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production of the type iv secretion system differs among brucella species as revealed with virb5- and virb8-specific antisera.expression of the virb operon, encoding the type iv secretion system required for brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic ph values. to analyze the production of virb proteins, polyclonal antisera against b. suis virb5 and virb8 were generated. western blot analysis revealed that virb5 and virb8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. unl ...200312595417
role of the brucella suis lipopolysaccharide o antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages.brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. these organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. however, the molecular mechanisms and the microbial factors involved are poorly understood. smooth lipopolysaccharide (lps) of brucella has b ...200312595466
evaluation of cattle for experimental infection with and transmission of brucella suis biovar 4.to determine whether cattle can become persistently infected with brucella suis biovar 4, whether the organism can be transmitted vertically or horizontally, and whether tests for bovine brucellosis are diagnostic.200312725315
type iii secretion homologs are present in brucella melitensis, b. ovis, and b. suis biovars 1, 2, and 3.protein sequences from characterized type iii secretion (tts) systems were used as probes in silico to identify several tts gene homologs in the genome sequence of brucella suis biovar 1 strain 1330. four of the genes, named flhb, flip, flir, and flif on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in pcr and hybridization assays to determine their distribution among other brucella nomen species and biovars. the results indicated that flhb, flip, flir and ...200312732970
subversion and utilization of the host cell cyclic adenosine 5'-monophosphate/protein kinase a pathway by brucella during macrophage infection.brucella spp. are intramacrophage pathogens that induce chronic infections in a wide range of mammals, including domestic animals and humans. therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity. however, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that brucella use to counteract these responses and secure thei ...200312759440
epitope mapping of the brucella melitensis bp26 immunogenic protein: usefulness for diagnosis of sheep brucellosis.sequencing of bp26, the gene encoding the brucella sp. immunogenic bp26 periplasmic protein, was performed in the reference strains of brucella abortus, b. suis, and b. ovis. the three bp26 sequences were almost identical to that published for b. melitensis 16m bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. the bp26 genes of the seven b. abortus biovar reference strains and b. abortus s19 and rb51 vaccine strains were also ...200312853399
characterization of new members of the group 3 outer membrane protein family of brucella spp.impairment of the omp25 gene in brucella spp. leads to attenuated strains and confers protection to the host. omp25 and omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (omps) (25 to 34 kda). recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in b. melitensis or b. abortus. in the present study, using protein microsequencing, we identified new members of group 3 o ...200312874309
purification and characterization of an immunogenic aminopeptidase of brucella melitensis.an immunogenic aminopeptidase was purified from brucella melitensis strain vtrm1. the purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. this procedure resulted in a yield of 29% and a 144-fold increase in specific activity. the aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kda and an isoelectric point of 4.8. its activity was optimal at ph 7.0 at 40 degrees c. the enzyme was strongly inhibited by edta, 1,10-phenathr ...200312933870
fluorescence polarization assay for diagnosis of human brucellosis.fluorescence polarization immunoassay (fpa) uses molecular rotational properties to measure antibody binding to antigen directly. the potential use of this method was assessed in comparison to a competitive enzyme immunoassay (celisa) and conventional serological tests for the diagnosis of brucellosis on a total of 587 human sera. based on 340 sera from asymptomatic blood donors with no evidence of brucellosis, the specificity of the fpa was 97.9 % using a cut-off value of 72 mp. sera from bruce ...200312972582
identification of species of brucella using fourier transform infrared spectroscopy.fourier transform infrared spectroscopy (ftir) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. the marked tendency of brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. we studied the type strains of the different species and biovar ...200314500003
molecular characterization of brucella abortus chromosome ii recombination.large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. the recently completed brucella melitensis 16m and brucella suis 1330 genomes have facilitated the investigation of such events in the brucella spp. suppressive subtractive hybridization (ssh) was employed in identifying genomic differences between b. melitensis 16m and brucella abortus 2308. analysis of 45 ssh clones revealed several deletions on chromosomes of b. abortus ...200314526025
[evaluation of 2 hemoculture media for the isolation of brucella spp.]].the diagnostic efficiency of two hemoculture media for the detection of different species of brucella strains was evaluated. strains of brucella melitensis, brucella suis, brucella abortus, brucella ovis, and brucella abortus s19 were used. each strain was diluted in phosphate buffer saline (pbs) to obtain a concentration of 10(5) colony forming units/ml (cfu/ml). blood from goats, pigs, cattle, and sheep was mixed with the bacterial suspension to obtain a final concentration minor or equal to 1 ...200314587372
wild boars (sus scrofa) as reservoirs of brucella suis biovar 2 in croatia.this work presents the results of findings for brucellosis in wild boars and domestic swine in two regions of croatia. in the region of djakovo the blood samples of 211 wild boars were analysed and in 29.4% of the samples serologically positive reactions were established. in the same region the blood samples of 1080 domestic swine on pastures were also analysed and positive serological reactions were established in 12.3%. in the regions around lonjsko polje the blood samples of 53 wild boars wer ...200314680058
