may also interfere with diagnosis. In this study, a duplex PCR was developed to distinguish Brucella suis vaccine strain S2 from field strains of B. suis biovar 1 and other Brucella spp. The PCR detected 60 fg genomic DNA of B. suis S2 or biovar 1 field strains and was able to distinguish B. suis S2 and wild-type strains of B. suis biovar 1 among 76 field isolates representing all the common species and biovars, as well as four vaccine strains, of Brucella. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Soroepidemiologia da brucelose canina causada por Brucella canis e Brucella abortus na cidade de Alfenas, MG Seroepidemiology of canine brucellosis caused by Brucella canis and Brucella abortus in Alfenas, MG, Brazil

    Directory of Open Access Journals (Sweden)

    A.C. Almeida

    2004-04-01

    Full Text Available The prevalence of canine brucellosis was evaluated in the city of Alfenas, MG through the technique of agarose gel imunodifusion for Brucella canis and slow serum agglutination test with 2-mercaptoetanol for Brucella abortus. The prevalence was of 14.2% and 2.8%, respectively, for B. canis and B. abortus. The positives, characterized by animals above one year of age (77.8%, and mongrel dogs (56.2%, showed a prevalence of 50 and 48% for males and females, respectively. The canine brucellosis was prevalent in the city principally in dogs of outskirts.

  7. Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria.

    Science.gov (United States)

    Sabrina, Rabehi; Mossadak, Hamdi Taha; Bakir, Mamache; Asma, Meghezzi; Khaoula, Boushaba

    2018-03-01

    The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR) assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. The results show that of the 65 DNA samples tested, 2 (3.08%) were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella -infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease.

  8. Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria

    Directory of Open Access Journals (Sweden)

    Rabehi Sabrina

    2018-03-01

    Full Text Available Aim: The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. Materials and Methods: In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. Results: The results show that of the 65 DNA samples tested, 2 (3.08% were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. Conclusion: The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella-infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease.

  9. Attenuation of Neuroinflammatory Responses in Lipopolysaccharide ...

    African Journals Online (AJOL)

    Chenopodiaceae) extract on neuroinflammatory responses induced by lipopolysaccharide (LPS) in BV-2 microglial cells and its antioxidant effects. Methods: Biochemical studies carried out include 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium ...

  10. Inhibition of lipopolysaccharide-induced neuroinflammatory events ...

    African Journals Online (AJOL)

    tetrazolium bromide (MTT) assay. Lipopolysaccharide (LPS) is used to activate BV-2 microglia. Nitric oxide (NO) levels were measured using Griess assay. Inducible NO synthase (iNOS) expressional levels were measured by Western blot analysis.

  11. Early Transcriptional Responses of Bovine Chorioallantoic Membrane Explants to Wild Type, ΔvirB2 or ΔbtpB Brucella abortus Infection

    Science.gov (United States)

    Mol, Juliana P. S.; Costa, Erica A.; Carvalho, Alex F.; Sun, Yao-Hui; Tsolis, Reneé M.; Paixão, Tatiane A.; Santos, Renato L.

    2014-01-01

    The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; Pabortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion. PMID:25259715

  12. Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy.

    Science.gov (United States)

    Adone, R; Ciuchini, F; La Rosa, G; Marianelli, C; Muscillo, M

    2001-03-01

    Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains.

  13. The Complete Genome of Brucella Suis 019 Provides Insights on Cross-Species Infection

    Directory of Open Access Journals (Sweden)

    Yuanzhi Wang

    2016-01-01

    Full Text Available Brucella species are the most important zoonotic pathogens worldwide and cause considerable harm to humans and animals. In this study, we presented the complete genome of B. suis 019 isolated from sheep (ovine with epididymitis. B. suis 019 has a rough phenotype and can infect sheep, rhesus monkeys and possibly humans. The comparative genome analysis demonstrated that B. suis 019 is closest to the vaccine strain B. suis bv. 1 str. S2. Further analysis associated the rsh gene to the pathogenicity of B. suis 019, and the WbkA gene to the rough phenotype of B. suis 019. The 019 complete genome data was deposited in the GenBank database with ID PRJNA308608.

  14. Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium.

    Science.gov (United States)

    Mohamed, Kettani-Halabi; Daniel, Tran; Aurélien, Dauphin; El-Maarouf-Bouteau, Hayat; Rafik, Errakhi; Arbelet-Bonnin, Delphine; Biligui, Bernadette; Florence, Val; Mustapha, Ennaji Moulay; François, Bouteau

    2015-01-01

    Lipopolysaccharides (LPS) are a component of the outer cell surface of almost all Gram-negative bacteria and play an essential role for bacterial growth and survival. Lipopolysaccharides represent typical microbe-associated molecular pattern (MAMP) molecules and have been reported to induce defense-related responses, including the expression of defense genes and the suppression of the hypersensitive response in plants. However, depending on their origin and the challenged plant, LPS were shown to have complex and different roles. In this study we showed that LPS from plant pathogens Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum induce common and different responses in A. thaliana cells when compared to those induced by LPS from non-phytopathogens Escherichia coli and Pseudomonas aeruginosa. Among common responses to both types of LPS are the transcription of defense genes and their ability to limit of cell death induced by Pectobacterium carotovorum subsp carotovorum. However, the differential kinetics and amplitude in reactive oxygen species (ROS) generation seemed to regulate defense gene transcription and be determinant to induce programmed cell death in response to LPS from the plant pathogenic Pectobacterium. These data suggest that different signaling pathways could be activated by LPS in A. thaliana cells.

  15. Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells.

    Science.gov (United States)

    Surendran, Naveen; Hiltbold, Elizabeth M; Heid, Bettina; Sriranganathan, Nammalwar; Boyle, Stephen M; Zimmerman, Kurt L; Makris, Melissa R; Witonsky, Sharon G

    2011-01-10

    Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu-Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th(1) and CD8+ Tc(1) adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production. Copyright © 2010. Published by Elsevier B.V.

  16. Evaluation of a New and Rapid Serologic Test for Detecting Brucellosis: Brucella Coombs Gel Test.

    Science.gov (United States)

    Hanci, Hayrunisa; Igan, Hakan; Uyanik, Muhammet Hamidullah

    2017-01-01

    Many serological tests have been used for the diagnosis of human brucellosis. A new serological method is identified as Brucella Coombs gel test based on the principle of centrifugation gel system similar to the gel system used in blood group determination. In this system, if Brucella antibodies were present in the serum, antigen and antibody would remain as a pink complex on the gel. Otherwise, the pink Brucella antigens would precipitate at the bottom of the gel card system. In this study, we aimed to compare the Brucella Coombs gel test, a new, rapid screen and titration method for detection of non-agglutinating IgG with the Brucella Coombs test. For this study, a total of 88 serum samples were obtained from 45 healthy persons and 43 individuals who had clinical signs and symptoms of brucellosis. For each specimen, Rose Bengal test, standard agglutination test, Coombs test and Brucella Coombs gel test were carried out. Sensitivity and specificity of Brucella Coombs gel test were found as 100.0 and 82.2%, respectively. Brucella Coombs gel test can be used as a screening test with high sensitivity. By the help of pink Brucella antigen precipitation, the tests' evaluation is simple and objective. In addition, determination of Brucella antibody by rapid titration offers another important advantage.

  17. Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

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    Yang Wang

    2017-06-01

    Full Text Available Brucellosis is caused by infection with Brucella species and exhibits diverse clinical manifestations in infected humans. Monocytes and macrophages are not only the first line of defense against Brucella infection but also a main reservoir for Brucella. In the present study, we examined the effects of Brucella infection on human peripheral monocytes and monocyte-derived polarized macrophages. We showed that Brucella infection led to an increase in the proportion of CD14++CD16− monocytes and the expression of the autophagy-related protein LC3B, and the effects of Brucella-induced monocytes are inhibited after 6 weeks of antibiotic treatment. Additionally, the production of IL-1β, IL-6, IL-10, and TNF-α from monocytes in patients with brucellosis was suppressed through the LC3-dependent autophagy pathway during Brucella infection. Moreover, Brucella infection inhibited macrophage polarization. Consistently, the addition of 3-MA, an inhibitor of LC3-related autophagy, partially restored macrophage polarization. Intriguingly, we also found that the upregulation of LC3B expression by rapamycin and heat-killed Brucella in vitro inhibits M2 macrophage polarization, which can be reversed partially by 3-MA. Taken together, these findings reveal that Brucella dysregulates monocyte and macrophage polarization through LC3-dependent autophagy. Thus, targeting this pathway may lead to the development of new therapeutics against Brucellosis.

  18. Identifikasi Brucella abortus Isolat Lokal dengan Brucella abortus Strain Specific-Polymerase Chain Reaction (IDENTIFICATION OF LOCAL ISOLATES OF BRUCELLA ABORTUS USING BRUCELLA ABORTUS STRAIN SPECIFIC-POLYMERASE CHAIN REACTION ASSAY

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    Susan Maphilindawati Noor

    2014-10-01

    Full Text Available Brucella abortus Strain Specific-Polymerase Chain Reaction (BaSS-PCR is a single multiplex PCRtechnique which able to identify and differentiate between Brucella abortus field strains (biovar 1, 2, and4, B. abortus vaccine strains, Brucella species, and non-Brucella species. In this study, BaSS-PCR wasapplied to identify local isolates of B. abortus in order to investigate the B. abortus strains that infectedcattle in Indonesia. Fifty local strains of B.abortus isolated from infected cattle in Java (Jakarta andBandung, South Sulawesi (Maros, East Nusa Tenggara (Kupang and Belu were used in this study. TheDNA bands were observed by agarose gel in the presence of ethidium bromide. Identification was performedbased on the size and number of DNA products amplified by PCR from each isolates. The results showedthat the 50 isolates were of B. abortus field strains. This finding showed that the cause of bovine brucellosisin Indonesia is B. abortus field strains.

  19. Seroprevalence of Brucella abortus in buffaloes and wildebeests in ...

    African Journals Online (AJOL)

    A sero-survey was conducted in buffalo and wildebeests in Ngorongoro Crater and Serengeti National Park (SNP) collectively known as Serengeti ecosystem to establish the level of exposure to Brucella arbortus. Rose Bengal Plate Agglutination test and Competitive ELISA were used serially in the analysis of 205 serum ...

  20. Molecular detection of Brucella spp. from broth culture of clinical ...

    African Journals Online (AJOL)

    PCR was employed to detect Brucella spp. from broth cultures of clinical samples using a group specific primer based on IS6501 sequence. The sensitivity and specificity of this assay was confirmed by Southern hybridization analysis using a digoxigenin-labeled DNA probe while reproducibility of the analysis was ...

  1. MLVA genotyping of human Brucella isolates from Peru

    NARCIS (Netherlands)

    Smits, Henk L.; Espinosa, Benjamin; Castillo, Rosa; Hall, Eric; Guillen, Alfredo; Zevaleta, Milagros; Gilman, Robert H.; Melendez, Paolo; Guerra, Carlos; Draeger, Angelika; Broglia, Alessandro; Nöckler, Karsten

    2009-01-01

    Recent human Brucella melitensis isolates from Peru were genotyped by multiple locus variable number repeat analysis. All 24 isolates originated from hospitalized patients living in the central part of Peru and consisted of six genomic groups comprising two to four isolates and nine unique

  2. Testing for Antibodies to Brucella abortus in Milk From Consumers ...

    African Journals Online (AJOL)

    Over 85% of all milk sales on Kenya pass through informal channels. The extent of the risk posed by the sale of this raw milk to human health in respect to brucellosis is unknown. This paper presents the results of a study on the occurrence of antibodies to Brucella abortus in milk from households consuming raw ...

  3. Brucella abortus agglutinins in dogs in Zaria, Nigeria | Osinubi ...

    African Journals Online (AJOL)

    Serum samples from dogs brought for routine physical examination, vaccination and other complaints at the Veterinary Teaching Hospital (VTH) of Ahmadu Bello University and other parts of Zaria were tested for Brucella abortus antibodies. Forty-three (21.5%) of the 200 dogs studied were positive for B. abortus agglutinins ...

  4. Effect of centrifugation and microagglutination techniques on Brucella agglutinin titers.

    OpenAIRE

    Klein, G C; Behan, K A; Brown, S L; Couch, E E

    1982-01-01

    The microagglutination technique without centrifugation was more effective than centrifugation of the standard tube test for increasing Brucella agglutinin titers of specimens with a titer greater than or equal to 160 but was less effective than centrifugation of the standard tube test for specimens with a titer less than 160.

  5. Prevalence of Brucella Antibodies in Migratory Fulani Cattle Herds ...

    African Journals Online (AJOL)

    Brucellosis is a major cause of economic losses such as abortion, infertility, low conception rate and low survival rate of neonates in the livestock industry and zoonoses of great public health significance. The prevalence of Brucella antibodies in migratory Fulani cattle in Kaduna State was determined using the Milk Ring ...

  6. BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

    Science.gov (United States)

    Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

    2007-01-01

    The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

  7. Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans.

    Science.gov (United States)

    Ohishi, Kazue; Bando, Takeharu; Abe, Erika; Kawai, Yasushi; Fujise, Yoshihiro; Maruyama, Tadashi

    2016-10-01

    In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994-2010 offshore surveys (21%, 285/1353) and in the 2006-2010 Japanese coastal surveys (20%, 86/436), in Bryde's whales (B. edeni brydei) in the 2000-2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002-2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000-2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000-2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde's whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level.

  8. Postreplication Roles of the Brucella VirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase

    Directory of Open Access Journals (Sweden)

    Erin P. Smith

    2016-11-01

    Full Text Available Brucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, the Brucella-containing vacuole (BCV. Initially of endosomal origin (eBCV, BCVs are remodeled into replication-permissive organelles (rBCV derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by the Brucella VirB type IV secretion system (T4SS. Following replication, rBCVs are converted into autophagic vacuoles (aBCVs that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of the Brucella intracellular cycle. To address this issue, we have generated a B. abortus strain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc-dependent complementation of a deletion of the virB11 gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression of virB11 prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in the Brucella intracellular cycle.

  9. Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans

    Science.gov (United States)

    OHISHI, Kazue; BANDO, Takeharu; ABE, Erika; KAWAI, Yasushi; FUJISE, Yoshihiro; MARUYAMA, Tadashi

    2016-01-01

    In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994–2010 offshore surveys (21%, 285/1353) and in the 2006–2010 Japanese coastal surveys (20%, 86/436), in Bryde’s whales (B. edeni brydei) in the 2000–2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002–2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000–2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000–2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde’s whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level. PMID:27320816

  10. Heat-killed and γ-irradiated Brucella strain RB51 stimulates enhanced dendritic cell activation, but not function compared with the virulent smooth strain 2308.

    Science.gov (United States)

    Surendran, Naveen; Hiltbold, Elizabeth M; Heid, Bettina; Sriranganathan, Nammalwar; Boyle, Stephen M; Zimmerman, Kurt L; Witonsky, Sharon G

    2010-11-01

    Brucella spp. are Gram-negative, facultative intracellular bacterial pathogens that cause abortion in livestock and undulant fever in humans worldwide. Brucella abortus strain 2308 is a pathogenic strain that affects cattle and humans. Currently, there are no efficacious human vaccines available. However, B. abortus strain RB51, which is approved by the USDA, is a live-attenuated rough vaccine against bovine brucellosis. Live strain RB51 induces protection via CD4(+) and CD8(+) T-cell-mediated immunity. To generate an optimal T-cell response, strong innate immune responses by dendritic cells (DCs) are crucial. Because of safety concerns, the use of live vaccine strain RB51 in humans is limited. Therefore, in this study, we analyzed the differential ability of the same doses of live, heat-killed (HK) and γ-irradiated (IR) strain RB51 in inducing DC activation and function. Smooth strain 2308, live strain RB51 and lipopolysaccharide were used as controls. Studies using mouse bone marrow-derived DCs revealed that, irrespective of viability, strain RB51 induced greater DC activation than smooth strain 2308. Live strain RB51 induced significantly (P≤0.05) higher DC maturation than HK and IR strains, and only live strain RB51-infected DCs (at multiplicity of infection 1:100) induced significant (P≤0.05) tumor necrosis factor-α and interleukin-12 secretion. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  11. Brucella abortus RB51 and hot saline extract from Brucella ovis as antigens in a complement fixation test used To detect sheep vaccinated with Brucella abortus RB51.

    Science.gov (United States)

    Adone, R; Ciuchini, F

    2001-01-01

    The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 x 10(9) CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect B. ovis-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with B. ovis also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type Brucella standard antigens.

  12. Genotyping of Brucella species using clade specific SNPs

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    Foster Jeffrey T

    2012-06-01

    Full Text Available Abstract Background Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus Brucella. The genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. We utilized two different genotyping strategies to characterize isolates. First, we developed a microarray-based assay based on 1000 single nucleotide polymorphisms (SNPs that were identified from whole genome comparisons of two B. abortus isolates , one B. melitensis, and one B. suis. We then genotyped a diverse collection of 85 Brucella strains at these SNP loci and generated a phylogenetic tree of relationships. Second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted 17 high value SNPs across 8 major branches of the phylogeny and determined their genotypes in a large collection ( n = 340 of diverse isolates. Results Our 1000 SNP microarray readily distinguished B. abortus, B. melitensis, and B. suis, differentiating B. melitensis and B. suis into two clades each. Brucella abortus was divided into four major clades. Our capillary-based SNP genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. Isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized. Conclusions We have identified clade-specific SNPs in Brucella that can be used for rapid assignment into major groups below the species level in the three main Brucella species. Our assays represent SNP genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates.

  13. [Characterization of Pantoea agglomerans lipopolysaccharides].

    Science.gov (United States)

    Varbanets, L D; Brovarskaya, O S; Bulygina, T N; Garkavaya, E G; Zhitkevich, N V

    2014-01-01

    Lipopolysaccharides (LPS) from seven Pantoea agglomerans strains isolated from various plants were purified and chemically identified. LPS of the studied P. agglomerans strains were heterogeneous in monosaccharide composition. Thus, the LPS of P. agglomerans 8606 differed considerably from the LPSs of other strains, containing mannose as the predominant monosaccharide (69.8%), as well as ribose (15.1%) and xylose (12.6%), while the content of rhamnose, one of the predominant monosaccharides in other LPS samples, was 2.5%. Analysis of the fatty acid composition revealed the presence of C12-C16 acids. In lipids A of all the studied strains, 3-OH-C14:0 was the predominant acid (31.7 to 39.1%, depending on the strain). C12:0 (8.2 to 31.5%), C14:0 (12.9 to 30.8%), and C16:0 acids (3.4 to 16.9%) were also revealed. The studied P. agglomerans strains fell into three groups according to their fatty acid composition. The differences stemmed from the presence or absence of two fatty acids, 2-OH-C14:0 and C16:1. Ouchterlony double immunodiffusion in agar revealed that all the LPS under study exhibited antigenic activity in homologous systems. The results of serological cross reactions indicated immunochemical heterogeneity of the species P. agglomerans. Comparative investigation of the complex of parameters of peripheral blood cells from a healthy donor before and after treatment with LPS solutions showed that the values of no parameters exceeded the normal range.

  14. An influenza viral vector Brucella abortus vaccine induces good cross-protection against Brucella melitensis infection in pregnant heifers.

