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M Grayon

Publications and source records attributed to M Grayon.

At least 19 recordsLinked to original sources
PubMed2006

A new variant of Brucella melitensis.

Brucella melitensis is highly pathogenic and constitutes a serious risk to public health. In Argentina, biovar 1 has been isolated from infected animals, but the Rev.1 strain vaccine is not authorised for use. This report describes nine atypical B. melitensis isolates obtained from humans. These isolates grew slowly, produced small colonies and were susceptible to penicillin and dyes, similar to the B. melitensis Rev.1 vaccine strain, but were inhibited by streptomycin 2.5 mg/L. The isolation of such atypical B. melitensis variants has never been reported from animals in Argentina, and could indicate the emergence of a new mutant variant.

Adolescent↗
PubMed2001

Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus.

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.

Animals↗
PubMed2001

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp.

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.

Amino Acid Sequence↗
PubMed2000

Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp.

Seven genes of the wb locus of Brucella melitensis 16M involved in the biosynthesis of the lipopolysaccharide O-side chain have been recently identified, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to mannosyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) the seven O-chain biosynthetic genes for polymorphism among Brucella spp. PCR-RFLP showed that the seven genes are highly conserved and occur even in the naturally rough species B. ovis and B. canis and also in rough strains of B. abortus and B. melitensis. Nevertheless, the few polymorphisms that were observed consisted of absence of additional restriction sites sometimes allowing differentiation at the species level (e.g. B. ovis) or at the biovar or strain level. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described.

Animals↗
PubMed2000

DNA polymorphism in the genus Brucella.

The genus Brucella has been described as consisting of six species, three of them including several biovars, which display a high degree of DNA homology by DNA-DNA hybridization. However, DNA polymorphism able to differentiate the six Brucella species and some of their biovars has been shown to exist. This work reviews the DNA variability in the genus Brucella and discusses the relationships between its members according to this genetic variability and a proposal for their evolution based on genetic diversity of the omp2 locus.

Animals↗
PubMed2000

An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals.

DNA polymorphism of the bp26 gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the bp26 gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized Brucella species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS711, downstream of the bp26 gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS711 insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS711 element downstream of the bp26 gene was a feature specific to the marine mammal Brucella strains. Thus, this PCR assay is able to differentiate Brucella terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.

Animals↗
PubMed1998

O-Polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry.

Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

Animals↗
PubMed1998

Effect of P39 gene deletion in live Brucella vaccine strains on residual virulence and protective activity in mice.

The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.

Animals↗
PubMed1997

Localization and characterization of a specific linear epitope of the Brucella DnaK protein.

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the alpha-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the alpha-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.

Adenosine Triphosphatases↗
PubMed1997

Rapid identification of rough Brucella isolates by a latex coagglutination assay with the 25-kilodalton outer membrane protein and rough-lipopolysaccharide-specific monoclonal antibodies.

A latex coagglutination assay was developed to identify rough (R) isolates of Brucella. Latex beads were coated, via protein A, with either an anti-Brucella rough-lipopolysaccharide (R-LPS) monoclonal antibody (MAb) or an anti-Brucella 25-kDa outer membrane protein (Omp25) MAb. Slide agglutination tests were done for 68 strains of Brucella spp., including type strains of all biovars as well as field isolates. Latex beads coated with MAb to R-LPS coagglutinated only R strains, whereas latex beads coated with MAb to Omp25 coagglutinated all the R Brucella isolates except Brucella ovis. Coagglutination was easier to read than agglutination with rabbit R-Brucella-specific antiserum. Thus, this assay accurately differentiates B. ovis from other R Brucella isolates. The latex coagglutination assay can substitute, to advantage, for the current anti-Brucella (R) rabbit monospecific serum.

Antibodies, Monoclonal↗
PubMed1996

Molecular and immunological characterization of the major outer membrane proteins of Brucella.

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s by selective extraction techniques and classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 kDa and 25-27 kDa OMPs which belong to the group 3 proteins. Variation in apparent molecular mass is essentially due to association with peptidoglycan subunits of different sizes. Two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of homology (> 85%), encode the 36 kDa porin proteins, now named Omp2a and Omp2b proteins respectively. Two genes code for the group 3 OMPs and are named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. Furthermore, all Brucella major OMPs share amino acid sequence homology with the major OMPs RopA or RopB of Rhizobium leguminosarum, which supports the close genetic relationship of brucellae with members of the alpha-2 subdivision of the class Proteobacteria. Another characteristic common to the major OMPs of R. leguminosarum and Brucella is that they are tightly, probably covalently, associated with the peptidoglycan. The major OMP genes display diversity among Brucella species, biovars and strains allowing their differentiation, and the polymorphic markers identified have brought new insights into the evolutionary development of the genus Brucella, antigenic variability of brucellae, and future prospects in the field of vaccine development.