antibody reactivity to omp31 from brucella melitensis in human and animal infections by smooth and rough brucellae.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 an ...200414715555
virb1 orthologs from brucella suis and pkm101 complement defects of the lytic transglycosylase required for efficient type iv secretion from agrobacterium tumefaciens.type iv secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells. the translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions. however, orthologs to proteins of the prototypical type iv system, virb of agrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complemen ...200414973016
brucella pathogenesis, genes identified from random large-scale screens.pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors. however, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship. bacteria that form these relationships have developed other strategies to infect and grow in their hosts. this is particularly true for brucella and othe ...200414979322
impairment of intramacrophagic brucella suis multiplication by human natural killer cells through a contact-dependent mechanism.brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. nk cells play a key role in host defense. they are implicated in an early immune response to a variety of pathogens. however, it was shown that they do not control brucella infection in mice. on the other hand, nk cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human nk cells mediate the killing of intramacrophagic ...200415039355
different responses of macrophages to smooth and rough brucella spp.: relationship to virulence.by comparing smooth wild-type brucella strains to their rough mutants, we show that the lipopolysaccharide (lps) o side chain of pathogenic brucella has a dramatic impact on macrophage activation. it favors the development of virulent brucella by preventing the synthesis of immune mediators, important for host defense. we conclude that this o chain property is firmly linked to brucella virulence.200415039375
brucella osteomyelitis of the proximal tibia: a case report.brucellosis is a disease of domestic and wild animals that is transmittable to humans. although endemic in some parts of the world, brucellosis is an uncommon human pathogen in the united states. the clinical presentation of brucellosis is nonspecific, and brucella osteomyelitis can produce lytic lesions on radiographs that resemble neoplasm. diagnosis can therefore be difficult unless a high index of suspicion is maintained. we present a case of brucella osteomyelitis of the proximal tibia that ...200415296202
dna polymorphism in the omp25/omp31 family of brucella spp.: identification of a 1.7-kb inversion in brucella cetaceae and of a 15.1-kb genomic island, absent from brucella ovis, related to the synthesis of smooth lipopolysaccharide.five genes homologous to the well-known omp25 and omp31 genes, that code for two major brucella spp. outer membrane proteins (omps), have been detected in the genome of brucella melitensis 16m and brucella suis 1330. in this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical brucella species reference strains and in representative strains of the recently proposed species brucella cetaceae and brucella pinniped ...200415374004
release of periplasmic proteins of brucella suis upon acidic shock involves the outer membrane protein omp25.the survival and replication of brucella in macrophages is initially triggered by a low intraphagosomal ph. in order to identify proteins released by brucella during this early acidification step, we analyzed brucella suis conditioned medium at various ph levels. no significant proteins were released at ph 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, b. suis released a substantial amount of soluble proteins. comparison of 13 n-terminal amino acid sequences determined by ed ...200415385468
essential role of vaccines in brucellosis control and eradication programs for livestock.brucellosis, in particular infections with brucella abortus, brucella melitensis or brucella suis, remains a significant human health threat in many areas of the world. the persistence of pathogenic brucella spp. in domestic livestock or free-ranging wildlife remains unresolved, despite decades of regulatory efforts worldwide. although vaccination is probably the most economic control measure, administration of currently available vaccines alone is not sufficient for elimination of brucellosis i ...200516372886
efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to yersinia enterocolitica o:9.yersinia enterocolitica o:9 bears a smooth lipopolysaccharide (s-lps) of brucella sp. o-chain a+c/y epitopic structure and is a cause of false-positive serological reactions (fpsr) in standard tests for cattle brucellosis. brucella s-lps, cross-reacting s-lpss representing several o-chain epitope combinations, brucella core lipid a epitopes (rough lps), brucella abortus s-lps-derived polysaccharide, native hapten polysaccharide, rough lps group 3 outer membrane protein complexes, recombinant bp2 ...200515642999
vgamma9vdelta2 t cells use a combination of mechanisms to limit the spread of the pathogenic bacteria brucella.human vgamma9vdelta2 t cells play a crucial role in early immune response to intracellular pathogens. in brucellosis infection, this population of cells is drastically increased in the peripheral blood of patients during the acute phase of infection. in vitro, vgamma9vdelta2 t cells exhibit strong cytolytic activity against brucella-infected cells and are able to impair intracellular growth of brucella suis in autologous macrophages. in this study, we have investigated the relative importance of ...200515668339
cloning, expression, and purification of brucella suis outer membrane proteins.brucella, an aerobic, nonsporeforming, nonmotile gram-negative coccobacillus, is a nih/cdc category b bioterror threat agent that causes incapacitating human illness. medical defense against the bioterror threat posed by brucella would be strengthened by development of a human vaccine and improved diagnostic tests. central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. outer membrane proteins (omps) are particularly attractive f ...200515721781
secondary structure in the target as a confounding factor in synthetic oligomer microarray design.secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. it is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from brucella su ...200515755320
