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PubMed1997

DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers.

The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.

Animals↗
PubMed1996

Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.

Amino Acid Sequence↗
PubMed1975

[Isolation of "Brucella suis" biotype 5 from a bitch, in Madagascar. Validity of the species name "Brucella canis" (author's transl)].

A Gram-negative organism isolated from a btich, in Madagascar, was examined by bacteriologic, immunologic and metabolic methods, in parallel with cultures representative of the Brucella species. The organism fits well into the genus Brucella on the basis of its growth, biochemical and antigenic characteristics and was found to have the metabolic pattern on L-asparagine (-), L-arginine (+) and DL-ornithine (+) that identifies and defines the species Brucella suis. It is of rough colonial morphology and electron microscopy showed a cell wall structure similar to that of other rough Brucella. By all the other recommended criteria for btotype identification it was found to be similar to Brucella suis biotype 5 best known as Brucella canis. In contrast to the strains of this biotype, it grows on basic fuchsin at 20 mug/ml and on safranine O at 200 mug/ml. These differences obtained with just one strain would not justify by now the proposal for a new biotype. We favor the designation Brucella suis biotype 5 proposed by Meyer, and the validity of Brucella canis (Carmichael and Bruner) as a separate species is discussed. It is the first strain of Brucella isolated in Madagascar.

Abortion, Induced↗
PubMed2001

Characterization of a Brucella species 25-kilobase DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide.

In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysaccharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide 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 detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells.

Base Sequence↗
PubMed1986

Antigenic differences between Brucella abortus and Brucella melitensis recognized by a monoclonal antibody.

By means of somatic hybridization of spleen cells from BALB/c mice inoculated with Brucella abortus and of a line of murine myeloma, a monoclone was obtained producing an antibody which specifically recognizes an epitope of Brucella abortus by means of the agglutination reaction. This epitope is not only on the surface of the homologous strains but on all the Brucella abortus biotype I, on the Brucella melitensis biotype II and on the Brucella suis biotype II tested. On the contrary it is not present on the Brucella melitensis biotype I and in just one of six Brucella melitensis biotype III. Very few strains of the other biotypes of Brucella suis were tested: the results are often negative.

Agglutination↗
PubMed2004

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 animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.

Animals↗
PubMed1958

Tissue culture studies on bacterial allergy in experimental brucellosis. I. The effect of Brucella suis whole antigen on cultures of spleen from normal and brucella-infected guinea pigs.

Tissue culture methods have been used to investigate infectious allergy in experimental brucellosis. A study was made of the effect of whole cell Brucella antigen on cultures of spleen from normal and Brucella-infected guinea pigs. The degree of toxicity was based upon the inhibition of migration of wandering cells and upon the morphologic appearance of stained sections of tissue cultures at different periods of incubation. A suspension of heat-killed Br. suis was more toxic for splenic cells from guinea pigs infected with Br. suis than for normal splenic cells. Macrophages were more sensitive than leucocytes to the toxic action of the antigen. The degenerative changes observed in Brucella-sensitive cells exposed to the antigen were similar to the degeneration previously observed in cultures of tuberculin-sensitive cells in the presence of tuberculin. The specific toxicity of the whole Brucella antigen, however, was more marked than that of tuberculin. Preliminary experiments indicate that serum and plasma containing specific antibodies obtained from Brucella-infected guinea pigs reduce the toxic effect of the antigen in cultures of both normal and Brucella-sensitive cells. The protective action of the homologous antiserum was greater for Brucella-sensitive cells than for normal cells.

Animals↗
PubMed2006

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 mutant of Sinorhizobium meliloti. The Rsh deletion mutants in Brucella suis and Brucella melitensis were characterized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evidenced that expression of virB, encoding the type IV secretion system, a major virulence factor of Brucella, was Rsh-dependent. All mutant phenotypes, including lack of VirB proteins, were complemented with the rsh gene of Brucella. In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella, Rsh might participate in the adaptation of the pathogen to low-nutrient environments, and indirectly in the VirB-mediated formation of the final replicative niche.

Animals↗
PubMed1999

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.

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.

Amino Acid Sequence↗
PubMed2000

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51.

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.

Animals↗
PubMed1975

[Brucella isolated in France: identification and typing. I. -- Brucella abortus (author's transl)].

Two hundred and eight strains of Brucella isolated from cattle and 5 isolated from man in various areas of France, during a two-year period from 1972 to 1974, were examined by all methods recommended by the Subcommittee on Taxonomy of the genus Brucella and were found to have the same characteristics that identify and define the species Brucella abortus. Of the 213 strains, 105 (49,29 p. 100) were biotype I, 103 strains (48,36 p. 100) biotype 3, three strains (1,41 p. 100) biotype 4 and 2 strains (0,94 p. 100) biotype 2. The distribution of Brucella abortus biotypes isolated from cattle and man in France is reviewed and compared with the data from our sample. It corroborates the epidemiologic importance of Brucella abortus biotype 3, just after type 1, and the absence of biotypes 5 to 9(1) in France. Five strains of Brucella abortus biotype 3, originating from a well-defined geographical area, are ureaseless. This new biochemical character distinguishes these strains from the main group of biotype 3 and is discussed from an epidemiological and methodological point of view. Data on the oxydative rates obtained on 12 substrates by the 213 strains are presented in graphic form which reveals a metabolic profile for the species Brucella abortus. The interest of this graphic presentation is discussed and its use should be recommended.

Animals↗