    Science.gov (United States)

    Tabynov, Kaissar; Ryskeldinova, Sholpan; Sansyzbay, Abylai

    2015-07-17

    Brucella melitensis can be transmitted and cause disease in cattle herds as a result of inadequate management of mixed livestock farms. Ideally, vaccines against Brucella abortus for cattle should also provide cross-protection against B. melitensis. Previously we created a novel influenza viral vector B. abortus (Flu-BA) vaccine expressing the Brucella ribosomal proteins L7/L12 or Omp16. This study demonstrated Flu-BA vaccine with adjuvant Montanide Gel01 provided 100% protection against abortion in vaccinated pregnant heifers and good cross-protection of the heifers and their calves or fetuses (90-100%) after challenge with B. melitensis 16M; the level of protection provided by Flu-BA was comparable to the commercial vaccine B. abortus S19. In terms of the index of infection and colonization of Brucella in tissues, both vaccines demonstrated significant (P=0.02 to P<0.0001) protection against B. melitensis 16M infection compared to the negative control group (PBS+Montanide Gel01). Thus, we conclude the Flu-BA vaccine provides cross-protection against B. melitensis infection in pregnant heifers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide

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    Neha eDabral

    2015-06-01

    Full Text Available Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s containing mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

  16. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide.

    Science.gov (United States)

    Dabral, Neha; Jain-Gupta, Neeta; Seleem, Mohamed N; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2015-01-01

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

  17. Brucella ceti Infection in a Common Minke Whale ( Balaenoptera acutorostrata ) with Associated Pathology.

    Science.gov (United States)

    Davison, Nicholas J; Perrett, Lorraine L; Dawson, Claire; Dagleish, Mark P; Haskins, Gary; Muchowski, Jakub; Whatmore, Adrian M

    2017-07-01

    There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales ( Balaenoptera acutorostrata ) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

  18. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples.

    Science.gov (United States)

    Mirnejad, Reza; Mohamadi, Mozafar; Piranfar, Vahbeh; Mortazavi, Seied Mojtaba; Kachuei, Reza

    2013-06-01

    To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  19. Observation of Serum Bactericidal Activity of Brucella abortus RB51 OMPs Combined with Brucella abortus RB51 Live Vaccine

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    Fahime Gholizadeh

    2013-06-01

    Full Text Available Background & objectives: vaccination is vital against brucellosis. Although current vaccines have low efficiency, some cell wall compartments such as Outer Membrane Proteins could be used as an immunogenic candidate in vaccine development. By this mean, our aim in this study was to evaluate the humoral immunity of the combination of Brucella abortus RB51 OMPs with the Brucella abortus RB51 live attenuated vaccine, by Serum Bactericidal Acitivity test. Materials and Methods: In this project, first Brucella abortus RB51 was cultivated in brucella agar. The OMPs were extracted by Sodium N-Lauryl Sarcosinate method, then added to the RB51 live attenuated vaccine. Immunization was done by injection of the vaccine to mice and rabbits. The blood was drawn on days 0, 15,30, and 45 from the rabbits and the sera were seperated. Brucella abortus 544 was also injected as challenge. Spleen colony count was also performed. Results: The data from Serum Bactericidal Assay has showed, there was a very high Humoral immunity and response as a bactericidal titre of the serum against Rb51 Live vaccine. There was a significant decrease of colonies in the group vaccinated with the combined vaccine in the Spleen colony count test. Statistical analysis of groups variances showed a significant difference between groups (P<0.05.Conclusions: The Serum Bactericidal Assay results showed despite previous studies, both the combine and live vaccine are capable to stimulate the Humoral immunity. greater activity of combined vaccine to boost the humoral activity might be due to the synergistic effect of this vaccine.

  20. Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis.

    Science.gov (United States)

    Kim, Ji-Yeon; Sung, So-Ra; Lee, Kichan; Lee, Hyang-Keun; Kang, Sung-Il; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Her, Moon

    2014-08-15

    The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3-5.6, 4-7 and 6-11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera - B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. A rare case of anterior mediastinal mass caused by Brucella infection.

    Science.gov (United States)

    Sabzi, Feridoun; Faraji, Reza

    2017-03-01

    A previously healthy man, who had undergone coronary artery bypass 10 years earlier and had been diagnosed with brucellosis due to Brucella septicemia after Brucella arthritis, presented with chest pain and high fever. Anti- Brucella antibiotics were started, but after 4 weeks, his high fever remained. An infected mass was confirmed by computed tomography, and surgical intervention was performed via a median sternotomy. A large amount of thick pus gushed from an abscess in the upper mediastinum. The abscess cavity had a thick granulation wall, and cultured pus was positive for Brucella only. The patient responded well to antibiotic therapy.

  2. Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice.

    Science.gov (United States)

    Vemulapalli, Ramesh; Contreras, Andrea; Sanakkayala, Neelima; Sriranganathan, Nammalwar; Boyle, Stephen M; Schurig, Gerhardt G

    2004-09-08

    Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.

  3. MLVA and MLST typing of Brucella from Qinghai, China.

    Science.gov (United States)

    Ma, Jun-Ying; Wang, Hu; Zhang, Xue-Fei; Xu, Li-Qing; Hu, Gui-Ying; Jiang, Hai; Zhao, Fang; Zhao, Hong-Yan; Piao, Dong-Ri; Qin, Yu-Min; Cui, Bu-Yun; Lin, Gong-Hua

    2016-04-13

    The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.

  4. Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

    Science.gov (United States)

    Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

    2009-07-01

    The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

  5. BRUCELLA ENDOCARDITIS IN IRANIAN PATIENTS: COMBINED MEDICAL AND SURGICAL TREATMENT

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    Ebrahim Nematipour

    1995-06-01

    Full Text Available Brucella endocarditis is a Tare but serious complication ofbrucellosis and is the main cause of death reuuedto thisdisease: Itis not rare in the endemic areas and aaualiy accounts for up to 8~lO% ofendocarditis infections: We report seven adult cases of brucella endocarditis in lmam-Khorneini Hospual: Contrary to previous independent reports, female patients were not rare in this study and accountedfor three out ofseven. Four patients were cared for by combined medical and surgical treatment and were recovered Three of the patients that did not receive the combined theraPl could not he saved This report confirms the necessity of prompt combined medical and surgical treatment ofbrucella endocarditis.

  6. Brucella abortus Cell Cycle and Infection Are Coordinated.

    Science.gov (United States)

    De Bolle, Xavier; Crosson, Sean; Matroule, Jean-Yves; Letesson, Jean-Jacques

    2015-12-01

    Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

    Science.gov (United States)

    Martínez, Diana; Thompson, Carolina; Draghi, Graciela; Canavesio, Vilma; Jacobo, Roberto; Zimmer, Patricia; Elena, Sebastián; Nicola, Ana M; de Echaide, Susana Torioni

    2014-09-17

    An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Garcinia kola extract reduced lipopolysaccharide activation of ...

    African Journals Online (AJOL)

    SERVER

    2008-03-18

    Mar 18, 2008 ... Key words: Garcinia kola, lipopolysaccharide, phorbol myristate acetate, macrophage, cytokines. INTRODUCTION. The seeds of Garcinia kola heckel commonly called bitter kola in Nigeria ... Ebola virus in its tract in laboratory tests and it has been ... medicine to successfully treat the virus that causes Ebola.

  9. Attenuation of Neuroinflammatory Responses in Lipopolysaccharide ...

    African Journals Online (AJOL)

    HP

    neuroinflammatory responses induced by lipopolysaccharide (LPS) in BV-2 microglial cells and its antioxidant effects. Methods: Biochemical studies ... [2] and as an anti-aging agent in cosmeceuticals [4]. However, its pharmacological actions on ... in LPS-stimulated BV-2 microglia and explored the possible mechanism.

  10. Inhibition of Lipopolysaccharide-Stimulated Neuro- Inflammatory ...

    African Journals Online (AJOL)

    Purpose: To investigate the in vitro antioxidant and anti-neuroinflammatory effects of Tetragonia tetragonoides (Pall.) Kuntze extract (TKE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Methods: To evaluate the effects of TKE, LPS-stimulated BV microglia were used and the expression and production of ...

  11. Carum carvi Linn (Umbelliferae) Attenuates Lipopolysaccharide ...

    African Journals Online (AJOL)

    Lipopolysaccharide (LPS) was used to activate BV-2 microglia. Nitric oxide (NO) levels were measured using Griess assay. Inducible NO synthase (iNOS) and cyclooxygenase (COX) levels were evaluated by Western blot analysis. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production were evaluated by ...

  12. Garcinia kola extract reduced lipopolysaccharide activation of ...

    African Journals Online (AJOL)

    The effect of Garcinia kola heckel seed extract on the promonocytic cell line U937 activated by lipopolysaccharide (LPS) was investigated. 200 l of U937 cells maintained in culture at 5 x 105 cells per ml was delivered into wells of a culture plate according to groups. Cells were pre-treated with 20 l of 100 ng/ml phorbol ...

  13. Seroprevalence of Brucella antibodies in camels in Katsina State, Nigeria.

    Science.gov (United States)

    Salisu, U S; Kudi, C A; Bale, J O O; Babashani, M; Kaltungo, B Y; Saidu, S N A; Asambe, A; Baba, A Y

    2017-06-01

    A cross-sectional study was carried out to determine the status of Brucella infection in one-humped (Dromedary) camels in the North and Central senatorial districts of Katsina State, Nigeria. Nine hundred and eighty serum samples from live and slaughtered camels were tested. Modified Rose Bengal plate test (RBPT) and serum agglutination test (SAT) with ethylenediaminetetraacetic acid, (EDTA) were used as screening and standard tests, respectively. The prevalence of Brucella antibodies were 110 (11.2%) and 103 (10.5%) for RBPT and SAT, respectively. Of the 472 and 508 serum samples tested from the herds and abattoirs, respectively, 63 (13.3%) and 47 (9.3%) were positive by RBPT while 62 (13.1%) and 41 (8.1%) were positive by SAT, respectively. Based on the results, it was concluded that Brucella antibodies were present in camels in the study area. Poor management practices and mixing of camels with other species of livestock as well as unrestricted movement of camels were proposed to be the reasons for the prevalence of the disease in the study area. In view of the public health importance of the disease, it is recommended that there is the need to develop a strategic plan to decrease spread of brucellosis in the study area.

  14. Validation of the multiplex PCR for identification of Brucella spp.

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    Lívia de Lima Orzil

    2016-05-01

    Full Text Available ABSTRACT: A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing biochemical tests, which greatly reduces the time for a final diagnosis, and provides more security for the analyst by reducing the time of exposure to microorganisms. The validation was performed in accordance with the Manual of Diagnostic Tests from OIE (2008 and following the requirements present in the ABNT NBR ISO/IEC 17025:2005. The mPCR validated in this study identified the different species of Brucella ( Brucella abortus , B. suis , B. ovis e B. melitensis of bacterial suspension obtained from the slaughterhouse samples, as well as distinguished the biovars (1, 2 e 4; 3b, 5, 6 e 9 of B. abortus in grouped form and differentiated the field strains from vaccine strains, as a quick, useful and less expensive technique in diagnosis of brucellosis in Brazil.

  15. In Vivo Differences in the Virulence, Pathogenicity, and Induced Protective Immunity of wboA Mutants from Genetically Different Parent Brucella spp.

    Science.gov (United States)

    Wang, Zhen; Niu, Jianrui; Wang, Shuangshan

    2013-01-01

    To explore the effects of the genetic background on the characteristics of wboA gene deletion rough mutants generated from different parent Brucella sp. strains, we constructed the rough-mutant strains Brucella melitensis 16 M-MB6, B. abortus 2308-SB6, B. abortus S19-RB6, and B. melitensis NI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks for B. abortus S19-RB6 to 11 weeks for B. abortus 2308-SB6 and B. melitensis NI-NB6. However, B. abortus S19-RB6 and B. melitensis 16 M-MB6, with a shorter survival time in mice, offered better protection against challenges with B. abortus 2308 in protection tests than B. abortus 2308-SB6 and B. melitensis NI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival time in vivo. In the cross-protection assay, both B. melitensis 16 M-MB6 and B. abortus S19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated with B. abortus S19-RB6 and B. melitensis 16 M-MB6, B. abortus S19-RB6 did not induce abortion, whereas B. melitensis 16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunity in vivo in the wboA deletion mutants from genetically different parent Brucella spp. and also indicated that future rough vaccine strain development could be promising if suitable parent Brucella strains and/or genes were selected. PMID:23239800

  16. Complement Fixation Test To Assess Humoral Immunity in Cattle and Sheep Vaccinated with Brucella abortus RB51

    Science.gov (United States)

    Adone, Rosanna; Ciuchini, Franco

    1999-01-01

    The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B. abortus 2308. The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS. The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in humans exposed to this strain. The purpose of this study was to evaluate the suitability of a complement fixation (CF) test performed with the rough strain B. abortus RB51, previously deprived of anticomplementary activity, in detecting anti-B. abortus RB51 antibodies in cattle and sheep experimentally vaccinated with this strain. The results of this study showed that a CF test with RB51 as the antigen is able to specifically detect antibodies following RB51 vaccination in cattle and sheep. In addition, this method could be a useful tool for detecting B. abortus RB51 infection in humans. PMID:10548564

  17. Development and validation of an indirect enzyme immunoassay for detection of antibody to Brucella abortus in milk.

    Science.gov (United States)

    Nielsen, K; Smith, P; Gall, D; Perez, B; Cosma, C; Mueller, P; Trottier, J; Cote, G; Boag, L; Bosse, J

    1996-09-01

    An indirect enzyme immunoassay for detection of antibody to Brucella abortus in bovine milk was developed and validated using 6238 milk samples from Canadian herds (brucellosis free) and 202 samples from herds infected with B. abortus (from Argentina and Chile). The assay utilized lipopolysaccharide as the antigen, immobilized on the polystyrene matrix, whole milk to test and a mouse monoclonal antibody, specific for an epitope of bovine IgG1, conjugated with horseradish peroxidase. The sensitivity of the assay was 95.2% +/- 3.7% at a confidence limit of 95% for samples from B. abortus infected herds obtained from chile and 98.7% +/- 0.3% at a confidence limit of 95% for samples from similar herds in Argentina. Of the negative milk samples tested, 77 gave a result above the threshold value of 0.200 optical density units. When the 77 false positive samples were retested using 7.5 mM (final concentration) of EDTA and ethyleneglycol-bis-aminoether-N,N,N', N'-tetraacetic acid (EGTA), the number of false positive reactions was reduced to 3, giving a diagnostic specificity of 99.95%. The divalent cation chelating agents did not affect positive reactions and the sensitivity remained the same. Based on control samples included with each assay, the performance of the assay was consistent.

  18. Summary of field trials using the direct and competitive enzyme immunoassays for detection of antibody to brucella abortus

    International Nuclear Information System (INIS)

    Nielsen, K.; Gall, D.

    1998-01-01

    Two indirect and two competitive enzyme immunoassays for detection of antibody to Brucella abortus, validated elsewhere, were field tested in five different Latin American laboratories. Testing was performed according to standardised protocols using sera obtained in each area. Sera from B. abortus infected herds, from vaccinated (but serologically negative in a screening test) and non-vaccinated cattle were tested in each assay and compared to the results obtained with conventional diagnostic tests used for diagnosis of brucellosis in each country. Relative sensitivity and specificity values were calculated for each country as well as a weighted summary combining the data from all the participating laboratories. The result demonstrate that all ELISAs performed as well as, or better than, the conventional aerological tests. Given the inherent errors in the use of the latter in the diagnosis of brucellosis, it is recommended that the ELISAs described here be considered as replacements for the conventional tests. The CELISA using the lipopolysaccharide antigen with the competing monoclonal antibody M84, should be considered as the most useful because of cross-species and vaccination considerations. (author)

  19. A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Brucella abortus Mediates Host-Pathogen Interactions.

    Science.gov (United States)

    Sheehan, Lauren M; Caswell, Clayton C

    2017-06-06

    In Brucella abortus , two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria , the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. IMPORTANCE Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the Alphaproteobacteria and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In Brucella abortus , AbcR1 and AbcR2 are functionally redundant, as only a double abcR1 abcR2 ( abcR1 / 2 ) deletion results in attenuation in

  20. Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9

    OpenAIRE

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

    2003-01-01

    Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

  1. Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus.

    Science.gov (United States)

    Caudill, Mitchell T; Budnick, James A; Sheehan, Lauren M; Lehman, Christian R; Purwantini, Endang; Mukhopadhyay, Biswarup; Caswell, Clayton C

    2017-07-01

    Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ΔputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.

  2. Serological Survey of Porcine Brucella Infection in SouthEast, Nigeria

    African Journals Online (AJOL)

    Porcine brucellosis, also called contagious abortion of swine is an infectious and zoonotic disease of swine caused by bacteria of the genus Brucella (Young, 1995). Brucella suis is the species found primarily in pigs. It is a zoonotic infection of domesticated and wild animals which humans (especially occupationally ...

  3. Serological response of cattle to Brucella allergen after repeated intradermal applications of this allergen

    NARCIS (Netherlands)

    Muskens, J.A.M.; Bercovich, Z.; Damen, C.P.R.M.

    1996-01-01

    A study was conducted to determine whether an allergen that has been prepared from a mucoid strain of Brucella abortus triggers a serum antibody response that interferes with the interpretation of serologic tests results. Fifteen cattle seronegative for Brucella antigen were tested with the SDTH

  4. Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas, USA

    Science.gov (United States)

    Various tissues, nasal swabs, urine, and blood samples were collected from 376 feral swine at two federally-inspected abattoirs in Texas during six separate sampling periods in 2015. Samples were tested for Brucella spp. by culture and serology. Brucella spp. were cultured from 13.0% of feral swin...

  5. Molecular Survey on Brucellosis in Rodents and Shrews - Natural Reservoirs of Novel Brucella Species in Germany?

    Science.gov (United States)

    Hammerl, J A; Ulrich, R G; Imholt, C; Scholz, H C; Jacob, J; Kratzmann, N; Nöckler, K; Al Dahouk, S

    2017-04-01

    Brucellosis is a widespread zoonotic disease introduced from animal reservoirs to humans. In Germany, bovine and ovine/caprine brucellosis were eradicated more than a decade ago and mandatory measures in livestock have been implemented to keep the officially brucellosis-free status. In contrast, surveillance of wildlife is still challenging, and reliable data on the prevalence of brucellae in small mammal populations do not exist. To assess the epidemiology of Brucella spp. in rodents and shrews, a molecular survey was carried out. A total of 537 rodents and shrews were trapped in four federal states located throughout Germany and investigated for the presence of Brucella. Using a two-step molecular assay based on the detection of the Brucella-specific bcsp31 and IS711 sequences in tissue samples, 14.2% (n = 76) of the tested animals were positive. These originated mainly from western and south-western Germany, where preliminary analyses indicate population density-dependent Brucella prevalence in voles (Myodes glareolus) and mice (Apodemus spp.). recA typing revealed a close relationship to a potentially novel Brucella species recently isolated from red foxes (Vulpes vulpes) in Austria. The molecular detection of brucellae in various rodent taxa and for the first time in shrew species shows that these animals may be naturally infected or at least have a history of exposure to Brucella spp. © 2015 Blackwell Verlag GmbH.