Antigenic Variation↗
PubMed1996

Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker.

The dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B. ovis dnaK gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for all species with all restriction endonucleases tested except for B. melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species. The absence of the second EcoRV site for B. melitensis 16M was confirmed by DNA sequence analysis. The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnaK sequence of B. ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence. The difference between B. ovis strain 63/290 and B. melitensis 16M was due to an additional base-pair in B. melitensis 16M. Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV. The presence of a unique EcoRV site was specific to B. melitensis strains. Southern blot analysis of whole genomic DNA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B. melitensis. These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B. melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well.

Base Sequence↗
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↗
PubMed1995

Comparison of the efficacy of Brucella suis strain 2 and Brucella melitensis Rev. 1 live vaccines against a Brucella melitensis experimental infection in pregnant ewes.

The comparative efficacy of Brucella suis strain 2 (S2) and Brucella melitensis strain Rev. 1 (Rev. 1) live vaccines in protecting sheep against B. melitensis infection was evaluated by clinical and bacteriological examination of ewes vaccinated conjunctivally with a dose of 1 x 10(9) c.f.u. when 4 months old and then challenged with 5 x 10(7) c.f.u. of the B. melitensis virulent strain 53H38 (H38) at the middle of the first or second pregnancy following vaccination. Animals were considered to be protected when no abortion, no excretion of the challenge strain and no infection at slaughter occurred. The percentages of protection in Rev. 1-vaccinated groups challenged during either first (80%) or second (62%) pregnancy were significantly different (p < 0.001 and p < 0.05, respectively) compared with those of the relevant unvaccinated control groups. In contrast no significant difference in protection was found between the S2-vaccinated and control groups.

Animals↗
PubMed1995

Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella.

Seventy-seven Brucella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an approximately 50 bp deletion at the 3' terminal end of the gene in all Brucella ovis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Brucella species and within the Brucella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. ovis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Brucella species as was previously suggested by Ficht et al. (1990; Mol Microbiol 4, 1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucellia species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.

Bacterial Outer Membrane Proteins↗
PubMed1993

Conjugative transfer and in vitro/in vivo stability of the broad-host-range IncP R751 plasmid in Brucella spp.

Escherichia coli strain K12 BM14, carrying plasmid R751 (51.4 kb; Tpr, Tra+, IncP), was used as donor in matings with reference strains of the six Brucella nomenspecies, not known so far to harbor naturally occurring plasmids. R751 was easily transferred to and between Brucella spp., at frequencies ranging between 10(-1) and 10(-4). All Brucella transconjugants stably maintained plasmid R751 both in vitro and in vivo in our experimental conditions. No genetic modification of the plasmid during and after the conjugal transfer process could be deduced from the comparative restriction analysis (BamHI, HindIII, and EcoRI) in each Brucella transconjugant and in the E. coli donor.

Brucella↗
PubMed1993

Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a strain of Brucella melitensis.

Brucella strains exhibit either a rough (R) or a smooth (S) colonial phase identifiable by bacteriological methods. This depends on the biosynthesis and translocation to the surface in S but not in R strains, of the O-polysaccharide chain of the lipopolysaccharide (LPS) molecule. B. melitensis biovar 1 strain EP exhibited simultaneously both S and R characteristics in relation to colonial morphology, agglutination by monospecific anti-M and anti-R sera, activity of bacteriophages lytic for rough Brucella spp. (phage R/C) and for smooth B. melitensis (phage Iz). B. melitensis strain EP expressed fewer O-chains with a similar distribution of molecular weights than B. melitensis reference strain 16M by SDS-PAGE and immunoblotting, but higher amounts of R-LPS. Quantitative determination of S-LPS by a turbidimetric latex inhibition immunoassay with monoclonal antibodies confirmed the limited expression of S-LPS in strain EP. As with other gram-negative bacteria, the phenomenon could be attributed to a deficiency in one step of the biosynthetic assembly of the O-chains.

Brucella melitensis↗