Molecular characterization of Brucella strains isolated from marine mammals

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B. melitensis, B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate.     

Sensitivity of Brucella spp to tetracycline and its analogues.

The sensitivity to eight tetracyclines of 100 strains of brucellae, comprising strains of Brucella abortus, Br. melitensis, and Br. suis, was determined. Demethylchlortetracycline was the most effecitve against all the groups of brucellae, whereas oxytetracycline and chlortetracycline were the least effective. The mean MIC value for demethylchlortetracycline, doxycycline, lymecycline, and tetracycline was less than or equal to 1 mug/ml. Strains of Br. abortus biotype 2 and Br. suis were the most sensitive strains examined.     

Binding of bacteria from the genus Brucella to human B lymphocytes.

In previous studies, we have shown that various lymphocyte subpopulations bind different strains of bacteria of different genera and species. Among these bacteria was a strain of Brucella melitensis which bound to all human B lymphocytes. To determine whether the binding of B. melitensis to human B lymphocytes was strain, species, or genus characteristic, we tested the binding of B. melitensis, Brucella abortus, Brucella ovis, Brucella suis, Brucella canis and Brucella neotomae to human normal and leukemic B lymphocytes. The binding of different Brucella species to B lymphocytes was determined by single- and double-labeling experiments in which a strain of Escherichia coli, coated with anti-light chain antibodies, was used as a marker for B cells. As in previous experiments, we found that B. melitensis and antibody-coated E. coli bound to the same cells. Also, we found that all the other species of bacteria tested bound to the B lymphocytes, normal or leukemic. B. ovis and B. neotomae, which are not human pathogens, bound to fewer B lymphocytes than did the human pathogens B. abortus, B. melitensis, B. suis, and B. canis. Furthermore, we found that the quality of rosettes formed by the nonpathogenic bacteria with the lymphocytes, i.e., the number of bacteria per lymphocytes, was lower than that of pathogenic Brucella species. We conclude that all of the Brucella species tested have the ability to bind to human B lymphocytes, but that only those which are human pathogens bind firmly to all B lymphocytes and may be used as reliable markers for these cells. We also suggest that the binding of Brucella species to B lymphocytes may have some bearing on the pathogenesis of brucellosis in humans.     

A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody.

A coagglutination test using monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 16M has been developed for the rapid identification of the smooth Brucella species. All reference strains of several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Brucella species, with the exception of B. abortus 45/20, were negative in the assay. Among the different organisms tested not belonging to the genus Brucella, serovar O9 of Yersinia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity, simplicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Brucella strains in diagnostic laboratories.     

Rapid identification of smooth Brucella species with a monoclonal antibody.

A colony blot enzyme-linked immunosorbent assay was developed for the rapid identification of smooth Brucella species, i.e., Brucella abortus, B. melitensis, and B. suis. Bacterial colonies from plates were blotted onto nitrocellulose disks, lysed by immersion in chloroform, and reacted with BRU 38, a rat monoclonal antibody with specificity for the O side chain of B. abortus. Reaction with anti-rat immunoglobulin G conjugated to horseradish peroxidase and development in 4-chloro-1-naphthol resulted in colonies of naturally occurring smooth Brucella species staining purple. Results could be obtained within 4 h after colonies were visible on plates and individual colonies could be detected. Yersinia enterocolitica serovar O:9 strains were the only other organisms tested which showed cross-reaction by using this procedure. Because of its speed, sensitivity, and specificity, this technique should be very useful for identifying smooth Brucella strains in diagnostic laboratories.     

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.     

Use of an enzyme immunoassay test for characterizing the A and M antigens of Brucella.

An enzyme immunoassay test was developed for detecting the A and M antigens of brucellae. One hundred fifteen isolates, including 96 strains of Brucella abortus, 5 strains of B. suis, 1 strain of B. melitensis and 1 strain of B. neotomae, were accurately serotyped with the enzyme immunoassay test. No reactions were observed with 11 isolates of B. canis and 1 isolate of B. ovis. This rapid serological test provides for considerable savings in time and materials as compared with the standard tube agglutination test.     

Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis.

Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.     

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.     

Occurrence of Quinovosamine in Lipopolysaccharides of Brucella Species

Lipopolysaccharides obtained from Brucella abortus, B. melitensis, and B. suis, but not B. canis, were found to contain amino sugars identified as glucosamine and quinovosamine by cation exchange and thin-layer cellulose chromatography and ninhydrin degradation.     

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.     

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.     

Limited genetic diversity of Brucella spp

Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis. Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici. The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.     

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.     

Development of a selective culture medium for primary isolation of the main Brucella species

Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.     

Characterization of new members of the group 3 outer membrane protein family of Brucella spp

Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.     

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.     

Broth microdilution susceptibility testing of Brucella species: quality control limits for ten antimicrobial agents against three standard quality control strains

Brucella broth without supplementation is the recommended medium for broth microdilution susceptibility tests of Brucella abortus, B. melitensis, and B. suis. Based on an eight-laboratory collaborative study using a pH-adjusted modification of this medium, we propose MIC quality control ranges for three control strains against 10 antimicrobials that are potentially efficacious for treating infections caused by these agents of bioterrorism.     

The IncP island in the genome of Brucella suis 1330 was acquired by site-specific integration

An 18,228-bp region containing open reading frames predicted to be derived from the IncP plasmid or phage ancestors is present in the genomes of Brucella suis biovars 1 to 4, B. canis, B. neotomae, and strains isolated from marine mammals, but not in B. melitensis, B. abortus, B. ovis, and B. suis biovar 5. The presence of circular excision intermediates and the results of an analysis of sequenced bacterial genomes suggest that the region downstream of the guaA gene is a hotspot for site-specific integration of foreign DNA mediated by a CP4-like integrase.     

Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR.

Several PCR assays which identify the genus Brucella but do not discriminate among species have been reported. We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella. Individual biovars within a species are not differentiated. The assay can identify three biovars (1, 2, and 4) of B. abortus, all three biovars of B. melitensis, biovar 1 of B. suis, and all B. ovis biovars. These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research. The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome. Identity is determined by the size(s) of the product(s) amplified from primers hybridizing at various distances from the element. The performance of the assay with U.S. field isolates was highly effective. When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods. Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhodospirillum rubrum) and two control bacteria (Bordetella bronchiseptica and Escherichia coli) tested negative by the assay.