J. M. Blasco

A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence

Two mutants showing increased sensitivity to polycations and surfactants were obtained by transposon mutagenesis of virulent Brucella abortus 2308 Nalr. These mutants showed no obvious in vitro growth defects and produced smooth‐type lipopolysaccharides. However, they hardly multiplied or persisted in mouse spleens, displayed reduced invasiveness in macrophages and HeLa cells, lost the ability to inhibit lysosome fusion and were unable to replicate intracellularly. Subsequent DNA analyses identified a two‐component regulatory system [Brucella virulence related (Bvr)] with a regulatory (BvrR) and sensory (BvrS) protein. Cloning of bvrR in the BvrR‐deficient mutant restored the resistance to polycations and, in part, the invasiveness and the ability to multiply intracellularly. BvrR and BvrS were highly similar (87–89% and 70–80% respectively) to the regulatory and sensory proteins of the chromosomally encoded Rhizobium meliloti ChvI–ExoS and Agrobacterium tumefaciens ChvI–ChvG systems previously shown to be critical for endosymbiosis and pathogenicity in plants. Divergence among the three sensory proteins was located mostly within a periplasmic domain probably involved in stimulus sensing. As B. abortus, R. meliloti and A. tumefaciens are phylogenetically related, these observations suggest that these systems have a common ancestor that has evolved to sense stimuli in plant and animal microbial environments.

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio- and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a One Health approach to be successful.

Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains

ABSTRACT An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9

ABSTRACT Yersinia enterocolitica O:9 bears a smooth lipopolysaccharide (S-LPS) of Brucella sp. O-chain A + C/Y epitopic structure and is a cause of false-positive serological reactions (FPSR) in standard tests for cattle brucellosis. Brucella S-LPS, cross-reacting S-LPSs representing several O-chain epitope combinations, Brucella core lipid A epitopes (rough LPS), Brucella abortus S-LPS-derived polysaccharide, native hapten polysaccharide, rough LPS group 3 outer membrane protein complexes, recombinant BP26, and cytosolic proteins were tested in enzyme-linked immunosorbent assays (ELISA) and precipitation tests to detect cattle brucellosis (sensitivity) and to differentiate it from FPSR (specificity). No single serological test and antigen combination showed 100% sensitivity and specificity simultaneously. Immunoprecipitation tests with native hapten polysaccharide, counterimmunoelectrophoresis with cytosolic proteins, and a chaotropic ELISA with Brucella S-LPS were 100% specific but less sensitive than the Rose Bengal test, complement fixation, and indirect ELISA with Brucella S-LPSs and native hapten or S-LPS-derived polysaccharides. A competitive ELISA with Brucella S-LPS and M84 C/Y-specific monoclonal antibody was not 100% specific and was less sensitive than other tests. ELISA with Brucella suis bv. 2 S-LPS (deficient in C epitopes), Escherichia hermannii S-LPSs [lacking the contiguous α-(1-2)-linked perosamine residues characteristic of Y. enterocolitica S-LPS], BP26 recombinant protein, and Brucella cytosolic fractions did not provide adequate sensitivity/specificity ratios. Although no serological test and antigen combination fully resolved the diagnosis of bovine brucellosis in the presence of FPSR, some are simple and practical alternatives to the brucellin skin test currently recommended for differential diagnosis.

Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

Background The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model.

ABSTRACT Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-d-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manBcore to distinguish it from the wbk manBO-Ag. The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

Neurobrucellosis in Stranded Dolphins, Costa Rica

Ten striped dolphins, Stenella coeruleoalba, stranded along the Costa Rican Pacific coast, had meningoencephalitis and antibodies against Brucella spp. Brucella ceti was isolated from cerebrospinal fluid of 6 dolphins and 1 fetus. S. coeruleoalba constitutes a highly susceptible host and a potential reservoir for B. ceti transmission.

A Brucella ovis antigenic complex bearing poly-ε-caprolactone microparticles confer protection against experimental brucellosis in mice

A hot saline antigenic extract (HS) from Brucella ovis was encapsulated in poly-epsilon-caprolactone microparticles (PEC), and tested as a vaccine against B. ovis and B. abortus infections in mice. Subcutaneous but not oral administration in BALB/c mice of the HS-PEC induced high amounts of IFN-gamma and IL-2 but low quantities of IL-4 suggesting a combined Th1/Th2 cellular immune response. The vaccine administered either subcutaneously or orally protected mice against B. ovis infection. Such protection was similar to that provided by the reference living attenuated B. melitensis Rev. 1 vaccine. By contrast, only the subcutaneous vaccination with HS-PEC was as effective as Rev. 1 in conferring protection against B. abortus infection. The use of free HS or empty PEC microparticles did not produce any protective effect.

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams.

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.

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

ABSTRACT 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. CITAs 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.