structures of two core subunits of the bacterial type iv secretion system, virb8 from brucella suis and comb10 from helicobacter pylori.type iv secretion systems (t4sss) are commonly used secretion machineries in gram-negative bacteria. they are used in the infection of human, animal, or plant cells and the propagation of antibiotic resistance. the t4ss apparatus spans both membranes of the bacterium and generally is composed of 12 proteins, named virb1-11 and vird4 after proteins of the canonical agrobacterium tumefaciens t4ss. the periplasmic core complex of virb8/virb10 structurally and functionally links the cytoplasmic ntpa ...200515764702
completion of the genome sequence of brucella abortus and comparison to the highly similar genomes of brucella melitensis and brucella suis.brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical brucella species in the alpha-2 subdivision of the proteobacteria. we report the complete genome sequence of brucella abortus field isolate 9-941 and compare it to those of brucella suis 1330 and brucella melitensis 16 m. the genomes of these brucella species are strikingly similar, with nearly identical genetic content and gene organization. however, a number of insertion-dele ...200515805518
from the discovery of the malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis.brucellosis is not a sustainable disease in humans. the source of human infection always resides in domestic or wild animal reservoirs. the routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. new brucella strains or species may emerge and existing brucella species adapt to changing social, cultural, travel and agricultural environment. brucella melitensis is the most important zoonotic agent, followed by brucella abortus and bruc ...200515845228
requirement of mgtc for brucella suis intramacrophage growth: a potential mechanism shared by salmonella enterica and mycobacterium tuberculosis for adaptation to a low-mg2+ environment.a brucella suis mgtc mutant is defective for growth within macrophages and in low-mg(2+) medium. these phenotypes are strikingly similar to those observed with mgtc mutants from salmonella enterica and mycobacterium tuberculosis, two other pathogens that proliferate within phagosomes. mgtc appears as a remarkable virulence factor that would have been acquired by distantly related intracellular pathogens to contribute to the adaptation to a low-mg(2+) environment in the phagosome.200515845525
isolation of brucella suis biovar 3 from horses in croatia. 200515866906
human brucellosis in a nonendemic country: a report from germany, 2002 and 2003.human brucellosis has become a rare disease in germany since the eradication of bovine and ovine/caprine brucellosis in this country. therefore, most physicians are unfamiliar with the illnesses clinical presentation, diagnostic tools, and therapeutic strategies. this retrospective study was carried out to evaluate the epidemiological, clinical, and laboratory features of human brucellosis in germany in the years 2002 and 2003. thirty-one bacterial isolates from 30 patients sent to the german na ...200515959815
putative outer membrane autotransporter protein influences survival of brucella suis in balb/c mice.in gram-negative bacteria, autotransporters are secreted proteins able to translocate themselves through the inner- and outer-membranes to the cell surface or to the extracellular environment. the influence of the putative outer membrane autotransporter (omaa) protein to the persistence of brucella suis was investigated. sequence analyses revealed that the omaa protein of b. suis strain 1330 consists of a signal peptide, a passenger alpha-domain, and a transporter beta-domain, which are the char ...200515970403
carboxyl-terminal protease regulates brucella suis morphology in culture and persistence in macrophages and mice.the putative carboxyl-terminal processing protease (ctpa) of brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. the b. suis ctpa protein shared up to 77% homology with ctpa proteins of other bacteria. a ctpa-deficient brucella strain (1330deltactpa), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growt ...200516077124
identification of a new virulence factor, bvfa, in brucella suis.we report the identification of bvfa (for brucella virulence factor a), a small periplasmic protein unique to the genus brucella, which is essential for the virulence of brucella suis. a bvfa knockout mutant was highly attenuated both in in vitro macrophage infection assays and in vivo in the murine model of brucellosis. fluorescence-activated cell sorting analysis with green fluorescent protein fusions showed that the expression of bvfa is induced within macrophages by phagosome acidification a ...200516113268
brucella abortus virb12 is expressed during infection but is not an essential component of the type iv secretion system.the brucella abortus virb operon, consisting of 11 genes, virb1 to virb11, and two putative genes, orf12 (virb12) and orf13, encodes a type iv secretion system (t4ss) that is required for intracellular replication and persistent infection in the mouse model. this study was undertaken to determine whether orf12 (virb12) encodes an essential part of the t4ss apparatus. the virb12 gene was found to encode a 17-kda protein, which was detected in vitro in b. abortus grown to stationary phase. mice in ...200516113325
targeting of the virulence factor acetohydroxyacid synthase by sulfonylureas results in inhibition of intramacrophagic multiplication of brucella suis.the acetohydroxyacid synthase (ahas) of brucella suis can be effectively targeted by the sulfonylureas chlorimuron ethyl and metsulfuron methyl. growth in minimal medium was inhibited, and multiplication in human macrophages was totally abolished with 100 microm of sulfonylureas. metsulfuron methyl-resistant mutants showed reduced viability in macrophages and reduced ahas activity.200516127072
absence of evidence for the participation of the macrophage cellular prion protein in infection with brucella suis.brucella spp. are stealthy bacteria that enter host cells without major perturbation. the molecular mechanism involved is still poorly understood, although numerous studies have been published on this subject. recently, it was reported that brucella abortus utilizes cellular prion protein (prp(c)) to enter the cells and to reach its replicative niche. the molecular mechanisms involved were not clearly defined, prompting us to analyze this process using blocking antibodies against prp(c). however ...200516177294
