Brucella Abortus Lipopolysaccharide Research Articles

Bactericidal Activity of Serum by Brucella Abortus RB51 Outer Membrane Protein's Combined by Brucella Abortus S99 Lipopolysaccharide Induction.

Brucellosis vaccines are designed to induce cellular immunity. An effective brucellosis vaccine could induce both cellular and humoral immunity. Serum Bactericidal Assay (SBA) is an important method for determining vaccine humoral immunity. This study is the first to observe humoral immunity in brucellosis by SBA. Extracted Brucella abortus (B. abortus) Lipopolysaccharide (LPS) and Outer Membrane Proteins (OMPs) were injected into rabbits. Group 1 was injected with 25 μg of LPS, Group 2 was injected with 50 μg of OMPs, and Group 3 was injected with 1 ml of combined vaccine, 3 times every 2 weeks. The groups were challenged with B. abortus 544 in the second injection. Sera were separated 2 weeks after the last injection. SBA was performed, and each well was streak-cultured into a plate of Brucella agar. A colony count was done for each plate. Results have shown, the third injection of the combined vaccine had the highest titer of , and the efficacy of the vaccine was 87.71%. As a conclusion, the results of this study showed that LPS and OMP's from B. abortus can provide acceptable immunity.

Immunization of guinea pigs with Salmonella delivered anti-Brucella formulation reduces organs bacterial load and mitigates histopathological consequences of Brucella abortus 544 challenge

With an objective to generate safe and effective anti-Brucella vaccine, an attenuated live Salmonella Typhimurium vector delivering heterologous Brucella immunogenic proteins SOD, Omp19, BLS, and PrpA formulated with purified Brucella abortus lipopolysaccharide was evaluated on a guinea pig model. This model represents high susceptibility to Brucella infections and showed similarities in reproducing human pathologies. On safety perspectives, the vaccine formulation induced no observable alterations on general health and histology of the vaccinated guinea pigs. Upon virulent strain 544 challenge, a protective index of 1.52 was observed based on differential splenic counts. Post-challenge histopathology revealed that Brucella induced microgranulomas and fatty degenerations were prominent in the organs of non-immunized animals as compared to immunized animals. With these findings, it is suggestive that this live Brucella-free vaccine formulation is safe and protective on a sensitive guinea pig model and may be suitable for further human-related vaccine trials.

Preparation and Evaluation of a New Lipopolysaccharide-based Conjugate as a Vaccine Candidate for Brucellosis

ObjectivesDevelopment of an efficacious vaccine against brucellosis has been a challenge for scientists for many years. At present, there is no licensed vaccine against human brucellosis. To overcome this problem, currently, antigenic determinants of Brucella cell wall such as Lipopolysaccharide (LPS) are considered as potential candidates to develop subunit vaccines. MethodsIn this study, Brucella abortus LPS was used for conjugation to Neisseria meningitidis serogroup B outer membrane vesicle (OMV) as carrier protein using carbodiimide and adipic acid–mediated coupling and linking, respectively. Groups of eight BALB/c mice were injected subcutaneously with 10 μg LPS alone, combined LPS + OMV and conjugated LPS–OMV on 0 days, 14 days, 28 days and 42 days. Anti-LPS IgG was measured in serum. ResultsThe yield of LPS to OMV in LPS–OMV conjugate was 46.55%, on the basis of carbohydrate content. The ratio for LPS to OMV was 4.07. The LPS–OMV conjugate was the most immunogenic compound that stimulated following the first injection with increased IgG titer of ∼5-fold and ∼1.3-fold higher than that produced against LPS and LPS in noncovalent complex to OMV (LPS + OMV), respectively. The highest anti-LPS IgG titer was detected 2 weeks after the third injection (Day 42) of LPS–OMV conjugate. The conjugated compound elicited higher titers of IgG than LPS + OMV, that showed a 100–120-fold rise of anti-LPS IgG in mice. ConclusionThese results indicate that our conjugated LPS–OMV can be used as a brucellosis vaccine, but further investigation is required.

Influence of Brucella abortus lipopolysaccharide as an adjuvant on the immunogenicity of HPV-16 L1VLP vaccine in mice.

