Characterization of three predicted zinc exporters in Brucella ovis identifies ZntR-ZntA as a powerful zinc and cadmium efflux system not required for virulence and unveils pathogenic Brucellae heterogeneity in zinc homeostasis.

Brucella ovis causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonotic Brucella species that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potential B. ovis zinc exporters, including the ZntA ortholog previously studied in B. abortus. Despite an in-frame deletion removing 100 amino acids from B. ovis ZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity for B. abortus ZntA, which, together with differences in ZntR-mediated regulation of zntA expression between B. ovis and B. abortus, could contribute to explaining why the ΔzntR mutant of B. abortus is attenuated while that of B. ovis is virulent. Additionally, B. ovis ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for the B. abortus ortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in B. abortus, behaved as the B. ovis parental strain in all tests performed. However, their overexpression in the ΔzntA mutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence of B. ovis ΔzntA, suggest that host-mediated zinc intoxication is not a relevant mechanism to control B. ovis infection.

A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams.

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

Preconception Care for Men and Women during the Pandemic, an Intervention Proposal.

The COVID-19 pandemic and its measures resulted in limited outdoor activities, reduced group meetings, etc., leading to unhealthy habits. Several studies showed how certain unhealthy habits can lead to serious consequences for both men and women, as well as affect future offspring. (1) Background: Therefore, we present a community intervention at the preconception stage to avoid future risks. The purpose of this intervention is to change lifestyles and beliefs about the health of men and women in the preconception period; (2) Methods: For the design of the intervention, a bibliographic search was performed both in English and Spanish in the main databases of health sciences and nursing (Cochrane, PubMed, Web of Science, CINAHL, LILACS, Dialnet), using descriptors in MeSH health for sciences; (3) Results: We proposed that a variety of lifestyles be analyzed, including aspects such as physical activity, nutrition, etc. In addition, stress management should be emphasized through a relaxation workshop, where three different techniques be proposed to reduce anxiety levels in stressful situations; (4) Conclusions: Due to the limited scientific results of interventions carried out in the preconception period simultaneously with men and women, more community interventions that address this topic are needed to assess the impact of these actions on the health of the population.

The Transcriptional Regulator MucR, but Not Its Controlled Acid-Activated Chaperone HdeA, Is Essential for Virulence and Modulates Surface Architecture and Properties in Brucella ovis PA.

Brucella ovis is a non-zoonotic bacterium causing contagious epididymitis and other genital lesions in rams and responsible for significant economic losses in sheep-breeding areas. It is a naturally rough (without O-chains in the lipopolysaccharide) Brucella species whose virulence mechanisms have been less explored than those of zoonotic smooth brucellae (bearing O-chains that mask other outer membrane molecules). Considering the rough nature of Brucella ovis, the influence of surface components other than O-chains on its biological properties may be greater than in smooth Brucella species. Here we describe the construction and characterization of the mucR deletion mutant of virulent B. ovis PA, which is defective in a transcriptional regulator, affecting surface properties and virulence in smooth brucellae. This mutant showed increased amounts of three proteins identified as HdeA (acid-activated chaperone), Omp25d (outer membrane protein undetectable in the parental strain), and BOV_A0299 (hypothetical protein of unknown function). This observation correlated with the enhanced transcription of the corresponding genes and constitutes the first report on this type of proteome alteration in Brucella ΔmucR mutants. The upstream regions of the three genes contained AT rich domains with T-A steps described as binding sites for MucR in the Brucella abortus 2308 babR promoter (gene also upregulated in B. ovis ΔmucR), which suggests that hdeA, omp25d, and BOV_A0299 expression could be repressed by MucR through a direct binding to their promoter regions. Relative quantification of transcripts of several other genes selected according to the transcriptome of smooth brucellae ΔmucR mutants revealed not only similarities but also relevant differences among strains, such as those detected in flagellar and virB genes. Periplasmic HdeA has been related to the resistance of B. abortus to acidic pH, conditions encountered by Brucella inside phagocytes, but the deletion of hdeA in B. ovis PA and the ΔmucR mutant did not modify any of the evaluated properties of these strains. The B. ovis PA ΔmucR and ΔmucRΔhdeA mutants had defective in vitro growth and altered surface properties and architecture, exemplified by detectable amounts of Omp25d. Moreover, they showed virulence attenuation but established persistent splenic infection in mice, which encourages their evaluation as specifical attenuated vaccines against B. ovis.

Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model.

Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate ⇌ phosphoenolpyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.

The Three Flagellar Loci of Brucella ovis PA Are Dispensable for Virulence in Cellular Models and Mice.

Brucella ovis is a facultative intracellular bacterium that causes a non-zoonotic ovine brucellosis mainly characterized by male genital lesions and is responsible for important economic losses in sheep farming areas. Studies about the virulence mechanisms of Brucella have been mostly performed with smooth (bearing O-polysaccharide in lipopolysaccharide) zoonotic species, and those performed with B. ovis have revealed similarities but also relevant differences. Except for few strains recently isolated from unconventional hosts, Brucella species are non-motile but contain the genes required to assemble a flagellum, which are organized in three main loci of about 18.5, 6.4, and 7.8 kb. Although these loci contain different pseudogenes depending on the non-motile Brucella species, smooth B. melitensis 16M builds a sheathed flagellum under particular culture conditions and requires flagellar genes for virulence. However, nothing is known in this respect regarding other Brucella strains. In this work, we have constructed a panel of B. ovis PA mutants defective in one, two or the three flagellar loci in order to assess their role in virulence of this rough (lacking O-polysaccharide) Brucella species. No relevant differences in growth, outer membrane-related properties or intracellular behavior in cellular models were observed between flagellar mutants and the parental strain, which is in accordance with previous results with B. melitensis 16M single-gene mutants. However, contrary to these B. melitensis mutants, unable to establish a chronic infection in mice, removal of the three flagellar loci in B. ovis did not affect virulence in the mouse model. These results evidence new relevant differences between B. ovis and B. melitensis, two species highly homologous at the DNA level and that cause ovine brucellosis, but that exhibit differences in the zoonotic potential, pathogenicity and tissue tropism.

Evaluation of human trophoblasts and ovine testis cell lines for the study of the intracellular pathogen Brucella ovis.

Since pathogenic Brucella survive and replicate inside phagocytes, cellular models of infection constitute important tools in brucellosis research. We describe the behavior of B. ovis PA (which causes a type of ovine brucellosis mainly affecting the male reproductive tract) and representative attenuated mutants in two commercially available cell lines of non-professional phagocytes related to Brucella tissue preference: OA3.Ts ovine testis cells and JEG-3 human trophoblasts. In comparison with J774.A1 macrophages and HeLa cells, intracellular bacteria were enumerated at several post-infection time points and visualized by confocal microscopy. Replication of B. ovis in OA3.Ts and JEG-3 cells was equivalent to that observed in J774.A1 macrophages-despite the more efficient internalization in the latter-and better than in HeLa cells. Multiplication and/or survival in all phagocytes was dependent on virB2 and vjbR but independent of cgs, despite the attenuation in mice of the Δcgs mutant. However, Omp25c was required for B. ovis internalization only in HeLa cells, and removal of Omp31 increased bacterial internalization in human HeLa and JEG-3 cells. The results presented here demonstrate variability in the interaction of B. ovis with different host cells and provide advantageous models of non-professional phagocytes to study the intracellular behavior of B. ovis.

Characterization of Cell Envelope Multiple Mutants of Brucella ovis and Assessment in Mice of Their Vaccine Potential.