analysis of the behavior of eryc mutants of brucella suis attenuated in macrophages.the facultatively intracellular pathogen brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. this property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. the ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. allelic exchange mutants in eryc of brucella suis were erythritol sensitive in ...200516177356
differential use of the two high-oxygen-affinity terminal oxidases of brucella suis for in vitro and intramacrophagic multiplication.expression of the high-oxygen-affinity cytochrome cbb3 and cytochrome bd ubiquinol oxidases of brucella suis was studied in vitro and in the intramacrophagic niche, which was previously proposed to be oxygen limited. the cytochrome cbb3 oxidase was exclusively expressed in vitro, whereas the cytochrome bd oxidase was preferentially used inside macrophages and contributed to intracellular bacterial replication.200516239582
the incp island in the genome of brucella suis 1330 was acquired by site-specific integration.an 18,228-bp region containing open reading frames predicted to be derived from the incp plasmid or phage ancestors is present in the genomes of brucella suis biovars 1 to 4, b. canis, b. neotomae, and strains isolated from marine mammals, but not in b. melitensis, b. abortus, b. ovis, and b. suis biovar 5. the presence of circular excision intermediates and the results of an analysis of sequenced bacterial genomes suggest that the region downstream of the guaa gene is a hotspot for site-specifi ...200516239585
peptidoglycan degradation by specialized lytic transglycosylases associated with type iii and type iv secretion systems.specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of gram-negative bacteria. specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. this paper reports the analysis of selected members of the specialized lytic transglycosylase family from type iii and type iv secretion systems. these proteins were analysed in vivo by assaying their ability to complement the dna transfer defect of ...200516272370
the putative lytic transglycosylase virb1 from brucella suis interacts with the type iv secretion system core components virb8, virb9 and virb11.virb1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type iv secretion systems via localized lysis of the peptidoglycan. this paper presents the biochemical analysis of interactions of purified brucella suis virb1 with core components of the type iv secretion system. genes encoding virb1, virb8, virb9, virb10 and virb11 were cloned into expression vectors; the affinity-tagged proteins were purified from escherichia coli, and analyses by gel filtrat ...200516272371
functional interactions between type iv secretion systems involved in dna transfer and virulence.this paper reports an analysis of the functional interactions between type iv secretion systems (t4ss) that are part of the conjugative machinery for horizontal dna transfer (ct4ss), and t4ss involved in bacterial pathogenicity (pt4ss). the authors' previous work showed that a conjugative coupling protein (t4cp) interacts with the virb10-type component of the t4ss in order to recruit the protein-dna complex to the transporter for conjugative dna transfer. this study now shows by two-hybrid analy ...200516272374
whole-genome analyses of speciation events in pathogenic brucellae.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 patho ...200516299333
high susceptibility of human dendritic cells to invasion by the intracellular pathogens brucella suis, b. abortus, and b. melitensis.bacteria from the brucella genus are able to survive and proliferate within macrophages. because they are phylogenetically closely related to macrophages, myeloid dendritic cells (dcs) constitute potential targets for brucella bacteria. here we report that dcs display a great susceptibility to brucella infection. therefore, dcs might serve as a reservoir and be important for the development of brucella bacteria within their host.200516299342
seroprevalence of brucellosis, tularemia, and yersiniosis in wild boars (sus scrofa) from north-eastern germany.brucellosis and tularemia are classical zoonotic diseases transmitted from an animal reservoir to humans. both, wildlife and domestic animals, contribute to the spreading of these zoonoses. the surveillance of the animal health status is strictly regulated for domestic animals, whereas systematic disease monitoring in wildlife does not exist. the aim of the present study was to provide data on the prevalence of anti-brucella, anti-francisella and anti-yersinia antibodies in wild boars from north ...200516364020
the brucella suis type iv secretion system assembles in the cell envelope of the heterologous host agrobacterium tumefaciens and increases incq plasmid pls1 recipient competence.pathogenic brucella species replicate within mammalian cells, and their type iv secretion system is essential for intracellular survival and replication. the options for biochemical studies on the brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. to overcome these problems, we heterologously expressed the brucella suis virb operon in the closely related alpha(2)-proteobacterium agrobacterium t ...200616368963
catabolism of 1,5-anhydro-d-fructose in sinorhizobium morelense s-30.7.5: discovery, characterization, and overexpression of a new 1,5-anhydro-d-fructose reductase and its application in sugar analysis and rare sugar synthesis.the bacterium sinorhizobium morelense s-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-d-fructose (af) as the sole carbon source. this strain metabolized af by a novel pathway involving its reduction to 1,5-anhydro-d-mannitol (am) and the further conversion of am to d-mannose by c-1 oxygenation. growth studies showed that the af metabolizing capability is not confined to s. morelense s-30.7.5 but is a more common feature among the rhizobiaceae. the af reducing enzyme wa ...200616461673
agrobacterium tumefaciens virb8 structure reveals potential protein-protein interaction sites.bacterial type iv secretion systems (t4ss) translocate dna and/or proteins to recipient cells, thus providing a mechanism for conjugative transfer of genetic material and bacterial pathogenesis. here we describe the first structure of a core component from the archetypal agrobacterium tumefaciens t4ss: the 2.2-a resolution crystal structure of the virb8 periplasmic domain (pvirb8(at)). virb8 forms a dimer in the crystal, and we identify residues likely important for stabilization of the dimer in ...200616481621