Brucella abortus lipopolysaccharide (LPS) has less toxicity and no pyrogenic properties in comparison with other bacterial LPS. It is a toll-like receptor 4 agonist and has been shown to have the potential use as a vaccine adjuvant. In this study, the immunostimulatory properties of LPS from smooth and rough strains of B. abortus (S19 and RB51) as adjuvants were investigated for the human papillomavirus type 16 (HPV16) L1 virus-like particles (L1VLPs) vaccines. C57BL/6 mice were immunized subcutaneously three times either with HPV-16 L1VLPs alone, or in combination with smooth LPS (S-LPS), rough LPS (R-LPS), aluminum hydroxide or a mixture of them as adjuvant. The humoral immunity was evaluated by measuring the specific and total IgG levels, and also the T-cell immune response of mice was evaluated by measuring different cytokines such as IFN-γ, TNF-α, IL-4, IL-10 and IL-17. Results showed that serum anti-HPV16 L1VLP IgG antibody titers was significantly higher in mice immunized with a combination of VLPs and R-LPS or S-LPS compared with other immunized groups. Co-administration of HPV-16 L1VLPs with R-LPS elicited the highest levels of splenocytes cytokines (IFN-γ, IL-4, IL-17 and TNF-α) and also effectively induced improvement of a Th1-type cytokine response characterized with a high ratio of IFN-γ/IL-10. The data indicate that B. abortus LPS particularly RB51-LPS enhances the immune responses to HPV-16 L1VLPs and suggests its potential as an adjuvant for the development of a potent prophylactic HPV vaccine and other candidate vaccines.

Improved immunogenicity of tetanus toxoid by Brucella abortus S19 LPS adjuvant.

Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund's adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.

RAPID IMMUNOFILTRATION ASSAY AS A FIELD DIAGNOSTIC TOOL FOR OVINE BRUCELLOSIS

This work describes the development of two rapid immunofiltration assays, enzymatic (ERIFA) and non-enzymatic (NERIFA), for the rapid detection of ovine anti-Brucella antibodies. Brucella abortus lipopolysaccharide and total bacterial extract were dotted separately as diagnostic antigens on a nitrocellulose filter-membrane of the individual assay unit along with a third dot of purified sheep IgG as an internal control. The assay's diagnostic performance was evaluated in comparison with a modified rose bengal test (mRBT) and an indirect enzyme-linked immunosorbent assay (ELISA) through usage of 590 serum samples from healthy, vaccinated, or infected sheep. The ERIFA and indirect ELISA were found to be significantly more sensitive than NERIFA, while mRBT was determined to be statistically equivalent to NERIFA. A perfect agreement (κ = 0.984) and a statistical equivalence to indirect ELISA suggest that the bi-antigenic ERIFA can be used as an “individual rapid ELISA” for screening ovine anti-Brucella antibody both in the field and in limited laboratory conditions.

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle-protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar's analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.

Effectiveness of Brucella abortus lipopolysaccharide as an adjuvant for tuberculin PPD

Bacterial lipopolysaccharide (LPS) has T-helper 1 (Th1) immunostimulatory activities but because of toxicity and pyrogenicity cannot be used as an adjuvant. Brucella abortus LPS has less toxicity and no pyrogenic properties in comparison to other bacterial LPS. In the current study, the immunostimulatory properties of B. abortus LPS were evaluated for its adjuvant activity. Tuberculin purified protein derivative (PPD) from Mycobacterium tuberculosis was extracted and after anion-exchange chromatography on Q-sepharose column, two fractions (17 and 23), which dominantly contained 30- and 70-kDa antigens, were collected for immunological studies. BALB/c mice were immunized with four different antigen preparations (BCG, PPD, 17th and 23rd PPD fractions) along with complete Freund’s adjuvant or B. abortus LPS. The T-cell immune response of mice was assessed by measurement of Th1-type cytokine (IFN-γ) and Th2-type cytokines (IL-5 and IL-10) levels. Also, the humoral immunity was evaluated by measuring the specific IgG levels. Our results showed that immunization of mice with 17th PPD fraction along with B. abortus LPS can induce a Th1-type cytokine response characterized with a high IFN-γ/IL-5 ratio, while immunization with PPD or 23rd PPD fraction along with the same adjuvant resulted to a mixed Th1/Th2-type cytokine response.

Evaluation of a fluorescence polarization assay for the detection of serum antibodies to Brucella abortus in water buffalo ( Bubalus bubalis)

The fluorescence polarization assay (FPA) was evaluated for the serological diagnosis of brucellosis in water buffalo ( Bubalus bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. Sera from 890 buffalos from the Campania Region – 526 positive sera and 364 negative sera according to the complement fixation test (CFT) – were evaluated in this study. All samples were tested with the Rose Bengal test (RBT), CFT, and FPA in parallel and in blind fashion. Sensitivities (Sn) were 84.5% and 92.6%, and specificities (Sp) were 93.1% and 91.2% for RBT and FPA, respectively, relative to CFT. Finally, receiver operating characteristic (ROC) analysis suggested a cut-off value of 117 millipolarization (mP) units. On the whole, these results suggested that FPA might replace RBT in the diagnosis of buffalo brucellosis for its better performance relative to CFT, its adjustable cut-off useful in different epidemiological situations, its reliability, ease of performance, and for its potential application in field and high-throughput laboratories.