Brucella ovis is a non-zoonotic Brucella species lacking specific vaccine. It presents a narrow host range, a unique biology relative to other Brucella species, and important distinct surface properties. To increase our knowledge on its peculiar surface and virulence features, and seeking to develop a specific vaccine, multiple mutants for nine relevant cell-envelope-related genes were investigated. Mutants lacking Omp10 plus Omp19 could not be obtained, suggesting that at least one of these lipoproteins is required for viability. A similar result was obtained for the double deletion of omp31 and omp25 that encode two major surface proteins. Conversely, the absence of major Omp25c (proved essential for internalization in HeLa cells) together with Omp25 or Omp31 was tolerated by the bacterium. Although showing important in vitro and in vivo defects, the Δomp10Δomp31Δomp25c mutant was obtained, demonstrating that B. ovis PA survives to the simultaneous absence of Omp10 and four out seven proteins of the Omp25/Omp31 family (i.e., Omp31, Omp25c, Omp25b, and Omp31b, the two latter naturally absent in B. ovis). Three multiple mutants were selected for a detailed analysis of virulence in the mouse model. The Δomp31Δcgs and Δomp10Δomp31Δomp25c mutants were highly attenuated when inoculated at 106 colony forming units/mouse but they established a persistent infection when the infection dose was increased 100-fold. The Δomp10ΔugpBΔomp31 mutant showed a similar behavior until week 3 post-infection but was then totally cleared from spleen. Accordingly, it was retained as vaccine candidate for mice protection assays. When compared to classical B. melitensis Rev1 heterologous vaccine, the triple mutant induced limited splenomegaly, a significantly higher antibody response against whole B. ovis PA cells, an equivalent memory cellular response and, according to spleen colonization measurements, better protection against a challenge with virulent B. ovis PA. Therefore, it would be a good candidate to be evaluated in the natural host as a specific vaccine against B. ovis that would avoid the drawbacks of B. melitensis Rev1. In addition, the lack in this attenuated strain of Omp31, recognized as a highly immunogenic protein during B. ovis infection, would favor the differentiation between infected and vaccinated animals using Omp31 as diagnostic target.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection.

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.

Quorum-sensing and BvrR/BvrS regulation, the type IV secretion system, cyclic glucans, and BacA in the virulence of Brucella ovis: similarities to and differences from smooth brucellae.

Brucella ovis is a rough bacterium--lacking O-polysaccharide chains in the lipopolysaccharide--that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguish B. ovis from smooth Brucella, which require O-polysaccharide chains for virulence, we have analyzed the significance in B. ovis of the main virulence factors described for smooth Brucella. Attempts to obtain strains of virulent B. ovis strain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system for in vitro survival, while the inactivation of bacA--in contrast to the results seen with smooth Brucella--did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake of B. ovis PA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by the virB operon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described for B. melitensis, VjbR regulates the transcription of the virB operon positively, and the N-dodecanoyl-dl-homoserine lactone (C(12)-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of the fliF flagellar gene was observed in B. ovis, probably due to the two deletions detected upstream of fliF. These results, together with others reported in the text, point to similarities between rough virulent B. ovis and smooth Brucella species as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics of B. ovis.

Differences in the outer membrane-related properties of the six classical Brucella species.

Outer membrane-related properties (such as auto-agglutination and susceptibility to various compounds) of strains representative of the six classical species of the genus Brucella were assessed. The differences identified could not be fully explained based on the smooth or rough phenotype of the strain. Smooth strains of the closely related species Brucella melitensis and B. abortus exhibited different susceptibility patterns and the rough, virulent B. ovis and B. canis strains were equally or more resistant to conditions such as pH, non-immune serum, hydrogen peroxide and bactericidal cationic peptides than smooth strains. Such heterogeneity in outer membrane characteristics could account for differences in pathogenicity and host tropism.

Cholesterol, ganglioside GM1 and class A scavenger receptor contribute to infection by Brucella ovis and Brucella canis in murine macrophages.

The establishment of infection by Brucella ovis and Brucella canis in J774.A1 macrophages was found to be dependent upon cholesterol and ganglioside GM(1), two components of lipid rafts. This process also required a class A scavenger receptor of macrophages, and was not inhibited by smooth and rough lipopolysaccharides from Brucella spp. In response to infection, both bacteria induced a weak degree of macrophage activation. These results demonstrate that B. ovis and B. canis use cell surface receptors common to smooth Brucella spp. for macrophage infection, thus limiting macrophage activation and favouring intracellular multiplication and/or the survival of both bacteria.

Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species.

