Brucella Lipopolysaccharide Research Articles

Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis.

BackgroundBrucellosis is the most common bacterial zoonosis, and serological tests are routinely used in brucellosis control and eradication programs. In order to improve the accuracy of serological diagnostic method used in bovine brucellosis detection, this study developed an improved competitive ELISA with higher specificity and good sensitivity.ResultsThis study prepared 12 monoclonal antibodies against smooth Brucella lipopolysaccharide. One monoclonal antibody 3 F9, presented C epitope specificity, was used to develop a competitive ELISA for the serological detection of bovine brucellosis. The competitive ELISA, a commercial competitive ELISA kit, the rose-bengal plate agglutination test, and a microplate agglutination test were all used in the detection of 6 hyperimmune antisera against other commonly cross-reacted bacterial pathogens and 110 clinical bovine serum samples. The results of the test comparisons indicated that the competitive ELISA had higher specificity than the commercial competitive ELISA kit and RBT, and comparable sensitivity with the commercial ELISA kit.ConclusionsThis study provided a valuable detection tool with high specificity and good sensitivity, which prevent the wrong-culling of bovines in the eradication campaigns of bovine brucellosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0436-3) contains supplementary material, which is available to authorized users.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

IMMUNOGENIC PROPERTIES OF ANTI-IDIOTYPIC ANTIBODIES AND THEIR FAB FRAGMENTS WITH SPECIFICITY TO IMMUNOGLOBULINS AGAINST BRUCELLA LYPOPOLISACCHARIDE

BALB/c mice have been immunized by 50 or 100 |ig of monoclonal anti-idiotypic antibodies (AIB) specific immunoglobulins to Brucella lipopolysaccharide antigen and their Fab-fragments on 1st, 7th, 11th, 12th and 13th days in amounts of 50 |ig and 100 |ig. After that, the titers ofAT3 and AT1 specific antibodies were determined in mice in the variants of immunization by AIB or LPS, respectively. The other scheme implied for the four-time immunization by 50 |ig of the antigen with two-week intervals. It was established that 50 |ig was an optimal dose for the immunization by AIB. When monoclonal anti-idiotypic antibodies were used as immunological reagents, the titers of immune sera were 1: 6400, whereas using the Brucella lipopolysaccharide antigen, the titer of sera was 1: 12800. Thus, it was demonstrated that the monoclonal anti-idiotypic antibodies and their Fab-fragments induce immune response to the lipopolysaccharide antigen.

The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

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.

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of Brucella abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure.

A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

Inactivation of the ABC transporter ATPase gene in Brucella abortus strain 2308 attenuated the virulence of the bacteria

Brucella abortus is a Gram-negative, facultative intracellular bacterial pathogen of human and other animals. Brucella lipopolysaccharide has been identified as an important virulence factor. In this study, the ABC transporter ATPase gene (BAB1_0542) of B. abortus strain S2308 was inactivated by deleting a 446-bp fragment from the gene, thereby generating the mutant strain, S2308ΔATP. Real time PCR analysis confirmed the inactivation of this gene with no polar effect on the transcription of adjacent genes on the chromosome. The mutant was identified as a rough phenotype strain using heat agglutination test and crystal violet staining. The mutant strain had a different growth rate in Tryptic Soy Broth (TSB), compared to the wild type S2308 strain. Moreover, the mutant strain showed attenuated virulence in vitro and in vivo in RAW264.7 macrophages and Balb/c mice, respectively. Complementation of the mutant strain recovered the smooth phenotype of the bacteria and the complemented strain C2308ΔATP survived for more than four weeks in Balb/c mice, comparable to wild type strain S2308. Furthermore, immunization with the mutant strain protected mice from virulent strain challenge, which suggests the potential for the mutant strain S2308ΔATP as a future vaccine candidate. MHC I, MHC II and co-stimulatory molecule expression levels in mice following infection of S2308ΔATP and S2308 were also investigated.