Comparison of fluorescence polarization assay with Rose Bengal (RB) test and complement fixation tests for the diagnosis of buffalo (Bubalus bubalis) brucellosis in a high-prevalence area

The fluorescence polarisation assay (FPA) was evaluated for the serological diagnosis of bovine brucellosis in buffalo (Bubalus Bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella Abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in short time. To measure the fluorescence polarization, a Tecan GENios Pro (Prionics) fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. 1996. A cut-off value of 117 millipolarization (mP) units was used for testing 912 buffalo sera from Campania Region (526 positive sera and 386 negative sera according to the complement fixation test; CFT). All samples were tested with the Rose Bengal plate (RB). Sensitivity and specificity (Sn) for RB were 84.0% and 87.8% and for FPA were 92.6% and 88.9 percent. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.755, while RB (relative to the CFT) gave coefficients of 0,715. Finally, ROC analysis suggested a cut-off value which did not agree with the one recommended in the test procedure for cow.

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation.

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide (20–30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test ( n = 490) and some with the rivanol-agglutination (RIV) test ( n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.

Binding of the Brucella abortus Lipopolysaccharide O-chain Fragment to a Monoclonal Antibody: QUANTITATIVE ANALYSIS BY FLUORESCENCE QUENCHING AND POLARIZATION

An antigenic O-chain polysaccharide fragment derived from Brucella abortus lipopolysaccharide was labeled with 14.8 +/- 1.8 (n = 5) and 52.3 +/- 2.4 (n = 3) micromol of fluorescein/g of polysaccharide (designated FL1 and FL2, respectively) for use in investigating the binding of O-chain to a specific murine antibody YsT9 under equilibrium conditions. Upon binding to YsT9, the fluorescence of FL1 and FL2 was quenched 45-57% with no shift in the excitation and emission spectra, and polarization of fluorescence increased by 300-335%. With fluorescence quenching and polarization as sensitive signals for antibody-bound labeled O-chains, the equilibrium constants for binding of FL1, FL2, and unlabeled O-chain to YsT9 were determined to be within a similar order (1.5 x 10(7) to 2.0 x 10(7) M-1) using a nonlinear curve fitting approach rather than Scatchard analysis. These results indicated that covalent attachment of fluorescein groups to the O-chain did not influence the recognition of the YsT9-defined epitope by the antibody. The reversibility of the O-chain-antibody reaction was also demonstrated by showing a rapid depolarization of the labeled O-chain-antibody complex in the presence of unlabeled O-chain, suggesting that this displacement experiment could be exploited to quantify the Brucella polysaccharide antigen. The study described here provides a useful model for characterization of the complex formation between a carbohydrate-binding protein and a carbohydrate ligand and also for the design of a homogeneous assay system to quantitate antigens or antibodies of clinical interest.

Age-dependent immune response in Merino sheep

The numerical and functional attributes of populations of lymphocytes were compared in the blood, lymph and skin of young and mature sheep. Young sheep, four to eight months old, had a lower proportion of CD4 + cells in blood, lymph and skin than mature sheep three to six years old. In contrast, B cells and T19 + cells were as prevalent or more prevalent in young sheep as in mature sheep. Blood lymphocytes from young sheep, cultured in vitro produced less interferon-γ, both spontaneously and in the presence of concanavalin A than did lymphocytes from older sheep. The serum antibody responses of adult sheep to the T cell-independent antigen Brucella abortus lipopolysaccharide (LPs) were greater over a range of antigen doses, suggesting that an apparent excess of antigen could not overcome the relative immune deficiency of young sheep. The adjuvant Quil A corrected the depressed antibody response of young sheep to B abortus LPs, but dextran sulphate did not. The skin contact hypersensitivity of mature sheep to dinitrochlorobenzene was greater. However, the T cell phenotypes present in infiltrates of lymphocytes elicited by the intradermal injection of tetanus and diphtheria, but not tuberculin antigens, were comparable for the two age groups. The capacity of Quil A to raise the antibody responses of both young and mature sheep to a similar titre suggests that it may be possible to overcome the immunological hyporesponsiveness that may contribute to the disease susceptibility of young sheep.