differentiation between serological responses to brucella suis and yersinia enterocolitica serotype o:9 after natural or experimental infection in pigs.false-positive serological reactions (fpsr) due to infections with yersinia enterocolitica serotype oratio9 (yeoratio9) are a problem in tests for brucellosis. in the present study, fpsr in classical and novel tests for brucellosis following experimental infections of pigs with yeoratio9 were compared with responses of b. suis biovar 2-inoculated pigs. fpsr were limited to 2-9 weeks post-yeoratio9 inoculation, while b. suis-infected pigs were test-positive throughout the 21-week period of invest ...200616490140
requirement of nord for brucella suis virulence in a murine model of in vitro and in vivo infection.a mutant of brucella suis bearing a tn5 insertion in nord, the last gene of the operon norefcbqd, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. the nord strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that nord is essential for brucella virulence.200616495577
dimerization and interactions of brucella suis virb8 with virb4 and virb10 are required for its biological activity.virb8-like proteins are essential components of type iv secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. based on cell biological, genetic, and x-ray crystallographic data, virb8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. here we report the results of a structure-function analysis of the periplasmic domain of virb8 from the ...200616648257
a large domain swap in the virb11 atpase of brucella suis leaves the hexameric assembly intact.virb11 atpases are hexameric assemblies that power type iv secretion systems in bacteria. the hexamer of brucella suis virb11 (bsb11), like that of the helicobacter pylori virb11 (hp0525), consists of a double ring structure formed by the n-terminal and c-terminal domains of each monomer. however, the monomer differs dramatically from that of hp0525 by a large domain swap that leaves the hexameric assembly intact but profoundly alters the nucleotide-binding site and the interface between subunit ...200616730027
the stringent response mediator rsh is required for brucella melitensis and brucella suis virulence, and for expression of the type iv secretion system virb.physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. one mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppgpp, synthesized by the action of rela/spot homologues. the intracellular pathogen brucella spp., causative agent of brucellosis, possesses a gene homologous to rela/spot, named rsh, encoding a (p)ppgpp synthetase as confirmed by heterologous complementation of a rela mut ...200616803581
the brucella abortus cyclic beta-1,2-glucan virulence factor is substituted with o-ester-linked succinyl residues.brucella periplasmic cyclic beta-1,2-glucan plays an important role during bacterium-host interaction. nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and deae-sephadex chromatography were used to characterize brucella abortus cyclic glucan. in the present study, we report that a fraction of b. abortus cyclic beta-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic beta-1,2-glucan. the oligosaccharide backbone is substituted ...200616816173
brucella outer membrane protein omp31 is a haemin-binding protein.the expression of haemin-binding proteins (hbps) in the outer membrane is one of the strategies used by gram-negative bacteria to obtain iron from the host. no hbp has been described in brucella spp. we investigated whether omp31, an outer membrane protein from brucella with homology to hbps from bartonella quintana, is an hbp. soluble recombinant omp31 bound specifically to haemin-agarose, while an unrelated brucella protein (sura) did not. a similar experiment showed that native omp31 found in ...200616517201
development of a multiplex pcr assay for polymorphism analysis of brucella suis biovars causing brucellosis in swine.swine brucellosis is caused by the biovars 1, 2 and 3 of brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. based on sequence variation of the omp2b gene, we have developed a four primer set multiplex pcr assay that was tested for polymorphism analysis of b. suis biovars causing brucellosis in swine. the assay exploits the single nucleotide polymorphisms found in omp2b g ...200616530357
swapping of periplasmic domains between brucella suis virb8 and a psb102 virb8 homologue allows heterologous complementation.a brucella suis mutant with a nonpolar deletion in the virb8 gene was attenuated in a macrophage infection model. complementation with the b. suis virb8 protein expressed from the virb promoter restored virulence. expression of traj, a virb8 homologue from plasmid psb102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the n terminus of the b. suis virb8 protein and the c-terminal periplasmic domain of traj.200616861687
parenteral vaccination of domestic pigs with brucella abortus strain rb51.to determine the immunogenicity and efficacy of brucella abortus strain rb51 (srb51) as a vaccine in domestic pigs.200617014337
release of ll-37 by activated human vgamma9vdelta2 t cells: a microbicidal weapon against brucella suis.human vgamma9vdelta2 t cells play a crucial role in early immune response to intracellular pathogens. moreover, in brucellosis, these cells are drastically increased in the peripheral blood of patients during the acute phase of infection. in vitro, vgamma9vdelta2 t cells are capable of inhibiting brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial ef ...200617015740
characterization of a brucella sp. strain as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in new zealand.naturally acquired infection of humans with a marine mammal-associated brucella sp. has only been reported once previously in a study describing infections of two patients from peru. we report the isolation and characterization of a strain of brucella from a new zealand patient that appears most closely related to strains previously identified from marine mammals. the isolate was preliminarily identified as brucella suis using conventional bacteriological tests in our laboratory. however, the re ...200617035490
the tolc homologue of brucella suis is involved in resistance to antimicrobial compounds and virulence.brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. in doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. members of the tolc family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. the role of proteins from the tolc family in brucella and other alpha-2-proteobacteria has b ...200717088356