Lipopolysaccharide as a target for brucellosis vaccine design

The gram-negative bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a world wide-distributed zoonotic disease that represents a serious problem for animal and human health. There is no human-to-human contagion and, since there is no human vaccine, animal vaccination is essential to control brucellosis. However, current vaccines (all developed empirically) do not provide 100% protection and are infectious in humans. Attempts to generate new vaccines by obtaining mutants lacking the lipopolysaccharide O-polysaccharide, in purine metabolism or in Brucella type IV secretion system have not been successful. Here we propose a new approach to develop brucellosis vaccines based on the concept that Brucella surface molecules evade efficient detection by innate immunity, thus delaying protective Th1 responses and opening a time window to reach sheltered intracellular compartments. We showed recently that a branch of the core oligosaccharide section of Brucella lipopolysaccharide hampers recognition by TLR4-MD2. Mutation of glycosyltransferase WadC, involved in the synthesis of this branch, results in a lipopolysaccharide that, while keeping the O-polysaccharide essential for optimal protection, shows a truncated core, is more efficiently recognized by MD2 and triggers an increased cytokine response. In keeping with this, the wadC mutant is attenuated in dendritic cells and mice. In the mouse model of brucellosis vaccines, the Brucella abortus wadC mutant conferred protection similar to that provided by S19, the best cattle vaccine available. The properties of the wadC mutant provide the proof of concept for this new approach and open the way for more effective brucellosis vaccines.

Comparison of total antibody and interferon-γ T-cell responses in patients following infection with brucellosis in Georgia

Brucellosis is an ancient disease that still remains a significant threat to humans and is typically linked to exposure to infected animals and/or consumption of unpasteurized animal products. Despite this history, we have a relatively limited understanding of the host characteristics of this disease; consequently, further research is necessary. In this study, we examined the humoral immune response in 43 Georgian individuals that had been diagnosed with brucellosis 3-12 months before enrollment in the study, many of whom still had symptoms after the completion of antibiotic therapy. In total, 35 of 43 (83%) of the patients had antibodies that bound to Brucella lipopolysaccharide (LPS) by COMPELISA, and 34 of 38 (89%) patients had demonstrable specific antibodies to Brucellergene™ antigens; the results from the two ELISAs were highly correlated (p=0.031, r=0.851). We also studied the cellular immune responses in 15 patients. All of the patients generated interferon (IFN)-γ in response to ex vivo stimulation with Brucella protein antigens, and the majority of the patients maintained measurable humoral responses to both LPS and protein antigens. From this initial study, we conclude that measurement of antibody and of cellular (IFN-γ) responses to brucellergene OCB protein epitopes may be worthy of further investigation as an alternative or adjunct to current diagnostics.

A potential novel Brucella species isolated from mandibular lymph nodes of red foxes in Austria

The wild red fox ( Vulpes vulpes) is a known indicator species for natural foci of brucellosis. Here, we describe phenotypic and molecular characteristics of two atypical Brucella strains isolated from two foxes hunted 2008 in Eastern Austria. Both strains agglutinated with monospecific anti- Brucella A serum and were positive in ELISA with monoclonal antibodies directed against various Brucella lipopolysaccharide epitopes. However, negative nitrate reductase- and negative oxidase-reaction were atypical traits. Affiliation to the genus Brucella was confirmed by 16S rRNA gene sequencing and by detection of the Brucella specific insertion element IS 711 and gene bcsp31 using real-time PCR. Both fox strains showed identical IS 711 Southern blot profiles but were distinct from known brucellae. The number of IS 711 copies detected was as high as found in B. ovis or marine mammal Brucella strains. Molecular analyses of the recA and omp2a/b genes suggest that both strains possibly represent a novel Brucella species.

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation.

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.