  6. Comparison of a New and Rapid Method: Brucella Coombs Gel Test With Other Diagnostic Tests.

    Science.gov (United States)

    Kalem, Fatma; Ergün, Ayşe Gül; Durmaz, Süleyman; Doğan, Metin; Ertuğrul, Ömür; Gündem, Seval

    2016-09-01

    The aim of this study was to detect reliability of Brucella Coombs gel test (BCGT) by comparing with with ELISA (IgG + IgM), Standard agglutination test, and Brucella immunocapture agglutination methods in serological diagnosis of brucellosis. Brucella Coombs gel test (BCGT), Brucella ELISA (IgG + IgM), Standard agglutination test, and Brucella immunocapture agglutination tests of 78 patients with presumptive diagnosis of brucellosis which were sent to Microbiology Laboratory of Konya Numune Hospital from various regions of Konya were studied. Of 78 patients with ELISA IgG and IgM, STA, BICA and BCGT; 26, 21, 10, 12 and 12 were positive. When compared with BICA, the sensitivity and specifity of BCGT were 100% and 100%, respectively. According to results BCGT can be used as a diagnostic test in routine laboratories after more comprehensive studies in control groups and patients. © 2016 Wiley Periodicals, Inc.

  7. MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain S2 from cattle in China.

    Directory of Open Access Journals (Sweden)

    Hai Jiang

    Full Text Available In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3 is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA. Among the B. melitensis isolates, the majority (74/82 belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal. The majority of B. abortus isolates (51/70 were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

  8. Identification of Secondary Mutations Which Enhance and Stabilize the Attenuation of Brucella HTRA Mutants: Improving Brucella HTRA-Based Strains as Vaccine

    National Research Council Canada - National Science Library

    Roop, R

    1999-01-01

    .... Studies completed to date under this contract indicate that although the Brucella HtrA contributes to resistance to oxidative killing in vitro and resistance to killing by cultured murine neutrophils...

  9. Lipopolysaccharide (LPS)-Induced Autophagy Is Responsible for Enhanced Osteoclastogenesis.

    Science.gov (United States)

    Sul, Ok-Joo; Park, Hyun-Jung; Son, Ho-Jung; Choi, Hye-Seon

    2017-11-30

    We hypothesized that inflammation affects number and activity of osteoclasts (OCs) via enhancing autophagy. Lipopolysaccharide (LPS) induced autophagy, osteoclastogenesis, and cytoplasmic reactive oxygen species (ROS) in bone marrow-derived macrophages that were pre-stimulated with receptor activator of nuclear factor-κB ligand. An autophagy inhibitor, 3-methyladenine (3-MA) decreased LPS-induced OC formation and bone resorption, indicating that autophagy is responsible for increasing number and activity of OCs upon LPS stimulus. Knockdown of autophagy-related protein 7 attenuated the effect of LPS on OC-specific genes, supporting a role of LPS as an autophagy inducer in OC. Removal of ROS decreased LPS-induced OC formation as well as autophagy. However, 3-MA did not affect LPS-induced ROS levels, suggesting that ROS act upstream of phosphatidylinositol-4,5-bisphosphate 3-kinase in LPS-induced autophagy. Our results suggest the possible use of autophagy inhibitors targeting OCs to reduce inflammatory bone loss.

  10. Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

    Science.gov (United States)

    Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

    2017-04-01

    The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

  11. Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Raziuddin, S.; Siegelman, H.W.; Tornabene, T.G.

    1983-01-01

    Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assay than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi. 37 references, 1 figure, 3 tables.

  12. Brucella Melitensis 16M Regulates the Effect of AIR Domain on Inflammatory Factors, Autophagy, and Apoptosis in Mouse Macrophage through the ROS Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Tiansen Li

    Full Text Available Brucellosis is a highly contagious zoonosis caused by Brucella. Brucella can invade and persist inside host cells, which results in chronic infection. We constructed AIR interference and overexpression lentiviruses to acquire AIR interference, overexpression, and rescue stable expression cell lines. We also established a Brucella melitensis 16M-infected macrophage model, which was treated with either the vehicle control or NAC (ROS scavenger N-acetylcysteine (NAC for 0, 3, 6, 12, and 24 h. Confocal laser microscopy, transmission electron microscopy, fluorescence quantitative PCR, flow cytometry, ELISA, and Western blot were used to detect inflammation, cell autophagy and apoptosis-related protein expression levels, ROS levels, and the distribution of mitochondria. It was found that after interference and overexpression of AIR, ROS release was significantly changed, and mitochondria became abnormally aggregated. B. melitensis 16M activated the NLRP3/AIM2 inflammatory complex, and induced RAW264.7 cells to secrete IL-1β and IL-18 through the ROS pathway. B. melitensis 16M also altered autophagy-related gene expression, increased autophagy activity, and induced cell apoptosis through the ROS pathway. The results showed that after B. melitensis 16M infection, ROS induced apoptosis, inflammation, and autophagy while AIR inhibited autophagosome maturation and autophagy initiation. Autophagy negatively regulated the activation of inflammasomes and prevented inflammation from occurring. In addition, mitophagy could promote cell apoptosis.

  13. A DNA Vaccine Encoding Cu,Zn Superoxide Dismutase of Brucella abortus Induces Protective Immunity in BALB/c Mice

    Science.gov (United States)

    Oñate, Angel A.; Céspedes, Sandra; Cabrera, Alex; Rivers, Rodolfo; González, Andrés; Muñoz, Carola; Folch, Hugo; Andrews, Edilia

    2003-01-01

    This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis. PMID:12933826

  14. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

    Science.gov (United States)

    Kim, Hye-Sook; Willett, Jonathan W; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-11-01

    In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology. © 2014 John Wiley & Sons Ltd.

  15. Radiology changes in brucella spondylitis, experience with 26 cases

    International Nuclear Information System (INIS)

    Abbassioun, K.; Amirjamshidi, A.; Taheri, B.

    2003-01-01

    Background/objective: brucellosis is an endemic zoonosis in the Middle East and despite all public health efforts it has not yet been eradicated in Iran. We aimed to highlight and categorize the imaging features of Brucella spondylitis. Material and method: twenty six cases of Brucella spondylitis were treated by the authors from 1982 up to 2003. The available imaging studies of all the cases are reviewed and include X-ray films, conventional myelography, computerized tomographic (CT Scan) and magnetic resonance imaging. Results: there were 21 male and 5 female patients with an age range of 5 to 62 years and the majority (60 %) in the 4 Th and 5 Th decades of life. Wright hemagglutination tests were positive in all cases. Plain x-ray films typically showed lysis of the end plates with osteophyte formation involving affected vertebrae, followed by narrowing of the inter spaces and destruction or collapse of the vertebral bodies in 7 cases. Myelography demonstrated various types of epidural filling defects and obstruction to the flow of contrast material in 10 cases. CT scan, available in 3 cases, showed erosion and cauliflower-like proliferation at the bony edges of the vertebral bodies and end plates. MRI findings varied depending upon the acute or chronic stages of the illness with hypo- or hyper-intense changes on T1 sequences, and primarily hyper-intense changes of T2 sequences in 8 cases. Conclusion: The findings in this series of patients suggest that imaging findings of Brucella spondylitis are scarcely specific. However when considering the medical history, place of origin of the patients, clinical presentation and laboratory findings, the early diagnosis of the illness may be possible before proceeding to surgical intervention

  16. First isolation and characterization of Brucella microti from wild boar.

    Science.gov (United States)

    Rónai, Zsuzsanna; Kreizinger, Zsuzsa; Dán, Ádám; Drees, Kevin; Foster, Jeffrey T; Bányai, Krisztián; Marton, Szilvia; Szeredi, Levente; Jánosi, Szilárd; Gyuranecz, Miklós

    2015-07-11

    Brucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary. The B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 μg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile. Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity

  17. Brucella Endocarditis as a Late Onset Complication of Brucellosis

    Directory of Open Access Journals (Sweden)

    Panagiotis Andriopoulos

    2015-01-01

    Full Text Available Brucella endocarditis (BE is a rare but life threatening complication of brucellosis. We present a case report of a patient with relapsing brucellosis complicated with aortic valve endocarditis. The patient underwent valve replacement and required prolonged antibiotic treatment because of rupture of the noncoronary leaflet and development of congestive heart failure. Since the onset of endocarditis in patients with brucellosis is not known, proper follow-up is required in order to identify any late onset complications, especially in endemic areas.

  18. Brucella antibody seroprevalence in Antarctic seals (Arctocephalus gazella, Leptonychotes weddellii and Mirounga leonina).

    Science.gov (United States)

    Jensen, Silje-Kristin; Nymo, Ingebjørg Helena; Forcada, Jaume; Hall, Ailsa; Godfroid, Jacques

    2013-09-03

    Brucellosis is a worldwide infectious zoonotic disease caused by Gram-negative bacteria of the genus Brucella, and Brucella infections in marine mammals were first reported in 1994. A serosurvey investigating the presence of anti-Brucella antibodies in 3 Antarctic pinniped species was undertaken with a protein A/G indirect enzyme-linked immunosorbent assay (iELISA) and the Rose Bengal test (RBT). Serum samples from 33 Weddell seals Leptonychotes weddelli were analysed, and antibodies were detected in 8 individuals (24.2%) with the iELISA and in 21 (65.6%) with the RBT. We tested 48 southern elephant seal Mirounga leonina sera and detected antibodies in 2 animals (4.7%) with both the iELISA and the RBT. None of the 21 Antarctic fur seals Arctocephalus gazella was found positive. This is the first report of anti-Brucella antibodies in southern elephant seals. The potential impact of Brucella infection in pinnipeds in Antarctica is not known, but Brucella spp. are known to cause abortion in terrestrial species and cetaceans. Our findings suggest that Brucella infection in pinnipeds is present in the Antarctic, but to date B. pinnipedialis has not been isolated from any Antarctic pinniped species, leaving the confirmation of infection pending.

  19. Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

    Science.gov (United States)

    Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

    2015-06-01

    Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Brucella placentitis and seroprevalence in northern fur seals ( Callorhinus ursinus) of the Pribilof Islands, Alaska.

    Science.gov (United States)

    Duncan, Colleen G; Tiller, Rebekah; Mathis, Demetrius; Stoddard, Robyn; Kersh, Gilbert J; Dickerson, Bobette; Gelatt, Tom

    2014-07-01

    Brucella species infect a wide range of hosts with a broad spectrum of clinical manifestations. In mammals, one of the most significant consequences of Brucella infection is reproductive failure. There is evidence of Brucella exposure in many species of marine mammals, but the outcome of infection is often challenging to determine. The eastern Pacific stock of northern fur seals (NFSs, Callorhinus ursinus) has declined significantly, spawning research into potential causes for this trend, including investigation into reproductive health. The objective of the current study was to determine if NFSs on St. Paul Island, Alaska have evidence of Brucella exposure or infection. Archived DNA extracted from placentas ( n = 119) and serum ( n = 40) samples were available for testing by insertion sequence (IS) 711 polymerase chain reaction (PCR) and the Brucella microagglutination test (BMAT), respectively. As well, placental tissue was available for histologic examination. Six (5%) placentas were positive by PCR, and a single animal had severe placentitis. Multilocus variable number tandem repeat analysis profiles were highly clustered and closely related to other Brucella pinnipedialis isolates. A single animal was positive on BMAT, and 12 animals had titers within the borderline range; 1 borderline animal was positive by PCR on serum. The findings suggest that NFSs on the Pribilof Islands are exposed to Brucella and that the organism has the ability to cause severe placental disease. Given the population trend of the NFS, and the zoonotic nature of this pathogen, further investigation into the epidemiology of this disease is recommended.

  1. Detection and differentiation of the six Brucella species by polymerase chain reaction.

    Science.gov (United States)

    Sifuentes-Rincón, A M; Revol, A; Barrera-Saldaña, H A

    1997-11-01

    Brucelosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Its current diagnosis is based on clinical observations that may be complemented by serology and microbiological culture tests; however, the former is limited in sensitivity and specificity, the latter is time consuming. To improve brucelosis diagnosis we developed a test which is specific and sensitive and is capable of differentiating the six species of Brucella. Four primers were designed from B. abortus sequences at the well-conserved Omp2 locus that are able to amplify the DNAs of all six species of Brucella. Our test detected all six species of Brucella. Their differentiation resulted directly from differences in the amplification patterns or was achieved indirectly using a RFLP present in one of the PCR products. The sensitivity and specificity of the new test were then determined; it was applied successfully in confirming the diagnosis of a patient whose clinical history and serology indicated infection with Brucella. The results make possible the use of a PCR test for Brucella detection and differentiation without relying on the measurement of the antibodies or microorganism culture. Our first results showed that the PCR test can confirm the presence of Brucella in blood samples of infected patients.

  2. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    Science.gov (United States)

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  3. The role of 'atypical' Brucella in amphibians: are we facing novel emerging pathogens?

    Science.gov (United States)

    Mühldorfer, K; Wibbelt, G; Szentiks, C A; Fischer, D; Scholz, H C; Zschöck, M; Eisenberg, T

    2017-01-01

    To discuss together the novel cases of Brucella infections in frogs with the results of published reports to extend our current knowledge on 'atypical' brucellae isolated from amphibians and to discuss the challenges we face on this extraordinary emerging group of pathogens. Since our first description, an additional 14 isolates from four different frog species were collected. Novel isolates and a subset of Brucella isolates previously cultured from African bullfrogs were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Fourier transform-infrared (FT-IR) spectroscopy and broth microdilution susceptibility testing. MALDI-TOF MS worked very efficiently for an accurate bacterial identification to the genus level. Within the cluster analysis, 'atypical' brucellae grouped distant from Brucella melitensis and were even more separated by FT-IR spectroscopy with respect to their geographical origin. Minimum inhibitory concentrations of 14 antimicrobial substances are provided as baseline data on antimicrobial susceptibility. The case history of Brucella infections in amphibians reveals a variety of pathologies ranging from localized manifestations to systemic infections. Some isolates seem to be capable of causing high mortality in zoological exhibitions putting higher demands on the management of endangered frog species. There is considerable risk in overlooking and misidentifying 'atypical' Brucella in routine diagnostics. Brucella have only recently been described in cold-blooded vertebrates. Their presence in frog species native to Africa, America and Australia indicates a more common occurrence in amphibians than previously thought. This study provides an extensive overview of amphibian brucellae by highlighting the main features of their clinical significance, diagnosis and zoonotic potential. © 2016 The Society for Applied Microbiology.

  4. Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model.

    Science.gov (United States)

    Nymo, Ingebjørg H; Arias, Maykel A; Pardo, Julián; Álvarez, María Pilar; Alcaraz, Ana; Godfroid, Jacques; Jiménez de Bagüés, María Pilar

    2016-01-01

    Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea.

  5. MLVA Genotyping of Brucella melitensis and Brucella abortus Isolates from Different Animal Species and Humans and Identification of Brucella suis Vaccine Strain S2 from Cattle in China

    Science.gov (United States)

    Xu, Liqing; Hu, Guiying; Ma, Junying; Xiao, Pei; Fan, Weixing; Di, Dongdong; Tian, Guozhong; Fan, Mengguang; Mi, Jingchuan; Yu, Ruiping; Song, Litao; Zhao, Hongyan; Piao, Dongri; Cui, Buyun

    2013-01-01

    In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the ‘East Mediterranean’ group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the ‘Americas’ group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs. PMID:24124546

  6. Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae.

    Directory of Open Access Journals (Sweden)

    Dina A Moustafa

    Full Text Available Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD, a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

  7. Brucella and Coxiella; if you don't look, you don't find.

    Science.gov (United States)

    Lambourne, Jonathan R; Brooks, Tim

    2015-02-01

    Brucella and Coxiella are similar; both are obligate intracellular, zoonotic pathogens with a broad geographic distribution. Infection in animals is usually asymptomatic, but causes fetal loss and therefore has significant economic impact. Human infection may be asymptomatic or give rise to either organ-specific or multi-system disease. Organism culture is challenging for Coxiella and can lack sensitivity for Brucella. Therefore, infection is most commonly diagnosed by serology, but this may be negative in early infection and serology results may be challenging to interpret. Both Brucella and Coxiella are typically susceptible to a wide range of antimicrobials, but long courses may be needed. © 2015 Royal College of Physicians.

  8. Brucella Causing Liver Abscess in a Child with Selective IgA Deficiency.

    Science.gov (United States)

    Mukherjee, Devdeep; Pal, Priyankar; Kundu, Ritabrata

    2017-07-15

    Brucella has been known to cause pyrexia of unknown origin. 9-year-old boy with fever and abdominal pain; multiple abscesses within the liver on ultrasonography. IgM Antibodies against Brucella were raised in his serum sample, and Brucella serum agglutination test was positive. Immunological work-up suggested selective IgA deficiency. Reduction in size following treatment with trimethoprim-sulphamethoxazole, amikacin and doxycycline. Brucellosis should be considered as an etiology of liver abscess in patients not responding to conventional antibiotics.

  9. Triad of infective endocarditis, splenic abscess, and septicemia caused by Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Shashank Purwar

    2017-01-01

    Full Text Available A 40-year-old farmer from the district of North Karnataka who had received treatment for high fever of 8 days duration was admitted with fever, dyspnea, and poor general condition. Ultrasonography and echocardiogram revealed multiple splenic abscesses, vegetation on atrioventricular valve, aortic regurgitation (Grade I–II, and mitral valve regurgitation (Grade II–III, respectively. Brucella melitensis was detected in blood culture, and high titers of IgM and IgG anti-Brucella antibodies were observed in Brucella specific serological tests. The patient developed fulminant septicemia and succumbed due to multi-organ failure.

  10. A safe and molecular-tagged Brucella canis ghosts confers protection against virulent challenge in mice.

    Science.gov (United States)

    Qian, Jing; Bu, Zhaoyang; Lang, Xulong; Yan, Guangmou; Yang, Yanling; Wang, Xiuran; Wang, Xinglong

    2017-05-01

    Canine brucellosis, caused by Brucella canis, is a persistent infectious reproductive disease in dogs. The absence of effective treatment to the intracellular pathogen and the irreversible consequence of infection makes the need of a specific vaccine urgent. Bacterial ghosts (BGs) are the empty envelopes of bacteria with no genome content inside, which emerge as a proper vaccine candidate due to its intact outer antigen. It is generally derived from a genetically engineered strain, through the expression of Bacteriophage phiX174 lysis E gene upon induction. In this study, we combined the homologous recombination (HR) and bacterial ghost technologies, generating a genetically stable B. canis ghost strain which bears no drug resistance gene. When the ghost strain grows to OD 600 of 0.6, 100% inactivation can be achieved under 42°C in 60h. The resultant BGs showed guaranteed safety and comparable immunogenicity to a live vaccine. The bacterial B0419 protein was depleted during HR process, which is subsequently proved to work as a molecular tag in distinguishing natural infection and BGs immunization through ELISA. Additionally, the BGs also conferred protection against B. canis RM6/66 and B. melitensis 16M. Therefore, the application of current BGs as a vaccine candidate and the corresponding serological diagnostic approach may provide better B. canis prevention strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Brucella ceti infection in dolphins from the Western Mediterranean sea.

    Science.gov (United States)

    Isidoro-Ayza, Marcos; Ruiz-Villalobos, Nazareth; Pérez, Lola; Guzmán-Verri, Caterina; Muñoz, Pilar M; Alegre, Fernando; Barberán, Montserrat; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; González-Barrientos, Rocio; Moreno, Edgardo; Blasco, José María; Domingo, Mariano

    2014-09-17

    Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis', subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea. We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses. All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.