different roles of the two high-oxygen-affinity terminal oxidases of brucella suis: cytochrome c oxidase, but not ubiquinol oxidase, is required for persistence in mice.the survival of brucella suis mutant strains in mice demonstrated different roles of the two high-oxygen-affinity terminal oxidases. the cbb3-type cytochrome c oxidase was essential for chronic infection in oxygen-deficient organs. lack of the cytochrome bd ubiquinol oxidase led to hypervirulence of bacteria, which could rely on nitrite accumulation inhibiting the inducible nitric oxide synthase of the host.200717101669
insight in to the phylogeny of polyhydroxyalkanoate biosynthesis: horizontal gene transfer.polyhydroxyalkanoates (phas) are gaining more and more importance the world over due to their structural diversity and close analogy to plastics. their biodegradability makes them extremely desirable substitutes for synthetic plastics. phas are produced in organisms under certain stress conditions. here, we investigated 253 sequenced (completely and unfinished) genomes for the diversity and phylogenetics of the pha biosynthesis. discrepancies in the phylogenetic trees for phaa, phab and phac gen ...200717113245
cell-mediated immune responses differentiate infections with brucella suis from yersinia enterocolitica serotype o:9 in pigs.due to almost identical lipopolysaccharide (lps) o-antigens, infections with yersinia enterocolitica serotype o:9 (yeo:9) cause false positive serological reactions (fpsr) in tests for brucella and thus cause problems in national brucella surveillance programs. as lps are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-lps antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. ...200717270281
prevalence of classical swine fever, aujeszky's disease and brucellosis in a population of wild boar in switzerland.during two survey rounds of a national surveillance system for infectious diseases in wild boar in switzerland, each lasting four months from november to february, between 2001 and 2003, 1949 blood samples and 62 tissue samples from the spleen and 50 from the reproductive organs were collected from hunted wild boar. the survey was designed so that freedom from infection could be detected with a probability of 95 per cent at a threshold prevalence of less than 1 per cent for classical swine fever ...200717369476
diagnostic characterization of a feral swine herd enzootically infected with brucella.eighty feral swine were trapped from a herd that had been documented to be seropositive for brucella and which had been used for brucella abortus rb51 vaccine trials on a 7,100-hectare tract of land in south carolina. the animals were euthanized and complete necropsies were performed. samples were taken for histopathology, brucella culture, and brucella serology. brucella was cultured from 62 (77.5%) animals. brucella suis was isolated from 55 animals (68.8%), and all isolates were biovar 1. bru ...200717459850
brucella suis histidinol dehydrogenase: synthesis and inhibition studies of a series of substituted benzylic ketones derived from histidine.brucella spp. is the causative agent of brucellosis (malta fever), which is the most widespread zoonosis worldwide. the pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. moreover, brucella is considered as a potential bioterrorism agent. histidinol dehydrogenase (hdh, ec 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. it therefore constitutes an original and ...200717481905
brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of balb/c mice.in prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. the brucella genomes contain two urease operons designated as ure1 and ure2. we investigated the role of the two brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-ph killing.200717578575
brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion.brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. in some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. the mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. we recently demonstrated that dendritic cells (dcs), which are critical components of adaptive immunity, are highly susceptible to brucella infection and are a prefere ...200717635859
targeting of the brucella suis virulence factor histidinol dehydrogenase by histidinol analogues results in inhibition of intramacrophagic multiplication of the pathogen.brucella suis histidinol dehydrogenase (hdh) can be efficiently targeted by substrate analogues. the growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. these effects have been shown to be correlated with the previously described inhibition of brucella hdh activity.200717698620
identification and isolation of brucella suis virulence genes involved in resistance to the human innate immune system.brucella strains are facultative intracellular pathogens that induce chronic diseases in humans and animals. this observation implies that brucella subverts innate and specific immune responses of the host to develop its full virulence. deciphering the genes involved in the subversion of the immune system is of primary importance for understanding the virulence of the bacteria, for understanding the pathogenic consequences of infection, and for designing an efficient vaccine. we have developed a ...200717709411
[construction and characterization of a brucella suis s2 strain with a chloramphenicol resistance marker].vaccination has not been used widely because of the interference in the discrimination between infected and vaccinated animals in immune-screening procedures. in the present study, chloramphenicol resistance gene (cm(r)) was cloned into the genomic dna of brucella suis s2 strain by homologous recombination with knocking out the wbkc gene, and obtained the recombinant rs2-wbkc. further study confirmed that rs2-wbkc was conversed into rough-phenotype form smooth-phenotype. the recombinant keeps th ...200718271249
interaction of brucella suis and brucella abortus rough strains with human dendritic cells.brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. we recently demonstrated that dendritic cells (dcs), which are critical components of adaptive immunity, are highly susceptible to brucella infection. furthermore, brucella prevented the infected dcs from engaging in maturation processes and impaired their capacity to present antigen to naive t cells and to secrete interleukin-12 (il-12). the lipopolysaccharide (lps) phenotype is largely ...200717938225