Intracellular trafficking of the Coxiella burnetii lipopolysaccharide

Coxiella burnetii is known to survive in monocytes ⁄macrophages and is the causative agent of Q fever. It undergoes an irreversible transition from virulent (phase I) to avirulent (phase II) forms that resembles smooth-to-rough transition in Enterobacteria [1]. C. burnetii inhibits phagosome conversion in professional phagocytes, while the phase II variants are eliminated within a phagolysosome [1]. The nature of bacterial components that inhibit phagosome–lysosome fusion has to be established. There are several indications that the C. burnetii lipopolysaccharide (LPS) could be a candidate molecule as it has long been considered a major determinant of virulence of the bacterium [2]. In addition, it plays an important role in the phase variation of the bacterium [1]. LPS I expressed in the phase I bacteria is of smooth (S) type and contains an O-polysaccharide chain [3] in contrast with LPS II present in the phase II bacteria that is of deeprough (R) type [3]. In LPS I, there are considerable amounts of two unique sugars, virenose and dihydrohydroxystreptose [3], in its O-polysaccharide chain, mainly in terminal positions. In this study, we elucidated the intracellular trafficking of Nine Mile LPS I and LPS II in murine bone marrow-derived macrophages (BMDM). The intracellular distribution of both LPSs with several markers of endocytic compartments was studied. We found that intracellular trafficking of LPS I was considerably similar to that of LPS II. BMDM were incubated with LPS I (1 mg ⁄L) and LPS II (1 mg ⁄L) [3] for 1 h at 4 C, followed by different periods of chase at 37 C. The co-localisation of LPSs with EEA1 (Early Endosome Antigen-1), a marker of early endosomes, and with Lamp-1 (Lysosomal Associated Membrane Protein-1), a marker of late endosomes and lysosomes, was determined by the quantification of immunofluorescence staining after the confocal microscopy. About 22% of LPS II co-localised with EEA-1 after 10 min; this percentage reached a maximum at 60 min and decreased thereafter (Fig. 1a). LPS II co-localised with Lamp-1. Indeed, the percentage of LPS II that co-localised with Lamp-1 was 33.6 ± 9% after 10 min, increased to 36.2 ± 3% at 30 min, reached a maximum of 66.5 ± 11% at 60 min, and decreased thereafter (Fig. 1b). It is evident that more than 40% of the internalised LPS II did not co-localise with the endosomal markers, suggesting that a considerable part of LPS II did not transit through early endosomes and the degradative pathway. Taken together, these data indicate that LPS II was rapidly internalised in early endosomes and transported thereafter to late endosomes and ⁄ or lysosomes. This time course is similar to that of fluorescein-conjugated albumin [4] and Shigella flexneri LPS [5]. The kinetics of LPS II trafficking differs from that of LPS of Brucella abortus as LPS II was localised in late compartments in 60 min while at least 5 h were required for the Brucella LPS [2]. In addition, when LPS II used the endosomal route, its trafficking was quite similar to that of the phase II C. burnetii [1]. LPS I co-localised with EEA1, and the percentage of co-localisation significantly decreased at 120 min (Fig. 1c). LPS I co-localised strongly with Lamp-1 (Fig. 1d). Indeed, the percentage of LPS I that co-localised with Lamp-1 was 33 ± 7% after 10 min, increased and reached a maximum of 60 ± 8% at 120 min, and decreased thereafter (Fig. 1d). Again, an important portion (40%) of LPS I did not co-localise with the endosomal markers, demonstrating that a considerable part of this LPS did not transit through both early endosomes and the degradative pathway. Trafficking of LPS I is similar to fluoresceinconjugated albumin [4], B. abortus LPS [2] and Corresponding author and reprint requests: E. Ghigo, URMITE CNRS UMR 6236 – IRD 3R198, Faculte de Medecine, 27 Bld J. Moulin, 13385 Marseille Cedex 05, France E-mail: eric.ghigo@univmed.fr

Immunogenic mimics of Brucella lipopolysaccharide epitopes

Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti- Brucella vaccines.