  12. DMPD: Structural and functional analyses of bacterial lipopolysaccharides. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12106784 Structural and functional analyses of bacterial lipopolysaccharides. Carof...html) (.csml) Show Structural and functional analyses of bacterial lipopolysaccharides. PubmedID 12106784 Ti...tle Structural and functional analyses of bacterial lipopolysaccharides. Authors

  13. Immunoelectron microscopy of lipopolysaccharide in Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

    1989-01-01

    Monoclonal antibodies (MAb) specific for Chlamydia trachomatis lipopolysaccharide (LPS) and major outer membrane protein (MOMP) were used for immunoelectron microscopy analysis. MAb specific for MOMP showed strong reaction with the chlamydial surface, whereas MAb specific for LPS showed strong...... association of gold particles with the periphery of the chlamydial body. After fixation of the chlamydia cells, the reactivity was, however, similar to the anti-MOMP reactivity. Enzyme-linked immunosorbent assay showed that MAb specific for LPS could remove LPS from the chlamydial membrane....

  14. Biological reactivity of Moraxella bovis lipopolysaccharide.

    Science.gov (United States)

    Johansen, K A; Wannemuehler, M J; Rosenbusch, R F

    1990-01-01

    Lipopolysaccharide (LPS) was isolated from Moraxella bovis 118F and ATCC 10900, M ovis ATCC 33078, and M phenylpyruvica ATCC 23333 by hot phenol-water extraction. In silver-stained sodium dodecyl sulfate polyacrylamide electrophoresis gels, M bovis 118F LPS had a smooth profile, whereas the other Moraxella preparations appeared to be rough. The LPS preparations induced pyrogenicity and dermal Shwartzman reactions in rabbits, and induced production of tumor necrosis factor and interleukin-1 in vitro. Induction of tumor necrosis factor appeared to be among the most potent biological activities of M bovis LPS.

  15. Coxiella burnetii Lipopolysaccharide: What Do We Know?

    Directory of Open Access Journals (Sweden)

    Prasad Abnave

    2017-11-01

    Full Text Available A small gram-negative bacterium, Coxiella burnetii (C. burnetii, is responsible for a zoonosis called Q fever. C. burnetii is an intracellular bacterium that can survive inside microbicidal cells like monocytes and macrophages by hijacking several functions of the immune system. Among several virulence factors, the lipopolysaccharide (LPS of C. burnetii is one of the major factors involved in this immune hijacking because of its atypical composition and structure. Thus, the aim of this mini-review is to summarize the repressive effects of C. burnetii LPS on the antibacterial immunity of cells.

  16. Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models.

    Science.gov (United States)

    Kwon, Ae Jeong; Moon, Ja Young; Kim, Won Kyong; Kim, Suk; Hur, Jin

    2016-11-01

    Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 10 8 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 10 8 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 10 9 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B-D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.

  17. Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation.

    Science.gov (United States)

    Beare, Paul A; Jeffrey, Brendan M; Long, Carrie M; Martens, Craig M; Heinzen, Robert A

    2018-03-01

    Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

  18. Host-Brucella interactions and the Brucella genome as tools for subunit antigen discovery and immunization against brucellosis

    Science.gov (United States)

    Gomez, Gabriel; Adams, Leslie G.; Rice-Ficht, Allison; Ficht, Thomas A.

    2013-01-01

    Vaccination is the most important approach to counteract infectious diseases. Thus, the development of new and improved vaccines for existing, emerging, and re-emerging diseases is an area of great interest to the scientific community and general public. Traditional approaches to subunit antigen discovery and vaccine development lack consideration for the critical aspects of public safety and activation of relevant protective host immunity. The availability of genomic sequences for pathogenic Brucella spp. and their hosts have led to development of systems-wide analytical tools that have provided a better understanding of host and pathogen physiology while also beginning to unravel the intricacies at the host-pathogen interface. Advances in pathogen biology, host immunology, and host-agent interactions have the potential to serve as a platform for the design and implementation of better-targeted antigen discovery approaches. With emphasis on Brucella spp., we probe the biological aspects of host and pathogen that merit consideration in the targeted design of subunit antigen discovery and vaccine development. PMID:23720712

  19. BrucellaCapt versus classical tests in the serological diagnosis and management of human brucellosis.

    Science.gov (United States)

    Casanova, Aurora; Ariza, Javier; Rubio, Manuel; Masuet, Cristina; Díaz, Ramón

    2009-06-01

    The BrucellaCapt test is an immunocapture agglutination test suggested as a possible substitute for the Coombs test in the diagnosis of human brucellosis. Here it is compared with classical tests using 321 samples from 48 patients with brucellosis (6.9 +/- 1.7 samples per patient), including 20 patients with focal disease and 8 patients with a total of 9 relapse episodes (mean follow-up, 18 months). The BrucellaCapt test was used according to the manufacturer's instructions, and we also used a variant of the BrucellaCapt test in which the microtiter plates were not coated with antibodies against total human immunoglobulin (BCAPV). The correlation between the BrucellaCapt and BCAPV tests was 0.982 (P < 0.001), with 260 coincident pairs of titers (81%). The areas under the receiver operating characteristic curve for the BrucellaCapt and BCAPV tests with respect to the Coombs test were 0.969 and 0.960, respectively. Upon admission, the BrucellaCapt, BCAPV, and Coombs tests and the microagglutination test (MAT) were positive for all cases: titers were 1/2,560 by the BrucellaCapt test, 1/2,560 by the BCAPV test, 1/1,280 by the Coombs test, and 1/320 by the MAT. The decreases in the BrucellaCapt and BCAPV titers over time were pronounced in comparison with the Coombs titers. Cumulative probabilities of persistence 12 months after therapy were as follows: 80% by the BrucellaCapt test, 80% by the BCAPV test, 87% by the Coombs test, and 35% by the MAT. Serological changes during relapse were detected in seven cases (88%) by the Coombs test, in five cases by the BrucellaCapt and BCAPV tests, and in three cases by the MAT. The BrucellaCapt test is a sensitive, specific, and simple test for routine use in human brucellosis. Similar results were obtained with the BCAPV test. However, in some cases of relapse and chronic forms of the disease, the slight changes observed in low-affinity antibodies alone are better detected by the Coombs test.

  20. Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

    Science.gov (United States)

    Papadopoulos, Alexia; Gagnaire, Aurélie; Degos, Clara; de Chastellier, Chantal; Gorvel, Jean-Pierre

    2016-01-01

    Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

  1. Differential phenotyping of Brucella species using a newly developed semi-automated metabolic system

    Directory of Open Access Journals (Sweden)

    Appel Bernd

    2010-10-01

    Full Text Available Abstract Background A commercial biotyping system (Taxa Profile™, Merlin Diagnostika testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. Results A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A, 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™ was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria. Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. Conclusions The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the

  2. Immune Responses and Protection against Experimental Challenge after Vaccination of Bison with Brucella abortus Strain RB51 or RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes▿

    OpenAIRE

    Olsen, S. C.; Boyle, S. M.; Schurig, G. G.; Sriranganathan, N. N.

    2009-01-01

    Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 (RB51) or a recombinant RB51 strain overexpressing superoxide dismutase (sodC) and glycosyltransferase (wboA) genes (RB51+sodC,wboA). Bison were vaccinated with saline only or with 4.6 × 101...

  3. Brucella canis: inquéritos sorológico e bacteriológico em população felina Brucella canis: serological and bacteriological surveys in the feline population

    Directory of Open Access Journals (Sweden)

    Maria Helena Matiko Akao Larsson

    1984-02-01

    Full Text Available De 134 soros de felinos domésticos examinados pela prova de soroaglutinação lenta em tubos, 4 (3% foram positivos para Brucella canis, todos com título igual a 100. Não se obteve êxito na tentativa de isolamento de Brucella canis através de hemocultura desses animais.Of the 134 feline sera tested by tube agglutination test, 4 (3% were positive for Brucella canis antibodies, all with titer 100. It was not possible to isolate Brucella canis by blood culture in the case of these animals.

  4. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

    Science.gov (United States)

    Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

    2015-01-01

    Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Exposure of harbour seals Phoca vitulina to Brucella in declining populations across Scotland.

    Science.gov (United States)

    Kershaw, Joanna L; Stubberfield, Emma J; Foster, Geoffrey; Brownlow, Andrew; Hall, Ailsa J; Perrett, Lorraine L

    2017-09-20

    Since 2000 there has been a major decline in the abundance of Scottish harbour seals Phoca vitulina. The causes of the decline remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n = 150). Serum samples collected from live capture-released seals (n = 343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). In total, 16% of seals cultured had Brucella isolated from one or more tissues, but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results, showing that overall 25.4% of seals were seropositive, with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.

  6. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

    Science.gov (United States)

    Sergueev, Kirill V.; Filippov, Andrey A.; Nikolich, Mikeljon P.

    2017-01-01

    For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types. PMID:28604602

  7. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

    Directory of Open Access Journals (Sweden)

    Kirill V. Sergueev

    2017-06-01

    Full Text Available For decades, bacteriophages (phages have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

  8. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood.

    Science.gov (United States)

    Sergueev, Kirill V; Filippov, Andrey A; Nikolich, Mikeljon P

    2017-06-10

    For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B . abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis . The addition of a simple short sample preparation step enabled the indirect phage-based detection of B . abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B . abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

  9. Enzymatic, immunological and phylogenetic characterization of Brucella suis urease

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    Sriranganathan Nammalwar

    2008-07-01

    Full Text Available Abstract Background The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. Results Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28–35°C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 ± 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 ± 0.31 mM and 26.12 ± 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130–135 kDa by gel filtration chromatography and 157 ± 7 kDa using 5–10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. Conclusion We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic

  10. Pasteurella multocida Heddleston Serovar 3 and 4 Strains Share a Common Lipopolysaccharide Biosynthesis Locus but Display both Inter- and Intrastrain Lipopolysaccharide Heterogeneity

    Science.gov (United States)

    Harper, Marina; St. Michael, Frank; John, Marietta; Vinogradov, Evgeny; Steen, Jennifer A.; van Dorsten, Lieke; Steen, Jason A.; Turni, Conny; Blackall, Patrick J.; Adler, Ben; Cox, Andrew D.

    2013-01-01

    Pasteurella multocida is a Gram-negative multispecies pathogen and the causative agent of fowl cholera, a serious disease of poultry which can present in both acute and chronic forms. The major outer membrane component lipopolysaccharide (LPS) is both an important virulence factor and a major immunogen. Our previous studies determined the LPS structures expressed by different P. multocida strains and revealed that a number of strains belonging to different serovars contain the same LPS biosynthesis locus but express different LPS structures due to mutations within glycosyltransferase genes. In this study, we report the full LPS structure of the serovar 4 type strain, P1662, and reveal that it shares the same LPS outer core biosynthesis locus, L3, with the serovar 3 strains P1059 and Pm70. Using directed mutagenesis, the role of each glycosyltransferase gene in LPS outer core assembly was determined. LPS structural analysis of 23 Australian field isolates that contain the L3 locus revealed that at least six different LPS outer core structures can be produced as a result of mutations within the LPS glycosyltransferase genes. Moreover, some field isolates produce multiple but related LPS glycoforms simultaneously, and three LPS outer core structures are remarkably similar to the globo series of vertebrate glycosphingolipids. Our in-depth analysis showing the genetics and full range of P. multocida lipopolysaccharide structures will facilitate the improvement of typing systems and the prediction of the protective efficacy of vaccines. PMID:23974032

  11. Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

    Science.gov (United States)

    Santiago-Rodríguez, María Del Rosario; Díaz-Aparicio, Efrén; Arellano-Reynoso, Beatriz; García-Lobo, Juan M; Gimeno, Miquel; Palomares-Reséndiz, Erika G; Hernández-Castro, Rigoberto

    2015-02-01

    The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×10⁸ colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×10⁸ CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

  12. Identification of Protective Brucella Antigens and their Expressions in Vaccinia Virus to Prevent Disease in Animals and Humans.

    Science.gov (United States)

    1996-05-01

    selected antigens is through fractionation of Brucella strain RB51 or E.coli recombinants expressing the appropriateBrucella antigen. Briefly, the method...animal species infected with Brucella spp. It is also able to induce the in vitro production of INF-y with lymphocytes of RB51 vaccinated mice (Table...SOD RB51 1IkDa 20 15 x0 0- 10 E 0- Uve Acetone Buffer Void 0-0.1 0.1-0-25 0.25->0.5 0.5-0.75 0.75->1.0 Klled 14 Preparation of new vaccinia/ Brucella

  13. Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China

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    Mingjun Sun

    2017-12-01

    Full Text Available Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2 was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2 and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS-single-nucleotide polymorphism (SNP-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

  14. Isolation of Aureimonas altamirensis, a Brucella canis-like bacterium, from an edematous canine testicle.

    Science.gov (United States)

    Reilly, Thomas J; Calcutt, Michael J; Wennerdahl, Laura A; Williams, Fred; Evans, Tim J; Ganjam, Irene K; Bowman, Jesse W; Fales, William H

    2014-11-01

    Microbiological and histological analysis of a sample from a swollen testicle of a 2-year-old Border Collie dog revealed a mixed infection of the fungus Blastomyces dermatitidis and the Gram-negative bacterium Aureimonas altamirensis. When subjected to an automated microbial identification system, the latter isolate was provisionally identified as Psychrobacter phenylpyruvicus, but the organism shared several biochemical features with Brucella canis and exhibited agglutination, albeit weakly, with anti-B. canis antiserum. Unequivocal identification of the organism was only achieved by 16S ribosomal RNA gene sequencing, ultimately establishing the identity as A. altamirensis. Since its first description in 2006, this organism has been isolated infrequently from human clinical samples, but, to the authors' knowledge, has not been reported from a veterinary clinical sample. While of unknown clinical significance with respect to the pathology observed for the polymicrobial infection described herein, it highlights the critical importance to unambiguously identify the microbe for diagnostic, epidemiological, infection control, and public health purposes. © 2014 The Author(s).

  15. Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

    Science.gov (United States)

    Kay, Gemma L.; Sergeant, Martin J.; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara

    2014-01-01

    ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. PMID:25028426

  16. An ultrasensitive electrochemical genosensor for Brucella based on palladium nanoparticles.

    Science.gov (United States)

    Rahi, A; Sattarahmady, N; Heli, H

    2016-10-01

    Palladium nanoparticles were potentiostatically electrodeposited on a gold surface at a highly negative potential. The nanostructure, as a transducer, was utilized to immobilize a Brucella-specific probe and the process of immobilization and hybridization was detected by electrochemical methods. The proposed method for detection of the complementary sequence and a non-complementary sequence was applied. The fabricated genosensor was evaluated for the assay of the bacteria in the cultured and human samples with and without PCR. The genosensor could detect the complementary sequence with a sensitivity of 0.02 μA dm(3) mol(-1), a linear concentration range of 1.0 × 10(-12) to 1.0 × 10(-19) mol dm(-3), and a detection limit of 2.7 × 10(-20) mol dm(-3). Copyright © 2016 Elsevier Inc. All rights reserved.

  17. [Brucella abortus endocarditis: survival of a 74 year old patient].

    Science.gov (United States)

    Olea M, Pilar

    2010-02-01

    Brucellosis is not frequent in Chile but it may present with life threatening complications like endocarditis. The case reported refers to a 74 year old man admitted to the Infectious Diseases Hospital Dr. Lucio Córdova in Santiago. He had been febrile for 3 months with no specific symptoms. The trans-esophageal echocardiography confirmed multiple large vegetations and important involvement of the aortic valve. Blood cultures yielded Brucella abortus. The patient required cardiac surgery, along with antibiotics, and he had a satisfactory outcome, being alive at the moment of this report???. Brucellosis can be the responsible for prolonged fever of unknown origin. It is necessary to take in mind brucellosis to obtain the specific laboratory tests. For a best prognosis an early treatment with associated antibiotics for at least 4 a 6 weeks is important. If endocarditis is present valve replacement is often necessary.

  18. Brucella abortus-infected B cells induce osteoclastogenesis.

    Science.gov (United States)

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2016-09-01

    Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7.

    Science.gov (United States)

    Cha, Seung Bin; Lee, Won Jung; Shin, Min Kyoung; Jung, Myung Hwan; Shin, Seung Won; Yoo, An Na; Kim, Jong Wan; Yoo, Han Sang

    2013-06-27

    Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.

  20. Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis.

    Science.gov (United States)

    Wang, Xiaolei; Wang, Yan; Ma, Limei; Zhang, Ran; De, Yanyan; Yang, Xiaowen; Wang, Chuanqing; Wu, Qingmin

    2015-05-21

    Brucellosis is the most common bacterial zoonosis, and serological tests are routinely used in brucellosis control and eradication programs. In order to improve the accuracy of serological diagnostic method used in bovine brucellosis detection, this study developed an improved competitive ELISA with higher specificity and good sensitivity. This study prepared 12 monoclonal antibodies against smooth Brucella lipopolysaccharide. One monoclonal antibody 3 F9, presented C epitope specificity, was used to develop a competitive ELISA for the serological detection of bovine brucellosis. The competitive ELISA, a commercial competitive ELISA kit, the rose-bengal plate agglutination test, and a microplate agglutination test were all used in the detection of 6 hyperimmune antisera against other commonly cross-reacted bacterial pathogens and 110 clinical bovine serum samples. The results of the test comparisons indicated that the competitive ELISA had higher specificity than the commercial competitive ELISA kit and RBT, and comparable sensitivity with the commercial ELISA kit. This study provided a valuable detection tool with high specificity and good sensitivity, which prevent the wrong-culling of bovines in the eradication campaigns of bovine brucellosis.

  1. Transcriptional profiling of the effect of lipopolysaccharide (LPS) pretreatment in blood from probiotics-treated dairy cows.

    Science.gov (United States)

    Adjei-Fremah, Sarah; Ekwemalor, Kingsley; Asiamah, Emmanuel; Ismail, Hamid; Worku, Mulumebet

    2016-12-01

    Probiotic supplements are beneficial for animal health and rumen function; and lipopolysaccharides (LPS) from gram negative bacteria have been associated with inflammatory diseases. In this study the transcriptional profile in whole blood collected from probiotics-treated cows was investigated in response to stimulation with lipopolysaccharides (LPS) in vitro. Microarray experiment was performed between LPS-treated and control samples using the Agilent one-color bovine v2 bovine (v2) 4x44K array slides. Global gene expression analysis identified 13,658 differentially expressed genes (fold change cutoff ≥ 2, P probiotics may stimulate the innate immune response of animal against parasitic and bacterial infections. We have provided a detailed description of the experimental design, microarray experiment and normalization and analysis of data which have been deposited into NCBI Gene Expression Omnibus (GEO): GSE75240.