comparison of multiple-locus variable-number tandem-repeat analysis with other pcr-based methods for typing brucella suis isolates.multiple-locus variable-number tandem-repeat analysis (mlva), multiplex pcr, and pcr-restriction fragment length polymorphism analysis were compared for typing brucella suis isolates. a perfect concordance was obtained among these molecular assays. however, mlva was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.200717942649
evaluation of genus-specific and species-specific real-time pcr assays for the identification of brucella spp.the identification of brucella isolates using conventional microbiological techniques is time-consuming and hazardous. we therefore assessed the performance of real-time pcr assays for the identification of members of the genus brucella to the genus and species level.200717970716
phylogenomics and signature proteins for the alpha proteobacteria and its main groups.alpha proteobacteria are one of the largest and most extensively studied groups within bacteria. however, for these bacteria as a whole and for all of its major subgroups (viz. rhizobiales, rhodobacterales, rhodospirillales, rickettsiales, sphingomonadales and caulobacterales), very few or no distinctive molecular or biochemical characteristics are known.200718045498
evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting gram-negative rods.to evaluate the activity of pyrrolidonyl arylamidase (pyr) for the differentiation and identification of nonfermenting gram negative rods (nfgnr), 293 isolates were tested. a 24 h culture of each test organism was prepared. from this a 108-109 cfu/ml suspension was added to 0.25 ml of sterile physiologic solution. a pyr disk was then added and the test was incubated for 30 minutes at 35-37 degrees c, at environmental atmosphere. reading was done by adding 1 drop of cinnamaldehyde reagent. strain ...200716822636
infection trials in pigs with a human isolate of brucella ( isolate 02/611 'marine mammal type' ).to determine if pigs could support infection of a human brucella isolate (brucella 02/611) from new zealand, and to study seroconversion to this isolate using a competitive elisa.200818322554
brucella suis identification and biovar typing by real-time pcr.fast and accurate identification of brucella suis at the biovar level is an important issue for public health laboratories because some of the biovars that infect suidae (boars and pigs) are pathogenic for humans while others are not. since classical biovar typing methods are often time-consuming, hard to standardize and require high-level biosafety containment, methodological improvements are desirable. this article describes new single nucleotide polymorphism (snp) signatures for the rapid ide ...200818499359
intracellular rescuing of a b. melitensis 16m virb mutant by co-infection with a wild type strain.brucella is a broad-range, facultative intracellular pathogen that can survive and replicate in an endoplasmic reticulum (er)-derived replication niche by preventing fusion of its membrane-bound compartment with late endosomes and lysosomes. this vacuolar hijacking was demonstrated to be dependent on the type iv secretion system virb but no secreted effectors have been identified yet. a virb mutant is unable to reach its er-derived replicative niche and does not multiply intracellularly. in this ...200818547782
brucella suis histidinol dehydrogenase: synthesis and inhibition studies of substituted n-l-histidinylphenylsulfonyl hydrazide.histidinol dehydrogenase (hdh, ec ec1.1.1.23) catalyses the final step in the biosynthesis of histidine and constitutes an attractive novel target for the development of new agents against the pathogenous, bacteria brucella suis. a small library of new hdh inhibitors based on the l-histidinylphenylsulfonyl hydrazide scaffold has been synthesized and their inhibitory activity investigated. the obtained results demonstrate that modification of the group between the histidinyl moiety and the phenyl ...200818569340
virb type iv secretory system does not contribute to brucella suis' avoidance of human dendritic cell maturation.dendritic cells (dcs), which are critical components of adaptive immunity, are highly susceptible to infection with the intracellular bacteria brucella. infection with living brucella prevents infected human dcs from engaging in maturation processes, thus impairing their capacity to present antigens to naïve t cells and to secrete il-12. recently, we have established that several attenuated mutants of brucella (rough, omp25, bvrr) are unable to control dcs maturation and thus effectively stimula ...200818625010
enzymatic, immunological and phylogenetic characterization of brucella suis urease.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.200818638408
brucella: a mr "hide" converted into dr jekyll.dr david bruce (1855-1931) first identified the causative agent of brucellosis as a small gram-negative alpha-proteobacterium, which was later on called brucella melitensis in his honor by meyer and shaw. nowadays, four strains exhibit pathogenicity in humans with b. melitensis being the least host specific and also the most infectious for humans. the other strains are brucella suis and brucella abortus and more recently human cases being infected with brucella cetaceae have been reported. why s ...200818664389
novel identification and differentiation of brucella melitensis, b. abortus, b. suis, b. ovis, b. canis, and b. neotomae suitable for both conventional and real-time pcr systems.we describe the development of a novel pcr assay for the rapid detection of members of the brucella genus, and the differentiation between six recognized brucella species. the assay has proven to be highly specific with the additional advantage of being suitable for use with both conventional and real-time pcr.200818675856
quantitative analysis of the intramacrophagic brucella suis proteome reveals metabolic adaptation to late stage of cellular infection.a 2-d dige approach allowed the characterization of the intramacrophagic proteome of the intracellular pathogen brucella suis at the late stage of in vitro infection by efficient discrimination between bacterial and host cell proteins. using a subtraction model, a total of 168 proteins showing altered concentrations in comparison with extracellularly grown, stationary-phase bacteria were identified. the majority of the 44 proteins significantly regulated at this stage of infection were involved ...200818704908
