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At least 19 recordsLinked to original sources
PubMed2001

Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis.

Real-time PCR-based assays specific for Brucella abortus, Brucella melitensis and Brucella suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS 711, whereas the downstream primers and probes are designed from signature sequences specific to a species or a biovar. The PCR reactions were monitored for fluorescence resonance energy transfer by including two adjacent labeled probes that hybridize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the downstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The assays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis. Therefore, specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies.

Brucella↗
PubMed1990

[The antigenic relationship between Brucella abortus, Brucella melitensis and Yersinia enterocolitica serotype 0:3 and 0:9].

Antigenic interrelationship between B. abortus, B. melitensis, Y. enterocolitica serotype 0:3 and serotype 0:9 were investigated in hyperimmün sera obtained from rabbits, by using agglutination and agglutinin absorbtion tests. Agglutination was determined at 1/2560 titer of B. abortus and B. melitensis hyperimmün sera when their own antigens were used. On the other hand, these sera agglutinated Y. enterocolitica serotype 0:9 antigen at 1/1280 and 1/320 titer respectively. As it was seen that high level antigenic relation was found between B. abortus and Y. enterocolitica serotype 0:9 while antigenic resemblance was found at lower level between B. melitensis and Y. enterocolitica serotype 0:9. There wasn't any significant interrelationship between B. abortus, B. melitensis and Y. enterocolitica serotype 0:3. In conclusion it is though that it is impossible to distinguish B. abortus and Y. enterocolitica serotype 0:9 infections from each other by using agglutination tests when 0 antigens are used.

Agglutination Tests↗
PubMed1994

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.

Animals↗
PubMed1982

[Development of a splenic infection in mice of 4 strains, inoculated by the venous route, with 3 doses of Brucella abortus].

Brucella abortus strain 544 was inoculated at three doses into outbred female Swiss mice CD-1 and OF1, and inbred strains CBA and DBA/2. Brucella were enumerated in the mice spleens which were first weighed, at 2, 7, 14, 21, 35 and 49 days post-challenge. A phase of multiplication occurred, reaching a maximum between 7 and 21 days according to the mice, but independently of challenge doses. A decreasing phase followed that was still going on in CD-1 and OF1 mice at 49 days, but ended by a plateau or recurrence in CBA and DBA/2. Spleen weights increased from the beginning to a maximum then decreased. This maximum was reached either before (DBA/2), after (CBA) or at the infection acme (CD-1, OF1). Hence, immune mechanisms may be turned on according to several sequences, but feed-back regulation would work similarly in different breeds.

Animals↗
PubMed1990

Characterization of a phosphomonoesterase from Brucella abortus.

Brucellae are facultative intracellular bacterial pathogens that reside primarily in cells of the reticuloendothelial system. The high-speed supernatant obtained after centrifuging a suspension of Brucella abortus that had been frozen-thawed and sonicated contained abundant phosphomonoesterase activity, determined by using 4-methylumbelliferylphosphate as the substrate; this enzyme was purified 2,900-fold (yield, 570%) by chromatography on DE-52 cellulose and hydroxylapatite columns and high-performance liquid chromatography-gel filtration. The native enzyme had a molecular mass of 120,000 daltons (+/- 10,000 daltons), as determined by gel filtration chromatography, and resolved into two bands (60,000 and 66,000 daltons) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The B. abortus phosphomonoesterase had the following properties: pH optimum, 6.0 to 6.5; isoelectric point, 3.0; substrate specificity, 5'-AMP greater than 3'-AMP greater than 3'-GMP greater than 5'-GDP greater than 5'-CDP greater than 5'-CTP greater than 5'-UPT greater than phosphotyrosine greater than phosphoserine greater than phosphothreonine. The Km for 5'-AMP was 0.37 mM. Phosphatidylinositol 4,5-bisphosphate and myo-inositol 1,3,4-trisphosphate were poor substrates for the B. abortus enzyme. The phosphomonoesterase did not inhibit superoxide anion production by human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. The phosphomonoesterase may be one of the bacterial enzymes in the pathway leading to the production of adenine, which is secreted by B. abortus and blocks the activation of neutrophils.

Acid Phosphatase↗
PubMed2004

Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus.

Brucella abortus is a facultative intracellular bacterium that can survive inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to the conjugative DNA transfer system. In this study, we showed that a class A scavenger receptor (SR-A) of macrophages is required to internalize B. abortus and contributes to the establishment of bacterial infection in mice. Macrophages from SR-A-deficient mice inhibited internalization and intracellular replication of both wild type strain and the virB4 mutant, and that bacterial proliferation was inhibited in SR-A-deficient mice. Adding lipopolysaccharide from B. abortus and Salmonella enterica serovar Typhimurium, but not from Escherichia coli, to macrophages inhibited bacterial internalization. VirB-dependent bacterial internalization induced localization of SR-A into detergent-resistant membrane lipid rafts. These results indicate that B. abortus internalizes into macrophages by using SR-A as a receptor and that the VirB type IV secretion system of B. abortus regulates signal transduction dependent on SR-A to form replicative phagosomes, and which is mediated by lipid rafts.

Animals↗
PubMed1996

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in the bacteria grown in the cell culture medium have been induced during intracellular growth in macrophages. In contrast, approximately 50 proteins that were expressed during growth in cell culture medium were completely repressed during intracellular growth. The level of expression of 19 proteins increases while that of 54 proteins decreases during intracellular growth. To understand these results, the protein synthesis pattern of B. abortus during intracellular growth was compared with those during other stress conditions. Under each stress condition studied, several new proteins were induced that were not present during regular growth conditions. Comparison of the protein synthesis pattern of B. abortus during intracellular growth with those obtained under various stress conditions has indicated that the response to intracellular growth was not just a simple sum of stress conditions studied so far.

Animals↗
PubMed2001

Immunity and protection against Brucella abortus.

Brucella abortus is an intracellular pathogen that causes disease in cattle and in humans. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4(+) and CD8(+) T cells, and B cells.

Animals↗
PubMed1974

Glucose transport in Brucella abortus.

Brucella abortus British strain 19 transported glucose with an apparent K(m) of 0.16 mM and an apparent V(max) of 250 nmol per min per mg of N. The only common glucose analogue transported was 2-deoxyglucose (2-DOG), with an apparent K(i) of 0.73 mM. Alpha- or beta-methyl glucosides and 3-O-methylglucose were not transported. Transport was linear for 70 to 90 s, depending on the concentration of substrate used. 2-Deoxyglucose was transported as the free sugar and was not further metabolized once inside the cell. There was no glucose phosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and there were no inhibitors present in Brucella cell-free extract that inhibited the Escherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (pCMB) completely inhibited transport of glucose and 2-DOG. Glutathione, dithiothreitol, and beta-mercaptoethanol reversed the effects of pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of 37 to 45 C were observed for both K(m) and V(max). The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, erythritol, or glucose. The electron transfer inhibitors carbonyl cyanide, m-chlorophenylhydrazone, NaN(3), 2,4-dinitrophenol, and KCN inhibited 2-DOG transport to a greater extent than did the metabolic energy inhibitors NaAsO(4), iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatases, inhibited transport by 100%.

Aerobiosis↗
PubMed2006

Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice.

Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The Deltaasp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The Deltaasp24 mutants also display superior protection compared to DeltamanBA and DeltavirB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.

Animals↗
PubMed2000

Lipopolysaccharides of Brucella abortus and Brucella melitensis induce nitric oxide synthesis in rat peritoneal macrophages.

Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucella in humans.

Animals↗
PubMed2004

Membrane topology analysis of cyclic glucan synthase, a virulence determinant of Brucella abortus.

Brucella abortus cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. B. abortus Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in B. abortus for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of B. abortus Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and beta-galactosidase (lacZ), and site-directed chemical labeling of lysine residues. We found that B. abortus Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.

Alkaline Phosphatase↗
PubMed2006

Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus.

Brucella abortus faces iron deprivation in both nature and the host. To overcome this limitation, Brucella secretes the siderophores 2,3-dihydroxybenzoic acid and brucebactin. A Fur-like protein named Irr has previously been characterized in B. abortus; this protein is present in the alpha-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model.

Bacterial Proteins↗
PubMed1988

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.

Animals↗