  2. A rapid minor groove binder PCR method for distinguishing the vaccine strain Brucella abortus 104M.

    Science.gov (United States)

    Nan, Wenlong; Qin, Lide; Wang, Yong; Zhang, Yueyong; Tan, Pengfei; Chen, Yuqi; Mao, Kairong; Chen, Yiping

    2018-01-24

    Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M. We developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006-0.022 and 0.012-0.044, respectively). A SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of

  3. Effects of equine metabolic syndrome on inflammatory responses of horses to intravenous lipopolysaccharide infusion.

    Science.gov (United States)

    Tadros, Elizabeth M; Frank, Nicholas; Donnell, Robert L

    2013-07-01

    To test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and horses with equine metabolic syndrome (EMS). Animals-6 healthy horses and 6 horses with EMS. Each horse randomly received an IV infusion of lipopolysaccharide (20 ng/kg [in 60 mL of sterile saline {0.9% NaCl} solution]) or saline solution, followed by the other treatment after a 7-day washout period. Baseline data were obtained 30 minutes before each infusion. After infusion, a physical examination was performed hourly for 9 hours and at 15 and 21 hours; a whole blood sample was collected at 30, 60, 90, 120, 180, and 240 minutes for assessment of inflammatory cytokine gene expression. Liver biopsy was performed between 240 and 360 minutes after infusion. Results-Following lipopolysaccharide infusion in healthy horses and horses with EMS, mean rectal temperature, heart rate, and respiratory rate increased, compared with baseline findings, as did whole blood gene expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The magnitude of blood cytokine responses did not differ between groups, but increased expression of IL-6, IL-8, IL-10, and tumor necrosis factor-α persisted for longer periods in EMS-affected horses. Lipopolysaccharide infusion increased liver tissue gene expressions of IL-6 in healthy horses and IL-8 in both healthy and EMS-affected horses, but these gene expressions did not differ between groups. Results supported the hypothesis that EMS affects horses' inflammatory responses to endotoxin by prolonging cytokine expression in circulating leukocytes. These findings are relevant to the association between obesity and laminitis in horses with EMS.

  4. Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M

    Directory of Open Access Journals (Sweden)

    Pradeepkiran JA

    2015-03-01

    Full Text Available Jangampalli Adi Pradeepkiran,1 Konidala Kranthi Kumar,1 Yellapu Nanda Kumar,2 Matcha Bhaskar11Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, 2Biomedical Informatics Centre, Vector Control Research Centre, Indian Council of Medical Research, Pondicherry, India Abstract: The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO as a template in MODELLER (v 9.10. The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9 and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC

  5. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

    Directory of Open Access Journals (Sweden)

    Gamal Wareth

    2016-04-01

    Full Text Available Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B. species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  6. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

    Science.gov (United States)

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-04-30

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  7. A rapid cycleave PCR method for distinguishing the vaccine strain Brucella abortus A19 in China.

    Science.gov (United States)

    Nan, Wenlong; Zhang, Yueyong; Tan, Pengfei; Xu, Zouliang; Chen, Yuqi; Mao, Kairong; Chen, Yiping

    2016-05-01

    Brucellosis is a widespread zoonotic disease caused by Brucella spp. Immunization with attenuated vaccines has proved to be an effective method of prevention; however, it may also interfere with diagnosis. Brucella abortus strain A19, which is homologous to B. abortus strain S19, is widely used for the prevention of bovine brucellosis in China. For effective monitoring of the control of brucellosis, it is essential to distinguish A19 from field strains. Single-nucleotide polymorphism-based assays offer a new approach to such discrimination studies. In the current study, we developed a cycleave PCR assay that successfully distinguished attenuated vaccine strains A19 and S19 from 22 strains of B. abortus and 57 strains of 5 other Brucella species. The assay gave a negative reaction with 4 non-Brucella species. The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 7.6 fg for the A19 strain and 220 fg for the single non-A19/non-S19 Brucella strain tested (B. abortus 104M). The assay was also reproducible (intra- and interassay coefficients of variation: 0.003-0.01 and 0.004-0.025, respectively). The cycleave assay gave an A19/S19-specific reaction in 3 out of 125 field serum samples, with the same 3 samples being positive in an alternative A19/S19-specific molecular assay. The cycleave assay gave a total of 102 Brucella-specific reactions (3 being the A19/S19-specific reactions), whereas an alternative Brucella-specific assay gave 92 positive reactions (all also positive in the cycleave assay). Therefore, this assay represents a simple, rapid, sensitive, and specific tool for use in brucellosis control. © 2016 The Author(s).

  8. Riboflavin attenuates lipopolysaccharide-induced lung injury in rats.

    Science.gov (United States)

    Al-Harbi, Naif O; Imam, Faisal; Nadeem, Ahmed; Al-Harbi, Mohammed M; Korashy, Hesham M; Sayed-Ahmed, Mohammed M; Hafez, Mohamed M; Al-Shabanah, Othman A; Nagi, Mahmoud N; Bahashwan, Saleh

    2015-01-01

    Riboflavin (vitamin B2) is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is therefore required by all flavoproteins. Riboflavin also works as an antioxidant by scavenging free radicals. The present study was designed to evaluate the effects of riboflavin against acute lungs injury induced by the administration of a single intranasal dose (20 μg/rat) of lipopolysaccharides (LPS) in experimental rats. Administration of LPS resulted in marked increase in malondialdehyde (MDA) level (p riboflavin in a dose-dependent manner (30 and 100 mg/kg, respectively). Riboflavin (100 mg/kg, p.o.) showed similar protective effects as dexamethasone (1 mg/kg, p.o.). Administration of LPS showed marked cellular changes including interstitial edema, hemorrhage, infiltration of PMNs, etc., which were reversed by riboflavin administration. Histopathological examinations showed normal morphological structures of lungs tissue in the control group. These biochemical and histopathological examination were appended with iNOS and CAT gene expression. The iNOS mRNA expression was increased significantly (p riboflavin significantly (p riboflavin caused a protective effect against LPS-induced ALI. These results suggest that riboflavin may be used to protect against toxic effect of LPS in lungs.

  9. Post-exposure serological and bacteriological responses of water buffalo (Bubalus bubalis) to Brucella abortus biovar 1 following vaccination with Brucella abortus strain RB51.

    Science.gov (United States)

    Diptee, M D; Asgarali, Z; Campbell, M; Fosgate, G; Adesiyun, A A

    2007-12-01

    Serological and bacteriological responses to Brucella abortus biovar 1 following vaccination with B. abortus strain RB51 (RB51) were evaluated in thirty domestic water buffalo (Bubalus bubalis) randomly divided into five treatment groups. Groups I to V received, respectively, the recommended dose (RD) of RB51 vaccine once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart, and saline once (control). Vaccination did not result in a serological response. Experimental animals released 27 weeks post initial inoculation (27 PIIW) into a brucellosis-positive herd failed to seroconvert after 29 weeks. Experimental challenge commenced at 57 PIIW. All animals received B. abortus biovar 1 intraconjunctivally at 0, 5 and 9 weeks post experimental exposure (PEEW). Serum samples collected at 4, 8 and 13 PEEW were negative. At 16 PEEW all animals received B. abortus biovar 1 subcutaneously (SC), and all seroconverted by 20 PEEW. Five of twenty-six animals were positive for Brucella infection on bacterial culture. Brucella abortus biovar 1 was isolated from three animals; B. abortus RB51 was isolated from two. Treatment group, age and sex had no effect on the isolation of Brucellae (P>0.05).

  10. Anticorpos anti-Brucella canis e anti-Brucella abortus em cães de Araguaína, Tocantins

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    Elaine Maria Seles Dorneles

    2011-04-01

    Full Text Available The aims of the present study were to determine the seroprevalence of infection by Brucella canis and Brucella abortus and to evaluate possible risk factors for infection in dogs from Araguaína, Tocantins, Brazil. Sera from 374 dogs, of the urban zones of the municipality, from both sexes, were submitted to the agar-gel immunodiffusion for Brucella canis-antibodies and to rose Bengal test (AAT and fluorescence polarization assay (FPA for Brucella abortus-antibodies. From the 374 tested dogs, 21 reacted in the AAT, but no one was positive in the FPA. The seroprevalence of B. canis infection found in Araguaína, Tocantins, Brazil, was 44.53% (95% IC; 39.43 to 49.72. No association was found among seropositivity for B. canis and the risk factors studied. Thus, data from the present study showed that there was no infection by B. abortus among dogs in the sample and that infection by B. canis is widespread and at high prevalence in Araguaína, Tocantins, Brazil.

  11. Th2-related immune responses by the Brucella abortus cellular antigens, malate dehydrogenase, elongation factor, and arginase.

    Science.gov (United States)

    Im, Young Bin; Shim, Soojin; Park, Woo Bin; Kim, Suk; Yoo, Han Sang

    2017-09-01

    Brucellosis is an important zoonotic disease caused by Brucella species. The disease is difficult to control due to the intracellular survival of the bacterium and the lack of precise understanding of pathogenesis. Despite of continuous researches on the pathogenesis of Brucella spp. infection, there is still question on the pathogenesis, especially earlier immune response in the bacterial infection. Malate dehydrogenase (MDH), elongation factor (Tsf), and arginase (RocF), which showed serological reactivity, were purified after gene cloning, and their immune modulating activities were then analyzed in a murine model. Cytokine production profiles were investigated by stimulating RAW 264.7 cells and naïve splenocytes with the three recombinant proteins. Also, immune responses were analyzed by ELISA and an ELIspot assay after immunizing mice with the three proteins. Only TNF-α was produced in stimulated RAW 264.7 cells, whereas Th1-related cytokines, IFN-γ and IL-2, were induced in naïve splenocytes. In contrast, Th2-type immune response was more strongly induced in antigen-secreting cells in the splenocytes obtained 28 days after immunizing mice with the three proteins, as were IgM and IgG. The induction of Th2-related antibody, IgG1, was higher than the Th1-related antibody, IgG2a, in immunized mice. These results suggest that the three proteins strongly induce Th2-type immune response in vivo, even though Th1-related cytokines were produced in vitro. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Effects of Opsonization and Gamma Interferon on Growth of Brucella Melitensis 16M in Mouse Peritoneal Macrophages In Vitro

    Science.gov (United States)

    2000-01-01

    those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis . 14. SUBJECT TERMS rjpsonlzatlon, Brucella... brucellosis . Brucella spp., short, nonmotile, nonsporulating, nonencap- sulated, gram-negative aerobic rods, are important facultative intracellular...heated bovine serum induces the produc- tion of oxidative products in bovine neutrophils (6). Although neutrophils represent an important first line

  13. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

    NARCIS (Netherlands)

    Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

    2003-01-01

    To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

  14. Whole-genome sequence of the first sequence type 27 Brucella ceti strain isolated from European waters

    DEFF Research Database (Denmark)

    Duvnjak, Sanja; Spicic, Silvio; Kusar, Darja

    2017-01-01

    Brucella spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of Brucella ceti CRO350, a sequence type 27 strain isolated from a bottlenose dolphin carcass found...

  15. WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System.

    Science.gov (United States)

    Herrou, Julien; Czyż, Daniel M; Willett, Jonathan W; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean

    2016-04-01

    The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes

  16. Evaluation of Brucella contamination in raw milk in Kerman dairy farms by PCR

    Directory of Open Access Journals (Sweden)

    Mohammad Khalili

    2016-10-01

    Full Text Available Background: Human brucellosis is a significant public health problem in many middle east countries including Iran. In endemic developing countries, dairy products produced from untreated milk are a potential threat to public health. The aim of this study was to detect brucellae in milk from dairy cattle farms in Kerman (Iran. Methods: Forthy and eight Bulk Tank Milk (BTM  were collected from 48 dairy cattle farm including 4200 cow. All milk samples were examined by PCR to detect Brucella-specific DNA. Results: Using IS711 primer were detected in 4 samples (8.3% Brucella spp from 48 BTM samples in this area.. Conclusions: The detection of Brucella DNA in milk for human consumption, especially the highly pathogenic species B. melitensis, is of obvious concern. The shedding of Brucella spp. in milk poses an increasing threat to consumers in Iran. Consumption of dairy products produced from non-pasteurized milk by individual farmers operating under poor hygienic conditions represents an unacceptable risk to public health.

  17. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR

    Directory of Open Access Journals (Sweden)

    Skjerve Eystein

    2009-12-01

    Full Text Available Abstract Background Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe. Findings Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates and biovar 2 (2 isolates were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2 and B. melitensis biovar 1, respectively. Conclusion We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.

  18. Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis).

    Science.gov (United States)

    Longo, Mariangela; Mallardo, Karina; Montagnaro, Serena; De Martino, Luisa; Gallo, Sergio; Fusco, Giovanna; Galiero, Giorgio; Guarino, Achille; Pagnini, Ugo; Iovane, Giuseppe

    2009-07-01

    The objective of this study was to determine if Brucella abortus rough mutant strain RB51 (SRB51) is eliminated in buffalo milk. Thirty Brucella-free female buffaloes were used in this study: ten 4-5 years old were inoculated with the triple of the recommended calfhood dose of SRB51 by subcutaneous route, ten 2-3 years old at the first lactation were previously vaccinated twice as calves with triple the recommended calf dose of RB51, while five 4-5 years old and five 2-3 years old not vaccinated Brucella-free female buffaloes served as controls. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on selective media for isolation of SRB51 and incubated for 11 days. Moreover, PCR analysis was also performed directly on milk samples. SRB51 was isolated from milk samples only during the first week post-vaccination while RB51 DNA was detected during the first week till the fourth week post-vaccination only in water buffaloes vaccinated as adults. The identification of Brucella RB51 in milk samples, strongly suggests that this Brucella vaccine could be excreted in milk of buffalo cows vaccinated as adults, while our data demonstrate that the vaccine is safe for use in buffaloes vaccinated as calves in which it was not excreted in milk.

  19. Meta-analysis of Brucella seroprevalence in dairy cattle of Ethiopia.

    Science.gov (United States)

    Asmare, Kassahun; Krontveit, Randi I; Ayelet, Gelagay; Sibhat, Berhanu; Godfroid, Jacques; Skjerve, Eystein

    2014-12-01

    This meta-analysis estimates a single-group summary (effect size) for seroprevalence of Brucella spp. exposure in dairy cattle of Ethiopia. It also attempts to identify study-level variables that could explain the variation in apparent seroprevalence. The literature search was restricted to studies published in English language from January 2000 to December 2013. A template was designed to retrieve the most biologically plausible and consistent variables from the articles. A total of 29 published papers containing 40 animal-level studies were used in the analyses. The single-group summary of Brucella seroprevalence in cattle was estimated to reach 3.3 % with 95 % confidence interval (CI) (2.6-4.2 %). Of all the variables considered, region was the only specific factor identified to explain about 20 % of between-study variation. Accordingly, the region-based meta-analysis forest plot revealed the highest prevalence in central Ethiopia followed by southern part. The lowest prevalence estimate was observed in the western part of the country. The visual inspection of the funnel plot demonstrated the presence of possible publication bias which might dictate shortage of studies with higher prevalences or variance inflation due to infectiousness of Brucella. In conclusion, the quantitative review showed the seroprevalence to be low but widely distributed. More importantly, the review underscores the need for isolation and characterization of the circulating Brucella spp. to capture the type of Brucella spp. involved and its distribution in cattle in Ethiopia.

  20. Neospora caninum versus Brucella spp. exposure among dairy cattle in Ethiopia: a case control study.

    Science.gov (United States)

    Asmare, Kassahun

    2014-08-01

    This case-control study aimed at assessing the relative association of Neospora caninum and Brucella species exposure with reproductive disorders. The study was carried out between October 2011 and June 2012 on 731 dairy cows sampled from 150 dairy farms in selected 17 conurbations of Ethiopia. Two hundred sixty-six of the cows were categorized as cases based on their history of abortion or stillbirth while the remaining 465 were controls. The presence of antibody to N. caninum was screened using indirect ELISA, while Brucella spp. exposure was assayed serially using Rose Bengal Plate Test and Complement Fixation Test. Exposure to N. caninum was more frequently observed among cases (23.8%) than controls (12.7%), while no significant difference (p > 0.05) was noted for Brucella exposure between the two groups. Moreover, the proportion of cows with disorders like retention of fetal membrane, endometritis and increased inter-calving period were significantly higher (p Brucella spp. exposure. However, neither N. caninum nor Brucella spp. could explain the majority (73.2%) of the reported abortions and stillbirths in cattle. Hence, this observation underscores the need for more intensive investigation on the identification of causes of the aforementioned disorders in dairy cattle of Ethiopia.

  1. [Detection of Brucella with an automatic hemoculture system: Bact/Alert].

    Science.gov (United States)

    Casas, J; Partal, Y; Llosá, J; Leiva, J; Navarro, J M; de la Rosa, M

    1994-12-01

    The ability of in vitro and in vivo detection of Brucella spp. with the Bact/Alert system was studied. Three strains of Brucella melitensis and two of Brucella abortus were used. Different dilutions of the five strains were performed in trypticase soy broth (TSB), achieving concentrations of 1 cfu/ml, 5 cfu/ml, 10 cfu/ml and 100 cfu/ml. Ten ml of each dilution and strain were inoculated into 5 aerobic bottles Bact/Alert and 5 biphasic Hemóline bottles. Furthermore, over a 9 month period, 8,216 bottles of Bact/Alert bottles from hospitalized patients and from the emergency department were processed in the authors' laboratory. The mean detection time for Brucella growth was from 2 to 3 days with the Bact/Alert system, and 14 days in the biphasic bottles. Former bottles processed in the authors' laboratory, 11 aerobic bottles belonged to 5 patients in whom brucelosis was confirmed by bloodculture. The Bact/Alert system detected Brucella melitensis in only on bottle at 2.9 days of incubation. In 7 bottles Bact/Alert detected B. melitensis by a blind pass of these bottles at 10 to 20 days of incubation. These results suggest that the Bact/Alert system does not totally solve the diagnosis of brucellosis. Blind passes of the bloodcultures are required.

  2. Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

    Science.gov (United States)

    De Santis, Riccardo; Ancora, Massimo; De Massis, Fabrizio; Ciammaruconi, Andrea; Zilli, Katiuscia; Di Giannatale, Elisabetta; Pittiglio, Valentina; Fillo, Silvia; Lista, Florigio

    2013-10-01

    Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

  3. Type IV Effector Secretion and Subversion of Host Functions by Bartonella and Brucella Species.

    Science.gov (United States)

    Dehio, Christoph; Tsolis, Renée M

    2017-01-01

    Bartonella and Brucella species comprise closely related genera of the order Rhizobiales within the class α-proteobacteria. Both groups of bacteria are mammalian pathogens with a facultative intracellular lifestyle and are capable of causing chronic infections, but members of each genus have evolved broadly different infection and transmission strategies. While Brucella spp. transmit in general via the reproductive tract in their natural hosts, the Bartonella spp. have evolved to transmit via arthropod vectors. However, a shared feature of both groups of pathogens is their reliance on type IV secretion systems (T4SSs) to interact with cells in their mammalian hosts. The genomes of Bartonella spp. encode three types of T4SS, Trw, Vbh/TraG, and VirB/VirD4, whereas those of Brucella spp. uniformly contain a single T4SS of the VirB type. The VirB systems of Bartonella and Brucella are associated with distinct groups of effector proteins that collectively mediate interactions with host cells. This chapter discusses recent findings on the role of T4SS in the biology of Bartonella spp. and Brucella spp. with emphasis on effector repertoires, on recent advances in our understanding of their evolution, how individual effectors function at the molecular level, and on the consequences of these interactions for cellular and immune responses in the host.