Outer membrane vesicle ofNeisseria meningitidis serogroup B as an adjuvant to induce specific antibody response against the lipopolysaccharide ofBrucella abortus S99

Brucellosis is a worldwide zoonosis and represents one of the most important public health problems in many countries, especially around the Mediterranean basin, Middle East, India and Central and South America. Currently subunit vaccines are being considered to develop effective vaccines for human which has been evidenced by vaccines currently available against the infections such as meningococcal diseases and influenza. The application of new adjuvants of microbial origins is also underway to design subunit vaccines promoting the immune responses to the antigenic determinant(s) of the vaccine. In order to explore the efficacy ofBrucella abortus lipopolysaccharide (LPS) combined with different adjuvants and proteins (as a vaccine candidate) in the induction of immune response as an effective and long-lasting immunity againstBrucella, we evaluated the outer membrane vesicle ofNeisseria meningitidis serogroup B (GBMOMV) as a subcutaneous adjuvant and a part of a brucellosis candidate vaccine to induce high titres of specific anti-Brucella abortus S99 LPS in animal model (mice). The obtained results were compared with complete and incomplete Freund’s adjuvants (CFA and IFA). LPS+GBMOMV was the most immunogenic compound that stimulated following the first injection and increase in IgG titre of about 3.90, 3.18 and 1.58 fold higher than that produced against LPS, LPS+IFA and LPS+CFA, respectively. The highest anti-LPS IgG titre was detected two weeks after the third injection (the day 42) of LPS+GBMOMV. Purified GBMOMV can be considered as a safe subcutaneous adjuvant and a part of a candidate vaccine when combined with lipopolysaccharide ofBrucella abortus S99.

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.

Rapid latex agglutination test for the serodiagnosis of human brucellosis

We developed and evaluated a user-friendly latex agglutination assay for the serodiagnosis of human brucellosis. The assay was obtained by coating colored latex beads with Brucella lipopolysaccharides and drying of the activated beads onto white agglutination cards. Individual cards were sealed in a protective foil to secure stability of the dried reagent and to obtain a test in a single assay format. The latex agglutination assay is simply performed by suspending the dried latex reagent in a drop of serum and looking for macroscopic agglutination of the latex beads by visual inspection. Results are obtained within 30 s after mixing the sample with the test reagent. The sensitivity of the assay was determined to be 89.1% (95% confidence interval [CI], 76–96) for the initial serum samples collected from patients with culture-confirmed brucellosis and the specificity is 98.2% (95% CI, 96–99). The assay is ideal for use as a field test in remote areas and as point-of-care test in hospitals and health care centers that lack the expertise and facilities to perform the more demanding classic serologic tests.

Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry

Brucella melitensis strains may occur as either smooth or rough variants depending on the expression of O-polysaccharides (OPS) as a component of the bacterial outer membrane lipopolysaccharide (LPS). The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth virulent B. melitensis 16M results in a rough mutant designated WRR51. We developed a flow cytometric method to determine the proportion of B. melitensis cells displaying surface O-polysaccharide (OPS) in liquid culture. OPS was detected using polyclonal antibodies from rabbits immunized with smooth (S) or rough (R) Brucella LPS. First, we evaluated the binding of these antibodies to 16M (S), WRR51 (R) and complemented WRR51 expressing the wboA gene (S) as well as to their corresponding GFP-expressing derivative strains 16M/GFP, WRR51/GFP and WRR51/GFP + wboA. The rough mutants did not react with anti-S-LPS nor did the smooth strains react with anti-R-LPS. Second, using different ratios of 16M/GFP and WRR51/GFP, we were able to detect the presence of 1% rough bacteria spiked into a sample of smooth organisms. Third, we evaluated the purity of cultures of B. melitensis strains grown in a fermenter. These flow cytometric methods may be useful for quality control of process development for large-scale vaccine production.

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl beta-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.

The effect of Brucella abortus on hydrogen peroxide and nitric oxide production by bovine polymorphonuclear cells.

The effect of Brucella on the generation of microbicidal reactive oxygen and nitrogen metabolites by bovine peripheral polymorphonuclear cells (PMNs) was investigated. The PMNs were recovered from the peripheral blood of control calves and experimental calves previously vaccinated against brucellosis. Significantly larger quantities of NO and H2O2 were generated by PMNs from control and experimental calves following activation by heat-killed whole cells or outer membrane protein of Brucella abortus than by non-activated cells (p<0.05-0.01). In contrast, generation of H2O2 and NO decreased when PMNs were exposed to the lipopolysaccharide of Brucella. However, the generation of H2O2 and NO by activated PMNs from the control and experimental calves did not differ significantly.