brucellosis complicating chronic non-infectious disorders: diagnostic and therapeutic dilemmas.there is little information in the literature on the clinical progress of brucellosis in patients affected by other non-infectious diseases; however, the infection can often trigger an exacerbation of existing underlying conditions in certain target organs. in this report we present four cases of brucellosis complicating previous diseases, and the difficulties in relation to their diagnosis and treatment. the study involved four patients with the following disorders: polycythaemia vera, pulmonar ...200818719189
activity of native vs. synthetic promoters in brucella.brucellosis caused by brucella species is reportedly the most common zoonotic infection worldwide. the bacterial pathogen is also classified by the centers for disease control and prevention as a category (b) pathogen that has the potential for development as a bioweapon. although eight genomes of brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in brucella spp. we therefore constructed a set of broad-host-range vecto ...200818811654
identification of vcea and vcec, two members of the vjbr regulon that are translocated into macrophages by the brucella type iv secretion system.survival and replication inside host cells by brucella spp. requires a type iv secretion system (t4ss), encoded by the virb locus. however, the identity of the molecules secreted by the t4ss has remained elusive. we hypothesized that proteins translocated by the t4ss would be co-regulated with the virb operon. the luxr family regulator vjbr, known to regulate virb, bound a fragment of the virb promoter containing an 18 bp palindromic motif (virb promoter box), showing that vjbr regulated the vir ...200819019140
improving the specificity of immunodiagnosis for porcine brucellosis.this report describes the use of cell mediated immunity to improve specificity of current diagnosis for brucella suis. diagnosis is problematic due to cross reactions that lead to false positive serological reactions (fpsr) in the standard diagnostic tests. a common cause of this cross reactivity is infection with the organism yersinia enterocolitica o:9. gottingen mini-pigs were experimentally infected with b. suis biovar i field strain or y. enterocolitica serotype o:9 biotype 3. infection was ...200817934790
interplay between two rnd systems mediating antimicrobial resistance in brucella suis.the rnd-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. also, rnd pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. we have previously shown that the bepc outer membrane factor of brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. in the present work, we characterize t ...200919201794
interactions between brucella suis virb8 and its homolog traj from the plasmid psb102 underline the dynamic nature of type iv secretion systems.the proteinvirb8 plays a critical role in the assembly and function of the agrobacterium tumefaciens virb type iv secretion system (t4ss). the structure of the periplasmic domain of both a. tumefaciens and brucella suis virb8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of virb8 and interactions with virb4 and virb10. we have shown previously that traj, the virb8 homologue from psb102, and the chimeric protein trajb8, encompassing the c ...200919251859
human cd4+ invariant nkt cells are involved in antibacterial immunity against brucella suis through cd1d-dependent but cd4-independent mechanisms.human invariant nkt (inkt) cells are a unique subset of t cells, which recognize glycolipids presented by the cd1d. among the inkt cells, several functionally distinct subsets have been characterized according to cd4 and/or cd8 co-receptor expression. the current study is focussed on the cd4(+) inkt cell subset and its role in an anti-infectious response. we have examined the role of cd4(+) inkt cells on the intracellular brucella suis growth. our results indicate that cd4(+) inkt cells impair t ...200919266487
wild boars as sources for infectious diseases in livestock and humans.wild boars (sus scrofa) are indigenous in many countries in the world. these free-living swine are known reservoirs for a number of viruses, bacteria and parasites that are transmissible to domestic animals and humans. changes of human habitation to suburban areas, increased use of lands for agricultural purposes, increased hunting activities and consumption of wild boar meat have increased the chances of exposure of wild boars to domestic animals and humans. wild boars can act as reservoirs for ...200919687039
evaluation of automated and manual commercial dna extraction methods for recovery of brucella dna from suspensions and spiked swabs.this study evaluated automated and manual commercial dna extraction methods for their ability to recover dna from brucella species in phosphate-buffered saline (pbs) suspension and from spiked swab specimens. six extraction methods, representing several of the methodologies which are commercially available for dna extraction, as well as representing various throughput capacities, were evaluated: the magna pure compact and the magna pure lc instruments, the it 1-2-3 dna sample purification kit, t ...200919846627
feral pig hunting: a risk factor for human brucellosis in north-west nsw?a multi-agency investigation followed the notification of four locally acquired human brucellosis cases in north-west nsw. feral pig hunting within a geographically discrete region was identified as the likely exposure with brucella suis the suspected cause. to test whether feral pigs in the region were infected with brucella, serological testing was performed on trapped feral pigs and testicular abscesses from condemned carcasses bound for export were cultured. although no brucella species were ...200920132743
development and characterization of mouse models of infection with aerosolized brucella melitensis and brucella suis.there is a need to identify vaccines that can protect against brucella, a potential bioterrorism agent. we have developed mouse models of infection with aerosolized brucella melitensis and brucella suis and demonstrated their utility for the evaluation of vaccines using the model live b. melitensis vaccine strain rev.1.200919321692
analysis of ten brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle.the facultative intracellular bacterial pathogen brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. currently, there are nine recognized brucella species based on host preferences and phenotypic differences. the availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order rhizobiales. phylogenomic analysis of ortholog families show ...200919346311
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