  4. Btk regulates macrophage polarization in response to lipopolysaccharide.

    Directory of Open Access Journals (Sweden)

    Joan Ní Gabhann

    Full Text Available Bacterial Lipopolysaccharide (LPS is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\\- mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/- macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/- macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/- macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/- mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

  5. Analysis of the cell wall and lipopolysaccharide of Spirillum serpens.

    Science.gov (United States)

    Chester, I R; Murray, R G

    1975-12-01

    Isolated walls of Spirillum serpens VHA contained lipid, lipopolysaccharide, and protein in amounts similar to those of other gram-negative organisms. The loosely bound lipids consisted mainly of phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Lipopolysaccharide was tightly bound to the wall and could only be removed in a substantial amount after digestion of the wall with Pronase. The lipopolysaccharide contained L-glycero-D-mannoheptose, rhamnose, glucosamine, ethanolamine, and phosphate in common with many of the lipopolysaccharides isolated from the Enterobacteriaceae. However, 2-keto-3-deoxyoctonic acid was not detected. Several unidentified sugars were present. The fatty acid composition resembled that found in lipopolysaccharides isolated from various pseudomonads. Two major regions were identified in the polysaccharide moiety, one apparently corresponding to the core polysaccharide and the other corresponding to the side-chain polysaccharide as in enterobacterial and pseudomonad lipopolysaccharides. The side chains were obtained as low-molecular-weight material and their structure was partially elucidated by the isolation and partial characterization of N-acetylglucosaminyl-(1 leads to 4)-rhamnose.

  6. The Myriad Properties of Pasteurella multocida Lipopolysaccharide

    Science.gov (United States)

    Harper, Marina; Boyce, John Dallas

    2017-01-01

    Pasteurella multocida is a heterogeneous species that is a primary pathogen of many different vertebrates. This Gram-negative bacterium can cause a range of diseases, including fowl cholera in birds, haemorrhagic septicaemia in ungulates, atrophic rhinitis in swine, and lower respiratory tract infections in cattle and pigs. One of the primary virulence factors of P. multocida is lipopolysaccharide (LPS). Recent work has shown that this crucial surface molecule shows significant structural variability across different P. multocida strains, with many producing LPS structures that are highly similar to the carbohydrate component of host glycoproteins. It is likely that this LPS mimicry of host molecules plays a major role in the survival of P. multocida in certain host niches. P. multocida LPS also plays a significant role in resisting the action of chicken cathelicidins, and is a strong stimulator of host immune responses. The inflammatory response to the endotoxic lipid A component is a major contributor to the pathogenesis of certain infections. Recent work has shown that vaccines containing killed bacteria give protection only against other strains with identical, or nearly identical, surface LPS structures. Conversely, live attenuated vaccines give protection that is broadly protective, and their efficacy is independent of LPS structure. PMID:28825691

  7. Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori.

    Science.gov (United States)

    Li, Hong; Liao, Tingting; Debowski, Aleksandra W; Tang, Hong; Nilsson, Hans-Olof; Stubbs, Keith A; Marshall, Barry J; Benghezal, Mohammed

    2016-12-01

    This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram-negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O-antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa-saccharide (Kdo-LD-Hep-LD-Hep-DD-Hep-Gal-Glc), the outer core composed of a conserved trisaccharide (-GlcNAc-Fuc-DD-Hep-) linked to the third heptose of the inner core, the glucan, the heptan and a variable O-antigen, generally consisting of a poly-LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O-antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium. © 2016 John Wiley & Sons Ltd.

  8. [Detection of Brucella canis by immunochromatography method in vague dogs captured in Temuco city, Chile, 2011].

    Science.gov (United States)

    Tuemmers, Christian; Lüders, Carlos; Rojas, Claudio; Serri, Michel; Castillo, Carolina; Espinoza, Rodrigo

    2013-08-01

    Brucella canis is responsible for brucellosis in dogs, causing reproductive disorders and is considered a zoonoses, as described in several countries. The epidemiological data are scarce in our country. To determine the prevalence of Brucella canis in vague dogs in Temuco city and housed in the Temuco Kennel. Quantitative and cross-section study. We used 400 samples of dogs of both sexes, different ages and mainly mixed race, which were tested by immunochromatography. Antibodies were detected in 4 samples Brucella canis which represented 1% of the population studied, 2 females (0.5%) and 2 males (0.5%). We conclude that dogs are infected by B. canis in a low range but remains a risk condition to the health of the human population if not maintained adequate sanitary control of pets, like vague dogs.

  9. Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda

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    Denis Rwabiita Mugizi

    2015-01-01

    Full Text Available Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

  10. Multipronged diagnostic approaches for monitoring the treatment of Brucella abortus infected patient: a case report

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    Rajeswari Shome

    2015-07-01

    Full Text Available Brucellosis caused by Brucella species is readily transmissible to humans, causing acute febrile illness and undulant fever which may progress to a more chronic form and can also produce serious complications affecting the musculoskeletal, cardiovascular, and central nervous systems. A veterinary livestock inspector presented to the institute with symptoms of intermittent fever, pain involving muscles and joints, loss of weight, anxiety and weakness for about three months has been investigated. The isolation, serological tests and PCR were performed for diagnosis of brucellosis. Based on history of constant professional association with animals, characteristic symptoms, hematological and biochemical, multiple serological and PCR assay results, the patient was diagnosed as brucellosis. Detection of Brucella abortus directly in the clinical samples by gel based PCRs were highly useful for diagnosis and monitoring of treatment. This diagnostic protocol will facilitate in a simple way to map major Brucella species infecting humans in a geographical region.

  11. An RNA Vaccine Based on Recombinant Semliki Forest Virus Particles Expressing the Cu,Zn Superoxide Dismutase Protein of Brucella abortus Induces Protective Immunity in BALB/c Mice

    Science.gov (United States)

    Oñate, Angel A.; Donoso, Gabriel; Moraga-Cid, Gustavo; Folch, Hugo; Céspedes, Sandra; Andrews, Edilia

    2005-01-01

    We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections. PMID:15908354

  12. Safety of Brucella abortus strain RB51 in deer mice.

    Science.gov (United States)

    Cook, W E; Williams, E S; Thorne, E T; Taylor, S K; Anderson, S

    2001-07-01

    Brucella abortus strain RB51 is an approved brucellosis vaccine for use in cattle that may have potential as an oral vaccine for use in elk (Cervus elaphus) and/or bison (Bison bison). This study was designed to determine effects of strain RB51 on deer mice (Peromyscus maniculatus), a nontarget species that could have access to treated baits in a field situation. In February 1994, 90 mice were orally dosed or intraperitoneally injected with 1 x 10(8) colony forming units strain RB51 and 77 controls were similarly dosed with sterile saline. At weekly intervals through early April 1994, 4 to 6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically. All orally inoculated mice cleared the infection by 6 wk post-inoculation (PI). While most of the injected mice cleared the infection by 7 wk PI, a few required 9 wk. There were minimal adverse effects attributable to strain RB51. Apparently, strain RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Also, deer mice appear to be able to clear the vaccine in 6 to 9 wk, thus the probability of these mice transmitting the vaccine to other animals is low.

  13. Seroprevalence of Brucella abortus and Leptospira hardjo in cattle

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    S. Jegaveera Pandian

    2015-02-01

    Full Text Available Aim: The aim was to assess the seroprevalence of B. abortus and Leptospira hardjo in the cattle population of Bihar, this work was carried out. Materials and Methods: Randomly selected 450 cattle from nine districts of Bihar were serologically screened for antibodies against L. hardjo and B. abortus. DAS-ELISA for leptospira and AB-ELISA for brucella were carried out. Based on the results prevalence in each district and the state are reported herewith. Results: In this study, it was found that the seroprevalence of L. hardjo was 9.11% and that of B. abortus was 12.2% in Bihar. Indigenous cattle were found to be less susceptible to leptospirosis and brucellosis even though they accounted for 83.11% of the study population. Conclusion: Although there was no acute disease, antibodies detected against L. hardjo and B. abortus in the cattle population indicated the presence of chronic and subclinical infection, which could challenge the fertility of the animals.

  14. The Effect of Irradiation on the Immunogenity of Brucella Abortus

    International Nuclear Information System (INIS)

    Arifin, M.; Tuasikal, Boky J.; Endhang Pudjiastuti; Ernawati Yulia

    2004-01-01

    An experiment was carried out to study the effect of irradiation on the immunogenity of B. abortus. The B. abortus were irradiated by Gamma Cells ( 60 Co). An experiment were divided into four groups. The first group (V1) was inoculated by irradiated B. abortus with the dose of 0.25 kGy. The second group (V2) was inoculated by irradiated B. abortus with the dose of 0.50 kGy. The third group (V3) was inoculated by irradiated B. abortus with the dose of 0.75 kGy. The fourth group (V4) was inoculated by Brucella vaccine 8.19. The observation respectively were included purely test, safety test, RBT serological test, diffusion test, development the colony of B. abortus in lien, and pathology anatomic inspection. The results obtained showed that 0.25 kGy was the expectantly dose of irradiation which could not only decreasing the infectivity of B. abortus but also has the ability to become a good immunogen for stimulating the immune response in the experiment animals. (author)

  15. Brucella abortus RB51 in milk of vaccinated adult cattle.

    Science.gov (United States)

    Miranda, Karina Leite; Poester, Fernando Padilla; Dorneles, Elaine Maria Seles; Resende, Thiago Magalhães; Vaz, Adil Knackfuss; Ferraz, Sandra Maria; Lage, Andrey Pereira

    2016-08-01

    The aim of this study was to evaluate the shedding of Brucella abortus in the milk of cows vaccinated with a full dose of RB51 during lactation. Eighteen cows, nine previously vaccinated with S19 as calves and nine non-vaccinated, were immunized subcutaneously with 1.3×10(10)CFU of B. abortus RB51, 30-60days after parturition. Milk samples from all animals were collected daily until day 7, and at weekly interval for the next 9 weeks after vaccination. To evaluate the shedding of B. abortus, milk samples were submitted for culture and PCR. No B. abortus was isolated from any sample tested. Only one sample, collected on first day after vaccination from a cow previously vaccinated, was faintly positive in the PCR. In conclusion, the public health hazard associated with milk consumption from cows vaccinated with RB51 in post-partum is very low, despite vaccination with the full dose and regardless of previous S19 vaccination. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Evaluation of oxidative stress in brucella infected cows

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    N. Kataria

    2010-05-01

    Full Text Available Oxidative stress can influence the metabolism of cells in vital organs of the body. Oxidative stress is extremely dangerous as it does not exhibit any symptom and is recognisable with great difficulty by means of laboratory methods. It can be monitored with several biomarkers like antioxidants and pro-oxidants which can be assessed in serum. The inexorableness of exposure of cows to brucella infection makes oxidative stress associated with this infection an appropriate field of investigation. There is paucity of work to detect stress, which is essential to take timely corrective measures and to save the animal population. Therefore the investigation was carried out to evaluate oxidative stress in the cows suffering from brucellosis. For this serum iomarkers of oxidative stress viz. vitamin C, vitamin E, catalase, monoamine oxidase, glutathione reductase, superoxide dismutase, glutathione, xanthine oxidase, oxidase and peroxidase were determined. Results indicated that vitamin C, vitamin E and glutathione activity decreased significantly in affected cows as compared to healthy cows. Serum catalase, superoxide dismutase, monoamine oxidase, glutathione reductase, xanthine oxidase, oxidase and peroxidase activities increased significantly in affected cows as compared to healthy cows. Decreased activity of vitamin C, vitamin E and glutathione indicated towards their depletion which generally occurs in the oxidative stress to scavenge the free radicals. It was concluded that oxidative stress was there in the animals. This study recommends the use of antioxidants in affected cows

  17. ENCUESTA EXPLORATORIA DE INFECCION POR Brucella canis EN PERROS DE

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    Ana Pardo D

    2009-08-01

    Full Text Available Objetivo. Determinar la presencia de anticuerpos a B. canis en perros domésticos y callejeros del municipio de Villavicencio, Colombia. Materiales y métodos. Se utilizó la prueba de aglutinación rápida en placa con antígeno menos mucoide (M- en 201 muestras de suero. En dos animales seropositivos se realizó intento de aislamiento por hemocultivo en medio selectivo para Brucella (oxoid®. En un animal seropositivo, con crecimiento bacteriano con características morfológicas sugestivas a B. canis se realizó histopatología de testículo, bazo e hígado. Resultados. La seropositividad general fue de 1.49% y correspondió a tres caninos machos, dos de los cuales presentaron signos clínicos de epididimitis y orquitis (unilateral. El cultivo y la histopatología no fueron concluyentes para el diagnostico de B. canis. Conclusiones. La seropositividad fue baja y sugiere que la población estudiada no ha estado en contacto con la bacteria. La presencia de reactores puede estar asociado con falsos positivos. El no aislamiento de la bacteria no indica que la enfermedad no exista por lo que se requiere de nuevos estudios.

  18. Expression of human serum albumin--L7/L12 (Brucella abortus ribosomal protein) fusion protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    Pakzad, Iraj; Rezaee, Abbas; Emaneini, Mohammad; Hosseini, Ahmad Zavaran; Tabbaraee, Bahman; Taherikalani, Morovat

    2009-01-01

    Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. It has already been expressed in several bacteria and has been used as DNA vaccine. In order to construct yeast expressing vector for the tHSA-L7/L12 fusion protein, the l7/l12 ribosomal gene was amplified by PCR. The expression plasmid pYtHSA-L7/L12 was constructed by inserting the L7/L12 gene into the pYHSA5 shuttle vector (containing inulinase signal sequence, HSA gene and Gal10 promoter). The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The secreted recombinant fusion protein was detected in supernatant by SDS-PAGE and confirmed by western blot analysis using anti-HSA and anti-L7/L12 antibodies. Fusion protein was purified by affinity chromatography and its amount was approximately 500 microg/liter.

  19. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    Science.gov (United States)

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  20. Exploring the Diversity of Field Strains of Brucella abortus Biovar 3 Isolated in West Africa

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    Moussa Sanogo

    2017-06-01

    Full Text Available Brucellosis is one of the most widespread bacterial zoonotic diseases in the world, affecting both humans and domestic and wild animals. Identification and biotyping of field strains of Brucella are of key importance for a better knowledge of the epidemiology of brucellosis, for identifying appropriate antigens, for managing disease outbreaks and for setting up efficient preventive and control programmes. Such data are required both at national and regional level to assess potential threats for public health. Highly discriminative genotyping methods such as the multiple locus variable number of tandem repeats analysis (MLVA allow the comparison and assessment of genetic relatedness between field strains of Brucella within the same geographical area. In this study, MLVA biotyping data retrieved from the literature using a systematic review were compared using a clustering analysis and the Hunter-Gaston diversity index (HGDI. Thus, the analysis of the 42 MLVA genotyping results found in the literature on West Africa [i.e., from Ivory Coast (1, Niger (1, Nigeria (34, The Gambia (3, and Togo (3] did not allow a complete assessment of the actual diversity among field strains of Brucella. However, it provided some preliminary indications on the co-existence of 25 distinct genotypes of Brucella abortus biovar 3 in this region with 19 genotypes from Nigeria, three from Togo and one from Ivory Coast, The Gambia, and Niger. The strong and urgent need for more sustainable molecular data on prevailing strains of Brucella in this sub-region of Africa and also on all susceptible species including humans is therefore highlighted. This remains a necessary stage to allow a comprehensive understanding of the relatedness between field strains of Brucella and the epidemiology of brucellosis within West Africa countries.

  1. Risk Factors Associated with Brucella Seropositivity in Sheep and Goats in Duhok Province, Iraq.

    Science.gov (United States)

    Alhamada, Ali G; Habib, Ihab; Barnes, Anne; Robertson, Ian

    2017-12-07

    Sera from 432 small ruminants (335 sheep and 97 goats) from 72 farms in Duhok Province, northern Iraq, were collected to investigate risk factors associated with brucellosis seropositivity. Serum samples were tested using the Rose Bengal test (RBT) and an indirect enzyme-linked immunosorbent assay (iELISA). Using parallel interpretation, RBT and iELISA results showed that 31.7% (95% confidence interval (CI): 26.1, 36.3) of sheep and 34.0% (95% CI: 24.7, 44.3) of goats had antibodies against Brucella in the study area. A random-effects multivariable logistic regression model indicated that a higher chance of being seropositive (odds ratio (OR) = 1.7; 95% 1.4; 2.2) was associated with an increase in the age of animals. The odds of Brucella seropositivity in flocks where sheep and goats grazed together was 2.0 times higher (95% CI: 1.08; 3.9) compared to flocks where sheep and goats grazed separately. The odds of Brucella seropositivity in small ruminants was 2.2 higher (95% CI: 1.2; 4.3) for animals originating from farms with a history of goat abortion in the preceding 12 months. In contrast, for every 1000 Iraqi Dinars (~0.85 US Dollar) spent by the farmers on control of Brucella in their flocks, the odds of Brucella seropositivity decreased significantly (OR = 0.9, p -value = 0.021). The final model also indicated significant differences in Brucella seropositivity between the different districts of Duhok Province. This study provides a contribution to the epidemiology of brucellosis in small ruminants in northern Iraq.

  2. Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

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    Lista Florigio

    2011-12-01

    Full Text Available Abstract Background The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. Results In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. Conclusions MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

  3. The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection.

    Science.gov (United States)

    Marim, Fernanda M; Franco, Miriam M Costa; Gomes, Marco Tulio R; Miraglia, Maria Cruz; Giambartolomei, Guillermo H; Oliveira, Sergio C

    2017-02-01

    The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response.

  4. Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis

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    Marianelli Cinzia

    2009-04-01

    Full Text Available Abstract Background Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Results Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. Conclusion In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

  5. Risk Factors Associated with Brucella Seropositivity in Sheep and Goats in Duhok Province, Iraq

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    Ali. G. Alhamada

    2017-12-01

    Full Text Available Sera from 432 small ruminants (335 sheep and 97 goats from 72 farms in Duhok Province, northern Iraq, were collected to investigate risk factors associated with brucellosis seropositivity. Serum samples were tested using the Rose Bengal test (RBT and an indirect enzyme-linked immunosorbent assay (iELISA. Using parallel interpretation, RBT and iELISA results showed that 31.7% (95% confidence interval (CI: 26.1, 36.3 of sheep and 34.0% (95% CI: 24.7, 44.3 of goats had antibodies against Brucella in the study area. A random-effects multivariable logistic regression model indicated that a higher chance of being seropositive (odds ratio (OR = 1.7; 95% 1.4; 2.2 was associated with an increase in the age of animals. The odds of Brucella seropositivity in flocks where sheep and goats grazed together was 2.0 times higher (95% CI: 1.08; 3.9 compared to flocks where sheep and goats grazed separately. The odds of Brucella seropositivity in small ruminants was 2.2 higher (95% CI: 1.2; 4.3 for animals originating from farms with a history of goat abortion in the preceding 12 months. In contrast, for every 1000 Iraqi Dinars (~0.85 US Dollar spent by the farmers on control of Brucella in their flocks, the odds of Brucella seropositivity decreased significantly (OR = 0.9, p-value = 0.021. The final model also indicated significant differences in Brucella seropositivity between the different districts of Duhok Province. This study provides a contribution to the epidemiology of brucellosis in small ruminants in northern Iraq.