Members of the Omp25/Omp31 family of surface proteins were previously shown to participate in the virulence of some Brucella species and a different distribution of the seven proteins of this family among species could be related to the difference in pathogenicity and host preference they exhibit. Accordingly, in this work we have analyzed the expression of the genes coding for the Omp25/Omp31 family in the six classical Brucella species and a set of B. ovis mutant strains with each omp gene inactivated. Immunoblot of whole-cell lysates with antibodies raised against the purified recombinant outer membrane proteins (OMPs) did not show the simultaneous presence of the seven OMPs in any of the Brucella strains studied, but different Omp25/Omp31 profiles were detected, in our experimental conditions, between the Brucella strains representative of the six classical species. Transcripts for omp31, omp25 and omp25c were, in general, the most abundant of the family and some hits were found in B. ovis for a posttranscriptional regulation mechanism and for a compensatory mechanism increasing the synthesis of a protein to compensate for the absence of another one. Finally, the potential interest of Omp25c and Omp31b as subcellular vaccines, considering their occurrence in the Brucella strains studied and their antigenic relatedness with other proteins of the family, is discussed.

Influence of brucellosis history on serological diagnosis and evolution of patients with acute brucellosis.

Serological diagnosis of human brucellosis is problematic in endemic brucellosis regions and with patients having a history of brucellosis. The aim of this study is to ascertain the serologic and evolutionary behavior of the tests of serum agglutination, Coombs anti-Brucella, immunocapture-agglutination, enzyme-linked immunosorbent assay (ELISA) IgG, IgA, IgM and ELISA-IgG avidity against Brucella lipopolysaccharide (S-LPS), in patients with acute brucellosis based on whether or not a history of brucellosis exists. Titers and seropositivity in all the tests assayed were higher in the patients having brucellosis history (from 90.9% in ELISA-IgM to 100% in ELISA-IgG) than in the patients lacking such history (from 79.3% in ELISA-IgM to 86.2% in Coombs, immunocapture-agglutination, and ELISA-IgG). IgG S-LPS avidity results in patients with brucellosis history were significantly higher (always over 84%) than in patients without brucellosis history (from 48.0% in the initial sera to 81% ten months later) (p<0.001). The titers of antibodies against Brucella in the initial sera and ELISA-IgG avidity against S-LPS may allow distinguishing patients with brucellosis caused by primary infection in the initial stages of the disease from patients seropositive due to prior infections from Brucella.

Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells.

The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Deltaomp25d mutant was unable to multiply, whereas the Deltaomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Deltaomp25d and Deltaomp22 mutants.

Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis.

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Deltaomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Deltaomp25d mutant were found, especially considering that the fully virulent Deltaomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies.

Ochrobactrum cytisi sp. nov., isolated from nodules of Cytisus scoparius in Spain.

Two strains named ESC1(T) and ESC5 were isolated from nodules of Cytisus scoparius growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene showed that these strains belong to the genus Ochrobactrum, their closest relatives being Ochrobactrum anthropi and Ochrobactrum lupini, with 100 and 99.9 % similarity to the respective type strains. Despite this high similarity, the results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses showed that these strains represent a novel species of genus Ochrobactrum. The DNA-DNA hybridization values were respectively 70, 66 and 55 % with respect to O. lupini LUP21(T), O. anthropi DSM 6882(T) and Ochrobactrum tritici DSM 13340(T). The predominant fatty acids were C(18 : 1)omega7c and C(18 : 1) 2-OH. Strains ESC1(T) and ESC5 were strictly aerobic and were able to reduce nitrate and to hydrolyse aesculin. They produced beta-galactosidase and beta-glucosidase and did not produce urease after 48 h incubation. The G+C content of strain ESC1(T) was 56.4 mol%. Both strains ESC1(T) and ESC5 contained nodD and nifH genes on megaplasmids that were related phylogenetically to those of rhizobial strains nodulating Phaseolus, Leucaena, Trifolium and Lupinus. From the results of this work, we propose that the strains isolated in this study be included in a novel species named Ochrobactrum cytisi sp. nov. The type strain is ESC1(T) (=LMG 22713(T)=CECT 7172(T)).