Immune responsiveness of lambs selected for resistance to haemonchosis

Several parameters of immune competence were measured in lambs with genetic resistance to Haemonchus contortus and in lambs with average susceptibility to H contortus (random-bred). Increase in skinfold thickness following intradermal injection was greater in resistant lambs for concanavalin A and to a lesser extent for phytohaemagglutinin. Both mitogens induced infiltration of superficial and deep dermis by eosinophils, CD4+, CD8+ and T19+ lymphocytes, though the responses did not differ between groups. Primary but not secondary antibody responses to the T cell-dependent antigen, ovalbumin was greater in resistant lambs. In contrast, antibody responses to the T cell-independent antigen, Brucella abortus lipopolysaccharide, did not differ between groups. Taken together the data suggest that enhanced T lymphocyte function may contribute to the greater resistance to H contortus of genetically resistant lambs.

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti- B. ovis sera and eliminated cross-reactivity with anti- B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

Bovine brucellosis: Evaluation of field sera by a competitive and superimposable ELISA utilising a monoclonal antibody against Brucella abortus lipopolysaccharide

With the aid of a horseradish peroxidase (HRP) tagged monoclonal antibody against smooth lipopolysaccharide from Brucella abortus (Bruce 1), a competitive and superimposable ELISA test procedure for bovine brucellosis has been evaluated for its ability to discriminate between Strain 19-vaccinated (S19-Vacc) and Biotype 1-infected (B1-Inf) cattle. In the competitive assay, all sera from S19-Vacc animals competed effectively against HRP-Bruce 1 (low HRP activity), while 10 out of 40 B1-Inf animals competed less effectively with Bruce 1 (high HRP activity). Successful competition by cattle antibodies would result in an increased proportion of cattle Igs binding to the assay antigen. This was confirmed by superimposing an alkaline phosphatase conjugated rabbit anti-cattle Ig after the competitive ELISA had been completed. With the superimposable assay, alkaline phosphatase activity was correspondingly high for S19-Vacc animals, and low for 36 out of 40 B1-Inf animals. The superimposable ELISA had therefore improved the discriminatory capabilities of the assay procedure from 75% to 90%.

Blastogenic response of bovine lymphocytes to Brucella abortus lipopolysaccharide.

Brucella abortus lipopolysaccharide was tested in a blastogenesis assay with unfractionated and nylon wool-separated peripheral blood lymphocytes of Brucella-naive cattle and cattle immunized with B. abortus. Our results indicated that in cattle the lipopolysaccharide of B. abortus is not a B-cell mitogen. In immunized animals it stimulated predominantly nylon wool-adherent cells. The lipopolysaccharide of Escherichia coli O128:B12, in contrast, induced a substantially greater proliferative response in circulating lymphocytes, predominantly those adherent to nylon wool, of the Brucella-naive cattle.

Comparative assessment of antibody isotypes to [formula omitted] by primary and secondary binding assays

Bovine isotypes IgM, IgG 1, IgG 2, and IgA, affinity purified via Brucella abortus lipopolysaccharide (LPS) attached covalently to agarose beads, were assessed for their antibody activity in various serological tests. In an enzyme linked immunosorbent test, using LPS as the antigen attached to a plastic matrix and goat anti-heavy chain specific IgG fractions conjugated to horseradish peroxidase, the limiting amount of antibody of each isotype detectable was 155 ng for IgM; 190 ng for IgG 1; 220 ng for IgG 2 and approximately 700 ng for IgA. In a hemolysis in gel test using LPS attached to bovine erythrocytes as the antigen, and guinea pig complement, the least amounts of detectable antibody were IgM:5500 ng; IgG 1:60 ng; IgG 2:4850 ng; IgA was not reactive. However, as preformed immune complexes all four isotypes were capable of activating bovine complement. For the indirect hemagglutination assay, both fresh sheep and chicken erythrocytes were sensitized with antigen. The least amounts of antibody detectable with LPS-coated sheep erythrocytes were IgG 2: 625 ng; IgG 1: 3750 ng; IgA: 4250 ng; and IgM: not reactive. Using LPS-coated chicken erythrocytes, only IgG 2 was reactive to a limit of 1250 ng. Using isotypes as the antibody in an antibody-dependent cell-mediated cytotoxicity assay, normal bovine mononuclear cells as effector cells and chicken erythrocytes sensitized with LPS as target cells, only IgG 2 was reactive. Specific activity was retained to approximately 1000 ng. The implication of these results in terms of diagnosis and protective immunity in brucellosis will be discussed.

Affinity purification of bovine antibodies to Brucella abortus Lipopolysaccharide.

Alkali-treated lipopolysaccharide from Brucella abortus S1119.3 coupled to agarose beads by cyanogen bromide activation resulted in an immunoadsorbent with which a large amount of B. abortus-specific antibodies could be purified. The method described gave alkali-treated lipopolysaccharide binding efficiencies of up to 98%. There was little loss of alkali-treated lipopolysaccharide from the column after several pH shifts, allowing the immunoadsorbent to be regenerated and used repeatedly.