  6. A Case of Brucella Endocarditis in Association with Subclavian Artery Thrombosis

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    Claudia Colomba

    2012-01-01

    Full Text Available Brucellosis is a common zoonosis, endemic in Mediterranean countries, and caused by bacteria of Brucella genus. Brucellosis is a systemic infection and the clinical presentation varies widely from asymptomatic and mild to severe disease. Cardiovascular complications are extremely rare. We present a case of arterial thrombosis in a previously healthy young patient with Brucella endocarditis. Careful attention must be paid to any sign or symptom of thrombosis in patients affected by brucellosis, regardless of the presence of endocarditis and cardiovascular risk factors.

  7. Rapid identification of Yersinia pestis and Brucella melitensis by chip-based continuous flow PCR

    Science.gov (United States)

    Dietzsch, Michael; Hlawatsch, Nadine; Melzer, Falk; Tomaso, Herbert; Gärtner, Claudia; Neubauer, Heinrich

    2012-06-01

    To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

  8. Brucella Antibodies in Alaskan True Seals and Eared Seals—Two Different Stories

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    Ingebjørg H. Nymo

    2018-01-01

    Full Text Available Brucella pinnipedialis was first isolated from true seals in 1994 and from eared seals in 2008. Although few pathological findings have been associated with infection in true seals, reproductive pathology including abortions, and the isolation of the zoonotic strain type 27 have been documented in eared seals. In this study, a Brucella enzyme-linked immunosorbent assay (ELISA and the Rose Bengal test (RBT were initially compared for 206 serum samples and a discrepancy between the tests was found. Following removal of lipids from the serum samples, ELISA results were unaltered while the agreement between the tests was improved, indicating that serum lipids affected the initial RBT outcome. For the remaining screening, we used ELISA to investigate the presence of Brucella antibodies in sera of 231 eared and 1,412 true seals from Alaskan waters sampled between 1975 and 2011. In eared seals, Brucella antibodies were found in two Steller sea lions (Eumetopias jubatus (2% and none of the 107 Northern fur seals (Callorhinus ursinus. The low seroprevalence in eared seals indicate a low level of exposure or lack of susceptibility to infection. Alternatively, mortality due to the Brucella infection may remove seropositive animals from the population. Brucella antibodies were detected in all true seal species investigated; harbor seals (Phoca vitulina (25%, spotted seals (Phoca largha (19%, ribbon seals (Histriophoca fasciata (16%, and ringed seals (Pusa hispida hispida (14%. There was a low seroprevalence among pups, a higher seroprevalence among juveniles, and a subsequent decreasing probability of seropositivity with age in harbor seals. Similar patterns were present for the other true seal species; however, solid conclusions could not be made due to sample size. This pattern is in accordance with previous reports on B. pinnipedialis infections in true seals and may suggest environmental exposure to B. pinnipedialis at the juvenile stage, with a

  9. Brucella ovis: invasion, traffic, virulence factors and immune responseBrucella ovis: invasão, tráfego, fatores de virulência e resposta imune

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    João Marcelo Azevedo de Paula Antunes

    2013-06-01

    Full Text Available Brucellosis remains an economic problem in animals and public health. Worldwide ovine brucellosis caused by Brucella ovis is considered a major cause of infertility in sheep. The factors responsible for persistence of the agent in these locations are not known, as well as the mechanisms involved in immune defense and possibly the persistence of the agent. Brucella spp. induces moderate inflammatory response. The nature of the intracellular agent stimulates immune response of the type 1 helper T lymphocytes. Studies of the pathogenesis of ovine brucellosis are scarce. Recent developments have shown that the inflammatory response induced by moderate brucelas represent probably the result of an attempt to escape the immune response and suppression of host immune response. Were reviewed by the mechanisms described by brucelas and Brucella ovis for penetration into the host, escape of the immune response and the immune response generated by the infection. A brucelose permanece como problema econômico em animais e de saúde pública. Em todo o mundo a brucelose ovina ocasionada pela Brucella ovis é considerada uma das principais causas de infertilidade em ovinos. Os fatores responsáveis pela persistência do agente nestes locais não são conhecidos, bem como os mecanismos imunes envolvidos na defesa e eventualmente na persistência do agente. Brucella spp. induz resposta inflamatória moderada. A natureza intracelular do agente estimula resposta imune celular do tipo linfócito T helper 1. Os estudos de patogenia da brucelose ovina são escassos. Recentes avanços demonstraram que a resposta inflamatória moderada induzida pelas brucelas representam provavelmente o resultado de tentativa de escape da resposta imune e supressão da resposta imune hospedeira. Foram revisados os mecanismos descritos pelas brucelas e pela Brucella ovis para penetração no hospedeiro, escape da resposta imune, bem como a resposta imunológica gerada pela infecção.

  10. Characterization of genomic island 3 and genetic variability of Chilean field strains of Brucella abortus.

    Science.gov (United States)

    Céspedes, Sandra; Salgado, Paulina; Valenzuela, Patricio; Vidal, Roberto; Oñate, Angel A

    2011-07-01

    One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains.

  11. Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus▿

    Science.gov (United States)

    Céspedes, Sandra; Salgado, Paulina; Valenzuela, Patricio; Vidal, Roberto; Oñate, Angel A.

    2011-01-01

    One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains. PMID:21543580

  12. Characterization and protective property of Brucella abortus cydC and looP mutants.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

    2014-11-01

    Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Brucella abortus 2308ΔNodVΔNodW double-mutant is highly attenuated and confers protection against wild-type challenge in BALB/c mice.

    Science.gov (United States)

    Li, Zhiqiang; Wang, Shuli; Zhang, Jinliang; Yang, Guangli; Yuan, Baodong; Huang, Jie; Han, Jincheng; Xi, Li; Xiao, Yanren; Chen, Chuangfu; Zhang, Hui

    2017-05-01

    Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Peripheral tumors alter neuroinflammatory responses to lipopolysaccharide in female rats.

    Science.gov (United States)

    Pyter, Leah M; El Mouatassim Bih, Sarah; Sattar, Husain; Prendergast, Brian J

    2014-03-13

    Cancer is associated with an increased prevalence of depression. Peripheral tumors induce inflammatory cytokine production in the brain and depressive-like behaviors. Mounting evidence indicates that cytokines are part of a pathway by which peripheral inflammation causes depression. Neuroinflammatory responses to immune challenges can be exacerbated (primed) by prior immunological activation associated with aging, early-life infection, and drug exposure. This experiment tested the hypothesis that peripheral tumors likewise induce neuroinflammatory sensitization or priming. Female rats with chemically-induced mammary carcinomas were injected with either saline or lipopolysaccharide (LPS, 250μg/kg; i.p.), and expression of mRNAs involved in the pathway linking inflammation and depression (interleukin-1beta [Il-1β], CD11b, IκBα, indolamine 2,3-deoxygenase [Ido]) was quantified by qPCR in the hippocampus, hypothalamus, and frontal cortex, 4 or 24h post-treatment. In the absence of LPS, hippocampal Il-1β and CD11b mRNA expression were elevated in tumor-bearing rats, whereas Ido expression was reduced. Moreover, in saline-treated rats basal hypothalamic Il-1β and CD11b expression were positively correlated with tumor weight; heavier tumors, in turn, were characterized by more inflammatory, necrotic, and granulation tissue. Tumors exacerbated CNS proinflammatory gene expression in response to LPS: CD11b was greater in hippocampus and frontal cortex of tumor-bearing relative to tumor-free rats, IκBα was greater in hippocampus, and Ido was greater in hypothalamus. Greater neuroinflammatory responses in tumor-bearing rats were accompanied by attenuated body weight gain post-LPS. The data indicate that neuroinflammatory pathways are potentiated, or primed, in tumor-bearing rats, which may exacerbate future negative behavioral consequences. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Effect of polymyxin B and environmental conditions on isolation of Brucella species and the vaccine strain RB51.

    Science.gov (United States)

    Jensen, Allen E; Halling, Shirley M

    2010-03-01

    Brucella are resistant to polymyxin B (PB), but their relative susceptibility to PB and its derivative, colistin (COL) has not been rigorously or systematically studied. Comparative susceptibility of Brucella reference strains, vaccine strain RB51, and Brucella isolates from marine mammals to these two cationic peptides were determined by Etest. Vast differences among Brucella species were found in susceptibility to both PB and COL. Brucella demonstrated similar pattern of relative susceptibility to PB as that of COL, but they were less susceptible to COL. Both B. melitensis and B. suis were the least susceptible to polymyxins and rough strains were more susceptible to both PB and COL than the smooth except for the BvrR mutant. Strains were generally less susceptible to PB when cultured in CO(2) rather than ambient air; some became more susceptible in acidified medium. Results show that environment cultural conditions must be considered when selecting for CO(2)-independent strains of Brucella especially the vaccine strain RB51 on selective media containing PB. Our observations extend basic knowledge of the differential resistance of Brucella to polymyxins. Published by Elsevier Ltd.

  16. The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

    Science.gov (United States)

    Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

    2016-12-01

    Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Deep-sequencing analysis of the mouse transcriptome response to infection with Brucella melitensis strains of differing virulence.

    Directory of Open Access Journals (Sweden)

    Fangkun Wang

    Full Text Available Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK pathways. Further analysis employed gene ontology (GO analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy.

  18. Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

    2016-01-04

    We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Detección de anticuerpos anti-Brucella spp. en cerdos mediante técnicas de aglutinación y ELISA indirecto en las provincias de Buenos Aires y La Pampa: Argentina Detection of anti-Brucella spp. antibodies in swine by agglutination techniques and indirect ELISA in the Buenos Aires and La Pampa provinces: Argentina

    Directory of Open Access Journals (Sweden)

    H.A. Castro

    2006-04-01

    Full Text Available En nuestro país no existe un programa de control sobre brucelosis porcina y su verdadera situación epidemiológica es desconocida. El objetivo de nuestro trabajo fue detectar la presencia de anticuerpos anti-Brucella spp. en porcinos provenientes de criaderos del sudoeste de la provincia de Buenos Aires y del este de la provincia de La Pampa. La toma de muestras de sangre se realizó en el momento del faenado de los animales. La detección de anticuerpos se efectuó mediante las técnicas de aglutinación con antígeno tamponado en placa (BPA, seroaglutinación en tubo (SAT, aglutinación con 2-ME (2-ME y ELISA indirecto, con dos antígenos diferentes: el antígeno CYT (fracción citoplasmática de B. abortus S19 y el antígeno CP (extracto citoplasmático libre de lipopolisacárido. Del total de las muestras analizadas (n=325, el 17,8% fue positivo para BPA, el 13,8% fue positivo para SAT y sólo el 8,0% fue positivo para 2-ME. Mediante ELISA-CYT, este porcentaje se elevó a 21,0%, mientras que a través del ELISA-CP sólo se halló un 10,0% de muestras reactivas. Estos resultados son compatibles con los informados en los escasos reportes previos para todo el país y sugieren la necesidad de extender los estudios a otras zonas, donde sea habitual la cría de cerdos.Porcine brucellosis is one of the most important zoonoses in this country. Currently, there is no control program for porcine brucellosis in Argentina and the epidemiological situation is still unknown. The purpose of our study was to detect anti-Brucella spp. antibodies in swine in the southwest of the Buenos Aires province and the east of the La Pampa province. Blood samples were obtained when animals were slaughtered. The presence of anti-brucella antibodies was studied by the buffered plate agglutination test (BPA, the tube agglutination test (SAT, the 2-mercaptoethanol (2-ME agglutination test and indirect ELISA tests, using the cytosolic fraction from Brucella abortus S19

  20. Epidemiology of Brucella infection in the human, livestock and wildlife interface in the Katavi-Rukwa ecosystem, Tanzania.

    Science.gov (United States)

    Assenga, Justine A; Matemba, Lucas E; Muller, Shabani K; Malakalinga, Joseph J; Kazwala, Rudovick R

    2015-08-08

    Brucellosis is a zoonosis of public health importance worldwide. In Tanzania, the disease is underreported due to insufficient awareness, inadequate diagnostic protocols, including lack of appropriate reagents for diagnosis. Livestock and wildlife are considered potential sources of infection to humans; however, the role played by these carriers in the epidemiology of the disease in the ecosystems in Tanzania is not fully understood. The objective of this study was to establish the prevalence of anti-Brucella antibodies in humans, wildlife and livestock; and molecular prevalence of Brucella spp in cattle and goats in the Katavi- Rukwa ecosystem. Anti-Brucella antibodies were detected in humans at 0.6 % (95 % CI: 0.1, 2.1 %); cattle at 6.8 % (95 % CI: 5.4, 8.5 %), goats at 1.6 % (95 % CI: 0.4, 4.1 %) and buffaloes at 7.9 % (95 % CI: 1.7, 21.4 %). One of the two sampled lions tested positive. Cattle had a significantly higher prevalence of anti-Brucella antibodies as compared to goats (P Brucella infection. Eight (3.5 %) out of 231 milk samples tested were positive for Brucella spp on Polymerase Chain Reaction (PCR), and Brucella abortus biovar 1 was detected in cattle milk. However, no Brucella spp were detected in goat milk. This study has shown the presence of anti- Brucella antibodies in humans, livestock, and wildlife in the Katavi- Rukwa ecosystem. Transmission of the infection between wildlife, livestock and humans is likely to continue due to increasing human activities in the human wildlife interface. This information is an important contribution to public health policy development in the human wildlife interface of the Katavi- Rukwa ecosystem.

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

    Science.gov (United States)

    Fernández, Andrea G.; Bonetto, Josefina; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.

    2015-01-01

    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. PMID:26448160

  2. Origins and global context of Brucella abortus in Italy.

    Science.gov (United States)

    Garofolo, Giuliano; Di Giannatale, Elisabetta; Platone, Ilenia; Zilli, Katiuscia; Sacchini, Lorena; Abass, Anna; Ancora, Massimo; Cammà, Cesare; Di Donato, Guido; De Massis, Fabrizio; Calistri, Paolo; Drees, Kevin P; Foster, Jeffrey T

    2017-02-02

    Brucellosis is a common and chronic disease of cattle and other bovids that often causes reproductive disorders. Natural infection in cattle is caused by Brucella abortus and transmission typically occurs during abortions, calving, or nursing. Brucellosis is also a major zoonotic disease due to contamination of dairy products or contact with the tissues of infected animals. Brucellosis has been eradicated from most of the developed world in the last 40 years but persists in many regions-the disease remains prevalent in portions of Africa, the Middle East, Asia, and Central and South America, as well as in the Mediterranean basin. In Italy, B. abortus has persisted in southern regions in both cattle and water buffalo. Previous attempts at analyzing the phylogenetics of B. abortus in Italy have been challenging due to limited genetic variability and unresolved global population genetic structure of this pathogen. We conducted genome-wide phylogenetic analyses on 11 representative strains of B. abortus from Italy, and compared these sequences to a worldwide collection of publically available genomes. Italian isolates belong to three clades that are basal to the main and global B. abortus lineage. Using six SNP-based assays designed to identify substructure within the Italian clades, we surveyed a collection of 261 isolates and found that one clade predominates throughout endemic districts in the country, while the other two clades are more geographically restricted to portions of southern Italy. Although related strains exist worldwide, B. abortus isolates from Italy are substantially different than those found in much of the rest of Europe and North America, and are more closely related to strains from the Middle East and Asia. Our assays targeting genetic substructure within Italy allowed us to identify the major lineages quickly and inexpensively, without having to generate whole genome sequences for a large isolate collection. These findings highlight the

  3. Antibody Production From Immunized Rabbits By Brucella Abortus

    International Nuclear Information System (INIS)

    Sadi, Suharni

    2002-01-01

    In this research Brucella abortus was used as antigen which was made by killing the bacteria in boiling water for 1 hour and then add 0.5% phenol. The suspension of bacteria of 6x10 8 cells/mm 3 was used as antigen. Rabbits of about 3 months old were injected with 0.50 mI of the antigen by intradermal route with an interval of two weeks. The animals were divided in three groups i.e. A (control group), B (immunization group) and C (immunization and irradiation group). In C group, the animals were first immunized by the antigen and then 2 days later were irradiated by a low dose of 0.50 Gy of gamma rays. Each group consisted of 3 animals. Parameters were observed by weighing the animals, counting leucocyte and lymphocyte cells, and anaIysing the antisera. The research were done two times, included immunization I x, boostered 4 x and analysed 5x. The results obtained were as follows: A (control group) yielded 2.34 g/dl of non specific antibody, B (immunization group) yielded 3.22 g/dI of specific antibody, C (immunization and irradiation group) yielded 3.50 g/dl of spesific antibody. The leucocyte cells of A, B , and C group were 8.240, 7.887, and 8.120 cells/mm 3, respectively. The lymphocyte cells of A, B, and C group were 69%, respectively. The weigh of A, B, and C group were 1.44; 1.53; and l.41 kg, respectively. The purpose of this research was prepared to produce the diagnostic reagen (RIA Kit) for a rapid detection of animals disease especially brucellosis. It seemed that C group (the combination of immunization and irradiation treatments) yielded the highest value of antibody production compared to another group

  4. Vaccination with a ΔnorD ΔznuA Brucella abortus mutant confers potent protection against virulent challenge.

    Science.gov (United States)

    Yang, Xinghong; Clapp, Beata; Thornburg, Theresa; Hoffman, Carol; Pascual, David W

    2016-10-17

    There remains a need for an improved livestock vaccine for brucellosis since conventional vaccines are only ∼70% efficacious, making some vaccinated animals susceptible to Brucella infections. To address this void, a vaccine capable of evoking protective immunity, while still being sufficiently attenuated to produce minimal disease, is sought. In this pursuit, the ΔnorD ΔznuA B. abortus-lacZ (termed as znBAZ) was developed to be devoid of functional norD and znuA B. abortus genes, and to contain the lacZ as a marker gene. The results show that znBAZ is highly attenuated in mouse and human macrophages, and completely cleared from mouse spleens within eight weeks post-vaccination. Producing less splenic inflammation, znBAZ is significantly more protective than the conventional RB51 vaccine by more than four orders of magnitude. Vaccination with znBAZ elicits elevated numbers of IFN-γ + , TNF-α + , and polyfunctional IFN-γ + TNF-α + CD4 + and CD8 + T cells in contrast to RB51-vaccinated mice, which show reduced numbers of proinflammatory cytokine-producing T cells. These results demonstrate that znBAZ is a highly efficacious vaccine candidate capable of eliciting diverse T cell subsets that confer protection against parenteral challenge with virulent, wild-type B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Brucella spp. in equines slaughtered in the south region of Brazil

    Directory of Open Access Journals (Sweden)

    R.F. Santos

    2016-08-01

    Full Text Available ABSTRACT Bacteria of the genus Brucella are widespread in many countries. These microorganisms can infect humans and many wild and domestic animal species. These bacteria have zoonotic potential, and can cause economic and public health problems since they can be transmitted by direct contact with sick animals, through consumption of contaminated milk, raw meat and its derivatives (Soares et al., 2015. Brucellosis is considered a chronic evolving disease, unusual in horses, predominantly caused by Brucella abortus. However, it is not characterized by reproductive disorders in horses, but primarily by abscess in the cervical region, bursa, tendons, and joints. Transmission is likely to occur via ingestion of contaminated water and pastures, especially in areas endemic for bovine brucellosis (Ribeiro et al., 2008. The slaughterhouse is a strategic point for obtaining information about the animal and animal products, edible or not. This study investigated the presence of anti-Brucella spp. immunoglobulins in the serum samples from horses slaughtered in a slaughterhouse in southern Brazil, to estimate the frequency of Brucella spp. antibodies and determine the spatial distribution of the cases.

