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PubMed1996

Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide.

Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1,2- plus alpha-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 degrees C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.

Brucella abortus↗
PubMed1998

Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

Bacterial Outer Membrane Proteins↗
PubMed1981

Effects of killed Brucella abortus strain 45/20 vaccine on reindeer later challenge exposed with Brucella suis type 4.

Six seronegative pregnant reindeer (Rangifer tarandus L) were vaccinated with killed Brucella abortus strain 45/20 with added adjuvant. These were challenge exposed with B suis type 4 after 90 days; at the same time, 4 seronegative, nonvaccinated, pregnant reindeer (controls) were given the challenge inoculum. Humoral antibodies were detected in the vaccinated reindeer by postvaccination day 14. A marked increase in antibody levels also occurred after they were challenge exposed, but did not reach the levels observed in control reindeer which seroconverted within 8 days after they were given the challenge inoculum. One control reindeer aborted at 45 days after challenge exposure (at 165 days of a normal 225-day gestation period), and the fawn of another lived only a few days after delivery. Brucella suis type 4 was isolated from tissues of 3 of 4 control reindeer and of 2 of their fawns. All vaccinated reindeer gave birth to live fawns which were culture negative, although 1 fawn lived only a few days. Brucella suis type 4 was isolated from tissues of 1 vaccinated reindeer. Under the conditions of this experiment, killed B abortus 45/20 vaccine provided increased resistance to brucellosis.

Abortion, Veterinary↗
PubMed1976

The serological response to rough and smooth brucella antigens in cattle vaccinated with Brucella abortus strain 45/20 adjuvant vaccine.

Cattle not previously exposed to Brucella abortus responded to a single dose of Br. abortus 45/20 adjuvant vaccine by producing low titres of agglutinins for rough brucella antigens and little or no agglutinins to the smooth antigens. Moderate titres of non-agglutinating antibodies reacting with both rough and smooth antigens were also produced. After a second dose of 45/20 vaccine higher titres of rough agglutinins and low titres of antibodies reacting with smooth antigens were produced. High titres of non-agglutinating antibodies reacting with both rough and smooth antigens were also produced. Animals previously exposed to smooth Br. abortus, either as strain 19 vaccine or virulent organisms, produced high titres of antibodies to smooth antigens soon after vaccination with 45/20 adjuvant vaccine. High titres of antibodies reacting with rough and smooth antigens were also produced. The evidence suggests that the Br. abortus strain 45/20 shares a surface antigenic component with smooth brucellae. The possible relationship of this to the protective antigen is discussed.

Animals↗
PubMed1989

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis.

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigen in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

Animals↗
PubMed1995

Evaluation of a potassium chloride extract of Brucella abortus in an ELISA for detecting Brucella antibodies in bulk tank milk samples from cows.

An enzyme-linked immunosorbent assay (ELISA) was developed using as antigen a potassium chloride extract of Brucella abortus strain 1119-3 for detecting Brucella antibodies in bulk tank samples of cow's milk. Three-hundred-thirty-four Milk Ring Test (MRT) suspicions milk samples originating from cattle herds in 13 states and 106 BRT negative milk samples were analyzed. Fifty-four of 334 MRT suspicious milk samples were positive on ELISA; bacteriologic examinations revealed B. abortus field strain was isolated from cows in 15 herds, B. abortus strain 19 was isolated from cows in 16 herds and serologic suspects were reported in 6 of the other 23 herds. Two-hundred-fifty-eight (85.6%) of the 301 MRT suspicious samples were negative on ELISA; field investigations and/or serologic tests on cattle failed to disclose Brucella infection in these herds. Suspicious ELISA reactions were detected in 22 MRT suspicious bulk tank milk samples; serologic suspects were reported in 8 of the 22 herds. No false positive ELISA reactions were detected in the 106 MRT negative bulk tank milk samples collected from dairy herds in 7 states.

Animals↗
PubMed1989

Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis.

Sheep infected with Brucella ovis produce antibody responses to the rough lipopolysaccharide and to proteins present in hot saline (HS) extracts of B. ovis (J. I. Riezu-Boj, I. Moriyón, J. M. Blasco, C. M. Marín, and R. Díaz, J. Clin. Microbiol. 23:938-942, 1986). The distribution and antigenic relatedness of proteins in HS extracts and in outer membrane blebs were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting for 41 strains of B. ovis and 26 strains of Brucella melitensis of diverse geographic origin. Five major groups of proteins were identified in HS extracts of B. ovis that had been freed of rough lipopolysaccharide: proteins of 43 kilodaltons (kDa), group A (25.5 to 32.0 kDa), group B (21.5 to 22.5 kDa); group C (18.0 to 19.5 kDa), and group D (13.0 to 15.5 kDa). Group A, B, C, and D proteins were also present in blebs. The profiles of proteins in HS extracts or blebs from strains of both Brucella species were very similar. Cross-reactions were demonstrated among HS extracts and blebs of all strains tested in immunoblots performed with an antiserum against the HS extract of a reference strain of B. ovis. Evidence was also provided of an antigenic relationship between group 3 proteins of the outer membrane and some of the proteins in groups A, B, and C. The conservation of these antigens and their immunogenicity in infected animals provide promise that they may serve as components of an effective subcellular vaccine for ovine brucellosis.

Animals↗
PubMed1993

Cytokine response of T-cell subsets from Brucella abortus-infected mice to soluble Brucella proteins.

Hot saline extracts of Brucella abortus 19 were separated by successive differential precipitation with 50 and 70% ammonium sulfate, yielding fractions SBP50, with predominantly 36-kDa proteins and a number of medium-sized proteins (26 to 33 kDa), and SBP70, with 14-kDa and lower-molecular-mass proteins. Both fractions stimulated specifically proliferation and cytokine production by spleen cells from brucella-infected mice, although the activity of SBP50 was much higher than that of SBP70. Further separation of SBP50 by a DEAE-Sepharose column resulted in three distinct subfractions which were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three subfractions were analyzed for their abilities to induce lymphocytes to proliferate and produce cytokines. The three subfractions were all active but with characteristic differences in magnitude. Subfraction 1 stimulated moderate proliferation, high interleukin 6 (IL-6) production, and relatively low production of gamma interferon (IFN-gamma). Subfraction 2 was the strongest stimulus for proliferation and production of IL-6 and IFN-gamma, while subfraction 3 stimulated moderate cell proliferation, a high level of IFN-gamma, and a low level of IL-6. IL-2 production stimulated by the three subfractions was similar. SBP50 and all three subfractions stimulated purified T cells of both CD4+ and CD8+ subsets to produce IFN-gamma. The production of IFN-gamma by CD8+ T cells to brucella antigens was enhanced with exogenous IL-2.

Animals↗
PubMed2004

Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M.

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.

Administration, Oral↗
PubMed1996

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.

Anti-Bacterial Agents↗
PubMed1968

Homologies of deoxyribonucleic acids from Brucella ovis, canine abortion organisms, and other Brucella species.

The bacterium that causes canine abortion has polynucleotide sequences similar, in deoxyribonucleic acid (DNA)-DNA homology studies, to those of Brucella suis and, by inference from previous data, those of B. abortus and B. melitensis as well as B. neotomae. Therefore, the organism causing canine abortion appears to be a member of the genus Brucella. DNA preparations from Serratia marcescens, Alcaligenes faecalis, and Bordetella bronchiseptica, 58, 62, and 66 mole% guanine plus cytosine, respectively, do not have detectable polynucleotide sequence homologies with B. suis DNA which is 56 mole% guanine plus cytosine. B. ovis DNA lacks some of the polynucleotide sequences present in B. suis DNA and appears to be a deletion mutant. However, a large proportion of B. ovis polynucleotides are similar to those of other Brucella species, which supports the inclusion of B. ovis in the genus.

Alcaligenes↗
PubMed2003

Evaluation of the Brucella abortus species-specific polymerase chain reaction assay, an improved version of the Brucella AMOS polymerase chain reaction assay for cattle.

In a blind test, 344 samples representing 80 bacterial isolates were analyzed by the Brucella abortus species-specific polymerase chain reaction (BaSS PCR) assay for the identification and discrimination of B. abortus field strains (wild-type biovars 1, 2, and 4) from 1) B. abortus vaccine strains, 2) other Brucella species, and 3) non-Brucella bacteria. Identical samples were tested in 2 laboratories. Half the samples were fully viable, and half were bacteria that had been killed by methanol fixation. The results in 1 laboratory correctly identified 100% of the samples, resulting in a predictive value of 100% for all categories and 100% sensitivity and specificity under the prescribed conditions. The second laboratory misidentified 31 samples, resulting in a range of 66.7-100% sensitivity, 93.2-99.7% specificity, and 77.3-98.2% predictive values depending on the category. There was no significant difference in viable versus fixed bacteria for either laboratory. Subsequent review of the protocol indicated that contamination was the likely cause of 26 of the 31 erroneous identifications. The results show that the BaSS PCR assay has the potential to be a very reliable screening tool for B. abortus identification. However, the data also provide a cautionary reminder of the importance of preventing contamination in diagnostic PCR.

Animals↗
PubMed1989

[Brucella clearance as a sensitive method for the detection of cross reactions of Brucella abortus with Yersinia enterocolitica 03, 06, 09 and Salmonella urbana and Salmonella abony].

Determination of the Brucella clearance rate has proved to enable assessment of Brucella immune reaction in rat, even after vaccination with Yersiniae and Salmonellae. Vaccination with Yersinia (Y.) enterocolitica O6 and O9 produced 95 per cent of "high responders", whereas 65 per cent of "high responders" and 25 per cent of "non-responders" were recorded in the wake of O3. Salmonella (S.) urbana vaccination gave 50 per cent of "high responders" and 27 per cent of "non-responders", while 100 per cent "non-responders" resulted from S. dublin. Vaccination, using Brucella abortus Buck 19, gave 100 per cent "high responders". The differentiated nature of immune reactions to Y. enterocolitica O3, S. urbana, and S. abony has been attributed to an individual genetic capability of reaction to the cross-reactive antigen.

Animals↗
PubMed1977

Enhancement activity of anti-brucella sera in experimental Brucella abortus infection in mice.

The passive transfer of rabbit anti-brucella sera promotes the multiplication of Brucella abortus in the spleen of mice infected intravenously with a low dose of this bacteria. The enhancement activity of the anti-brucella sera is related to their modes of preparation in rabbits. The agglutinin titers of all these antisera were relatively high, while there was some discrepancies in their content in haemagglutinating, immune adherence and complement fixing antibodies.

Animals↗
PubMed1997

Brucella abortus siderophore 2,3-dihydroxybenzoic acid protects brucellae from killing by macrophages.

Addition of 2,3-dihydroxybenzoic acid, a siderophore produced by Brucella abortus, to macrophage cultures prevented intracellular killing of brucellae during the first 12 h after infection and increased the number of intracellular brucellae recovered at 48 h after infection. The protective effect could be demonstrated with inflammatory macrophages, interferon-gamma-activated macrophages and with macrophages supplemented with iron, shown elsewhere to facilitate killing of B abortus.

Animals↗
PubMed1977

Agglutinins to Coxiella burnetii and Brucella spp, with particular reference to Brucella canis, in wild animals of southern Texas.

The prevalence of agglutinins to Coxiella burnetii and Brucella spp, particularly Brucella canis, was determined in 269 wild animals (14 species) in southern Texas. Serologic evidence of coxiellosis and brucellosis, including B canis infection, was shown for coyotes, raccoons, opossums, badgers, jackrabbits, and feral hogs. Using the microagglutination test, the seroprevalence of C burnetii, phases I and II (titer greater than or equal to 4) was 4.1 and 27.9%, respectively. For brucella agglutinins, prevalence rates were 7.1, 8.9, and 6.7%, as determined by the brucellosis card test, the rapid slide agglutination test, and the salt 2-mercaptoethanol tube agglutination (titer greater than or equal to 50) test, respectively.

Agglutinins↗
PubMed1996

Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs.

Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium. As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B. Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR. Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside. The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody. Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins. In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion. Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb. The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants. The recombinant B. abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs. Moreover, the results of this study suggest that B. abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.

Animals↗