Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard serological tests in diagnosis of sheep brucellosis.
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Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera from Brucella-free, Brucella melitensis-infected, and B. melitensis Rev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis. (+info)
In vitro activities of antibiotics alone and in combination against Brucella melitensis at neutral and acidic pHs.
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Brucellae survive acidic pHs in phagolysosomes. Azithromycin, streptomycin, and quinolones were active against Brucella melitensis at pH 7.0 but not at pH 5.0; rifampin and doxycycline retained activity at pH 5.0. Regardless of pH, azithromycin-rifampin and ofloxacin-rifampin showed less synergy than established streptomycin-doxycycline and rifampin-doxycycline combinations. (+info)
Effect of early antibiotic treatment on the antibody response to cytoplasmic proteins of Brucella melitensis in mice.
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To test whether antibiotic therapy hampers the antibody response to Brucella antigens, 30 BALB/c mice were infected with Brucella melitensis H38 and randomized for treatment with doxycycline administered intraperitoneally for 42 days starting at 7 or 28 days postinfection (p.i.) (groups DOX7 and DOX28, respectively) or for no treatment (control group). Antibodies to smooth lipopolysaccharide (LPS) reached peak levels (mean optical density [OD] = 2.618) between days 56 and 70 p.i. in the control group, and similar peak levels (mean OD = 2.486) were observed in the DOX28 group, but significantly lower peak levels (mean OD = 0.821) were observed at 28 days p.i. in the DOX7 group. The antibody response against cytoplasmic proteins depleted of LPS (CPs) reached maximal levels (mean OD = 2.402) between days 56 and 70 p.i. in the control group, but no response was detected in the DOX7 group. Anti-CP antibodies were detected in only three animals from the DOX28 group, at levels significantly lower than those in the control group (mean maximal OD = 0.791). The pattern of antibody response to an 18-kDa cytoplasmic protein of Brucella spp. was similar to that against the CP antigen. This study shows that early antibiotic treatment affects the antibody response of mice to cytoplasmic proteins of Brucella and, to a lesser extent, to LPS. (+info)
Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains: evidence for a locus involved in the synthesis of a polysaccharide.
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A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment. (+info)
Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains.
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Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested. (+info)
Protection of mice against brucellosis by vaccination with Brucella melitensis WR201(16MDeltapurEK).
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Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of the conjunctiva or traumatized skin by infected animal products. A vaccine to protect humans from occupational exposure or from zoonotic infection in areas where the disease is endemic would reduce an important cause of morbidity worldwide. Vaccines currently used in animals are unsuitable for human use. We tested a live, attenuated, purine-auxotrophic mutant strain of Brucella melitensis, WR201, for its ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M. Mice inoculated intraperitoneally with WR201 made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 (IL-2), gamma interferon, and IL-10 when cultured with Brucella antigens. Immunization led to protection from disseminated infection but had only a slight effect on clearance of the challenge inoculum from the lungs. These studies suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis. (+info)
Brucellar spondylitis: review of 35 cases and literature survey.
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Thirty-five patients aged 14-74 years (average, 54 years) who had brucellar spondylitis were treated between January 1991 and December 1997. The time from onset of symptoms to diagnosis of spondylitis ranged from 1 week to 8 months (median, 9 weeks). Back or neck pain (100% of patients), fever (66%), and constitutional symptoms (57%) were the most common symptoms. Cultures of blood specimens from 26 patients (74%) were positive for Brucella melitensis. The duration of antimicrobial therapy (median, 120 days; range, 45-535 days) varied according to clinical response and the presence of epidural and paravertebral masses. One of the 35 patients underwent surgical treatment of a spinal epidural abscess. Therapy failed for 9 patients (26%; 95% confidence interval [CI], 12%-43%), and 5 (14%; 95% CI, 5%-30%) had a relapse. There were no deaths or severe sequelae in this study. Brucellar spondylitis causes considerable suffering and absenteeism from work, but long-term clinical responses are favorable. (+info)
Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro.
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Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0. 01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 microg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N(G)-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis. (+info)