  6. Modeling the survivability of brucella to exposure of Ultraviolet radiation and temperature

    Science.gov (United States)

    Howe, R.

    Accumulated summation of daily Ultra Violet-B (UV-B = 290? to 320 ? ) data? from The USDA Ultraviolet Radiation Monitoring Program show good correlation (R^2 = 77%) with daily temperature data during the five month period from February through June, 1998. Exposure of disease organisms, such as brucella to the effects of accumulated UV-B radiation, can be modeled for a 5 month period from February through June, 1998. Estimates of a lethal dosage for brucell of UV-B in the environment is dependent on minimum/maximum temperature and Solar Zenith Angle for the time period. The accumulated increase in temperature over this period also effects the decomposition of an aborted fetus containing brucella. Decomposition begins at some minimum daily temperature at 27 to 30 degrees C and peaks at 39 to 40C. It is useful to view the summation of temperature as a threshold for other bacteria growth, so that accumulated temperature greater than some value causes decomposition through competition with other bacteria and brucella die from the accumulated effects of UV-B, temperature and organism competition. Results of a study (Cook 1998) to determine survivability of brucellosis in the environment through exposure of aborted bovine fetuses show no one cause can be attributed to death of the disease agent. The combination of daily increase in temperature and accumulated UV-B radiation reveal an inverse correlation to survivability data and can be modeled as an indicator of brucella survivability in the environment in arid regions.

  7. In vitro bactericidal activity of aminoglycosides, including the next-generation drug plazomicin, against Brucella spp.

    Science.gov (United States)

    Plazomicin is a next-generation aminoglycoside with a potentially improved safety profile compared to other aminoglycosides. This study assessed plazomicin MICs and MBCs in four Brucella spp. reference strains. Like other aminoglycosides and aminocyclitols, plazomicin MBC values equaled MIC values ...

  8. Immunogenicity and efficacy of a rough Brucella suis vaccine delivered orally or parenterally to feral swine

    Science.gov (United States)

    Brucella suis strain 353-1 is a stable vaccine strain that is clinically safe, does not cause positive serologic responses on conventional brucellosis surveillance tests, and induces humoral and cellular immunity in swine after vaccination. In this study, we evaluated tissue clearance and immunologi...

  9. Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

    NARCIS (Netherlands)

    Maio, E.; Begeman, L.; Bisselink, Y.J.W.M.; Tulden, van P.W.; Wiersma, L.; Hiemstra, S.; Ruuls, R.; Gröne, A.; Roest, H.I.J.; Willemsen, P.T.J.; Giessen, van der J.

    2014-01-01

    The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265)

  10. Prevalence of Brucella,/i> antibodies in donkeys ( Equus asinus ) in ...

    African Journals Online (AJOL)

    A cross-sectional study was designed to study the serological prevalence of antibodies against Brucella spp by using Rose Bengal Plate Test (RBPT) and Microtitre Serum Agglutination Test (MSAT). A total of six hundred (600) adult donkeys comprising of 393 males and 207 females were sampled from three local ...

  11. Colony-level assessment of Brucella and Leptospira in the Guadalupe fur seal, Isla Guadalupe, Mexico.

    Science.gov (United States)

    Ziehl-Quirós, E Carolina; García-Aguilar, María C; Mellink, Eric

    2017-01-24

    The relatively small population size and restricted distribution of the Guadalupe fur seal Arctocephalus townsendi could make it highly vulnerable to infectious diseases. We performed a colony-level assessment in this species of the prevalence and presence of Brucella spp. and Leptospira spp., pathogenic bacteria that have been reported in several pinniped species worldwide. Forty-six serum samples were collected in 2014 from pups at Isla Guadalupe, the only place where the species effectively reproduces. Samples were tested for Brucella using 3 consecutive serological tests, and for Leptospira using the microscopic agglutination test. For each bacterium, a Bayesian approach was used to estimate prevalence to exposure, and an epidemiological model was used to test the null hypothesis that the bacterium was present in the colony. No serum sample tested positive for Brucella, and the statistical analyses concluded that the colony was bacterium-free with a 96.3% confidence level. However, a Brucella surveillance program would be highly recommendable. Twelve samples were positive (titers 1:50) to 1 or more serovars of Leptospira. The prevalence was calculated at 27.1% (95% credible interval: 15.6-40.3%), and the posterior analyses indicated that the colony was not Leptospira-free with a 100% confidence level. Serovars Icterohaemorrhagiae, Canicola, and Bratislava were detected, but only further research can unveil whether they affect the fur seal population.

  12. Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast

    NARCIS (Netherlands)

    Maio, Elisa; Begeman, Lineke; Bisselink, Yvette; van Tulden, Peter; Wiersma, Lidewij; Hiemstra, Sjoukje; Ruuls, Robin; Gröne, Andrea; Roest, Hendrik-Ido-Jan; Willemsen, Peter; van der Giessen, Joke

    2014-01-01

    The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265)

  13. Immuogenicity and safety of a natural rough mutant of Brucella suis as a vaccine for swine

    Science.gov (United States)

    The objective of the current study was to evaluate the safety, immunogenicity and clearance of the natural rough mutant of Brucella suis strain 353-1 (353-1) as a vaccine in domestic swine. In three studies encompassing 155 animals, pigs were inoculated with 353-1 by conjunctival (5 x 10**7 CFU), p...

  14. Seroprevalence of Brucella antibodies in harbor seals in Alaska, USA, with age, regional, and reproductive comparisons.

    Science.gov (United States)

    Hoover-Miller, A; Dunn, J L; Field, C L; Blundell, G; Atkinson, S

    2017-09-20

    Populations of harbor seal Phoca vitulina in the Gulf of Alaska have dramatically declined during the past 4 decades. Numbers of seals in Glacier Bay, in southeast Alaska, USA, have also declined despite extensive protection. Causes of the declines and slow recovery are poorly understood. Brucellosis is a zoonotic disease that adversely affects reproduction in many domestic species. We measured the seroprevalence of Brucella antibodies in 554 harbor seals in 3 Alaska locations: Prince William Sound (PWS), Glacier Bay (GB), and Tracy Arm Fords Terror (TAFT) Wilderness Area. Objectives included testing for regional, sex, age, and female reproductive state differences in Brucella antibody seroprevalence, persistence in titers in recaptured seals, and differences in titers between mother seals and their pups. Overall, 52% of adults (AD), 53% of subadults (SA), 77% of yearlings (YRL), and 26% of Brucella. Results show higher seroprevalence (64%) for AD and SA seals in the depressed and declining populations in PWS and GB than in TAFT (29%). Lactating females were less likely to be seropositive than other AD females, including pregnant females. Further research is needed to seek evidence of Brucella infection in Alaskan harbor seals, identify effects on neonatal viability, and assess zoonotic implications for Alaska Natives who rely on harbor seals for food.

  15. Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

    NARCIS (Netherlands)

    Lista, F.; Reubsaet, F.A.G.; Santis, R. de; Parchen, R.R.; Jong, A.L. de; Kieboom, J.; Laaken, A.L. van der; Voskamp-Visser, I.A.I.; Fillo, S.; Jansen, H.J. de; Plas, J. van der; Paauw, A.

    2011-01-01

    Background: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks.

  16. Molecular epidemiology and antibiotic susceptibility of livestock Brucella melitensis isolates from Naryn Oblast, Kyrgyzstan.

    Science.gov (United States)

    Kasymbekov, Joldoshbek; Imanseitov, Joldoshbek; Ballif, Marie; Schürch, Nadia; Paniga, Sandra; Pilo, Paola; Tonolla, Mauro; Benagli, Cinzia; Akylbekova, Kulyash; Jumakanova, Zarima; Schelling, Esther; Zinsstag, Jakob

    2013-01-01

    The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.

  17. The attenuation effect of UVc radiation doses in gram-negative bacteria (Brucella, Yersinia, Escherichia coli)

    International Nuclear Information System (INIS)

    Al-Mariri, A.

    2006-06-01

    The gram-negative bacteria Yersinia enterocolitica sero group O:3 and O:9, and Brucella (Melitensis and abortus) together with Escherichia coli (O:157, DH5α-pEt15b), were investigated to evaluate their susceptibility to UV radiation at 254 nm. If the dose of UVc was 18.7 mW/cm 2 , the time required for inactivation of Y. enterocolitica and E. coli DH5α-pEt15b and O:157 was 240s and 360s in the dark and light respectively; where if the dose was 19.5 mW/cm 2 , the time required was 60s in the dark and 120s in light respectively. The time required for inactivation of Brucella strains (melitensis and abortus) if the dose was 18.7 mW/cm 2 was 240s in both dark and light, whereas it was 120s(dark) and 240s (light) respectively, when the dose was 19.5 mW/cm 2 . Using E. coli O:157 as control, it appears that Y. enterocolitica sero group O:3 and O:9 and vaccinal strains of Brucella (Rev. 1 and S19) are more sensitive to UV than wild Brucella strains. No relation was found between the sensitivity of Y. enterocolitica to UV and the presence or absence of a pYV + virulence plasmid. (author)

  18. Brucella abortus is Prevalent in Both Humans and Animals in Bangladesh.

    Science.gov (United States)

    Rahman, A K M A; Saegerman, C; Berkvens, D; Melzer, F; Neubauer, H; Fretin, D; Abatih, E; Dhand, N; Ward, M P

    2017-08-01

    To determine the role of different Brucella (B.) spp. in Bangladesh, 62 animal samples and 500 human sera were tested. Animal samples from cattle, goats and sheep (including milk, bull semen, vaginal swabs and placentas) were cultured for Brucella spp. Three test-positive human sera and all animal samples were screened by Brucella genus-specific real-time PCR (RT-PCR), and positive samples were then tested by IS711 RT-PCR to detect B. abortus and B. melitensis DNA. Only B. abortus DNA was amplified from 13 human and six animal samples. This is the first report describing B. abortus as the aetiological agent of brucellosis in occupationally exposed humans in Bangladesh. Of note is failure to detect B. melitensis DNA, the species most often associated with human brucellosis worldwide. Further studies are required to explore the occurrence of Brucella melitensis in Bangladesh. © 2017 Blackwell Verlag GmbH.

  19. Molecular epidemiology of Brucella genotypes in patients at a major hospital in central Peru

    NARCIS (Netherlands)

    Nöckler, Karsten; Maves, Ryan; Cepeda, David; Draeger, Angelika; Mayer-Scholl, Anne; Chacaltana, Jesus; Castañeda, María; Espinosa, Benjamin; Castillo, Rosa; Hall, Eric; Al Dahouk, Sascha; Gilman, Robert H.; Cabeza, Franco; Smits, Henk L.

    2009-01-01

    The multiple-locus variable-number repeat analysis of 90 human Brucella melitensis isolates from a large urban area in central Peru revealed variations at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) out of 16 loci investigated, of which 1 (Bruce42) also is used for species identification. Ten

  20. Brucella β 1,2 cyclic glucan is an activator of human and mouse dendritic cells.

    Directory of Open Access Journals (Sweden)

    Anna Martirosyan

    Full Text Available Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+ and CD8(+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

  1. The attenuation effect of UVc radiation doses in gram-negative bacteria (Brucella, Yersinia, Escherichia coli)

    International Nuclear Information System (INIS)

    Al-Mariri, A.

    2007-01-01

    The gram-negative bacteria Yersinia enterocolitica sero group O:3 and O:9, and Brucella (Melitensis and abortus) together with Escherichia coli (O:157, DH5alpha-pEt15b), were investigated to evaluate their susceptibility to UV radiation at 254 nm. If the dose of UVc was 18.7 mW/cm2, the time required for inactivation of Y. enterocolitica and E. coli DH5alpha-pEt15b and O:157 was 240s and 360s in the dark and light respectively. Where if the dose was 19.5 mW/cm2, the time required was 60s in the dark and 120s in light respectively. The time required for inactivation of Brucella strains (melitensis and abortus) if the dose was 18.7 mW/cm2 was 240s in both dark and light, whereas it was 120s (dark) and 240s (light) respectively, when the dose was 19.5 mW/cm2. Using E. coli O:157 as control, it appears that Y. enterocolitica sero group O:3 and O:9 and vaccinal strains of Brucella (Rev. 1 and S19) are more sensitive to UV than wild Brucella strains. No relation was found between the sensitivity of Y. enterocolitica to UV and the presence or absence of a pYV+ virulence plasmid. (author)

  2. Et tilfælde af brucella spondylodiscitis efter rejse til Libanon

    DEFF Research Database (Denmark)

    Nielsen, Stig Lønberg; Johansen, Isik Somuncu

    2012-01-01

    Brucellosis is a widespread endemic zoonotic infection affecting more than 500,000 people per year. The disease is very uncommon in Denmark and almost always imported. We present a case of a 57 year-old male with blood culture and magnetic resonance imaging verified brucella spondylodiscitis. Prior...

  3. Clinical and Diagnostic Aspects of Brucellosis and Antimicrobial Susceptibility of Brucella Isolates in Hamedan, Iran.

    Science.gov (United States)

    Torkaman Asadi, Fatemeh; Hashemi, Seyyed Hamid; Alikhani, Mohammad Yousef; Moghimbeigi, Abbas; Naseri, Zahra

    2017-05-24

    Current drug regimens for brucellosis are associated with relatively high rates of therapeutic failure or relapse. Reduced antimicrobial susceptibility of Brucella spp. has been proposed recently as a potential cause of therapeutic failure. The aim of this study was to evaluate the antibiotic resistance pattern of Brucella melitensis clinical isolates by E-test method in Hamadan, west of Iran. In a 15-month period, all patients with suspected brucellosis were enrolled. Blood specimens were collected for diagnosis of brucellosis by BACTEC system and serological tests. Antimicrobial susceptibility of clinical isolates to 7 antibiotics was assessed by the E-test method. One hundred forty-nine patients with brucellosis were evaluated. 38.3% of cultures of clinical samples were positive for BACTEC system, of which 91.2% were associated with a positive serological test result. No significant associations were found between serology and the culture method. All Brucella isolates were susceptible to doxycycline, streptomycin, gentamicin, ciprofloxacin, and moxifloxacin. However, decreased sensitivity to rifampin and trimethoprim-sulfamethoxazole was found in 35.1% and 3.5% of isolates, respectively. Because of the high rates of intermediate sensitivity to rifampin among Brucella isolates, this drug should be prescribed with caution. We recommend restricting the use of rifampin for treatment of brucellosis except as an alternative drug for special situations.

  4. Brucella microti sp. nov., isolated from the common vole Microtus arvalis

    Czech Academy of Sciences Publication Activity Database

    Scholz, H. C.; Hubálek, Zdeněk; Sedláček, I.; Vergnaud, G.; Tomaso, H.; Al Dahouk, S.; Melzer, F.; Kämpfer, P.; Neubauer, H.; Cloeckaert, A.; Maquart, M.; Zygmunt, M. S.; Whatmore, A. M.; Falsen, E.; Bahn, P.; Göllner, C.; Pfeffer, M.; Huber, B.; Busse, H.-J.; Nöckler, K.

    2008-01-01

    Roč. 58, č. 2 (2008), s. 375-382 ISSN 1466-5026 Institutional research plan: CEZ:AV0Z60930519 Keywords : common vole * Brucella microti * rodent brucellosis Subject RIV: EE - Microbiology, Virology Impact factor: 2.222, year: 2008

  5. High throughput MLVA-16 typing for Brucella based on the microfluidics technology

    Directory of Open Access Journals (Sweden)

    Di Giannatale Elisabetta

    2011-03-01

    Full Text Available Abstract Background Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology. Results The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created. Conclusion In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to

  6. Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

    Directory of Open Access Journals (Sweden)

    Meritxell García-Quintanilla

    Full Text Available The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010, one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/106 cells compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

  7. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo

    Directory of Open Access Journals (Sweden)

    Vincenzo Caporale

    2010-03-01

    Full Text Available Approximately 250 000 water buffalo (Bubalus bubalis live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150 000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51 has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19. The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

  8. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo.

    Science.gov (United States)

    Caporale, Vincenzo; Bonfini, Barbara; Di Giannatale, Elisabetta; Di Provvido, Andrea; Forcella, Simona; Giovannini, Armando; Tittarelli, Manuela; Scacchia, Massimo

    2010-01-01

    Approximately 250,000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150,000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

  9. The Antibacterial Activity of Selected Labiatae (Lamiaceae Essential Oils against Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Ayman Al-Mariri

    2013-03-01

    Full Text Available Background: Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. The major cause of brucellosis worldwide is brucella melitensis. Medicinal plants are considered as new antibacterial sources that could replace conventional antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the efficacy of some native plants, alone and in combination with some antibiotics, in the treatment of brucellosis.Methods: The present experimental in vitro study was carried out to evaluate the anti-brucella activities of essential oils of Rosmarinus officinalis L., Origanum syriacum, Thymus syriacus, Salvia palaestina Benth, Mentha piperia, and Lavandula stoechas L., alone and in combination with some antibiotics. The activity against 16 tetracycline-resistant B. melitensis isolates was determined by disc diffusion method incorporating a concentration of 5%. Antibiotic discs were also used as a control. Microdilution brucella broth susceptibility assay was used in order to determine the MICs of essential oils and five antibiotics.Results: Among all the herbs evaluated, only the essential oils of O. syriacum and T. syriacus plants demonstrated most effective anti-brucella activity, and were then chosen for MIC study. The minimal inhibitory concentrations (MIC50 of essential oils of O. syriacum and T. syriacus against tetracycline-resistant B. melitensis were 3.125 µl/ml and 6.25 µl/ml, respectively. Conclusion: Among the essential oils studied, those of O. syriacum and T. syriacus were most effective. Since a combination of levofloxacin and Thymus syriacus essential oil increased the efficacy of this antibiotic, O. syriacum and T. syriacus are recommended to be used as bactericidal agents against B. melitensis.

  10. Serological survey of Brucella canis in dogs in urban Harare and selected rural communities in Zimbabwe

    Directory of Open Access Journals (Sweden)

    Simbarashe Chinyoka

    2014-02-01

    Full Text Available A cross-sectional study was conducted in order to detect antibodies for Brucella canis (B. canis in dogs from urban Harare and five selected rural communities in Zimbabwe. Sera from randomly selected dogs were tested for antibodies to B. canis using an enzyme-linked immunosorbent assay. Overall, 17.6% of sera samples tested (57/324, 95% CI: 13.5–21.7 were positive for B. canis antibodies. For rural dogs, seroprevalence varied from 11.7% – 37.9%. Rural dogs recorded a higher seroprevalence (20.7%, 95% CI: 15.0–26.4 compared with Harare urban dogs (12.7%, 95% CI: 6.9–18.5 but the difference was not significant (p = 0.07. Female dogs from both sectors had a higher seroprevalence compared with males, but the differences were not significant (p > 0.05. Five and two of the positive rural dogs had titres of 1:800 and 1:1600, respectively, whilst none of the positive urban dogs had a titre above 1:400. This study showed that brucellosis was present and could be considered a risk to dogs from the studied areas. Further studies are recommended in order to give insight into the epidemiology of brucellosis in dogs and its possible zoonotic consequences in Zimbabwe. Screening for other Brucella spp. (Brucella abortus, Brucella melitensis and Brucella suis other than B. canis is also recommended.

  11. Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

    Science.gov (United States)

    Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

    2015-10-05

    We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight co