Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle (original) (raw)
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Veterinary research, 2015
Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and pre...
Veterinary World, 2021
Background and Aim: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. Materials and Methods: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. Results: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). Conclusion: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.
Veterinary Microbiology, 2000
An O-polysaccharide (O-chain) and a hot-water extracted polysaccharide (PS), both obtained from Brucella abortus 1119-3, and a B. melitensis 16 M native hapten (NH) were evaluated by indirect enzyme linked immunosorbent assay (ELISA) on three groups of cattle sera. The sera tested were: (a) 75 sera from cows naturally infected with B. abortus; (b) 130 sera from noninfected and non-vaccinated cattle; and (c) 61 sera from non-infected heifers recently vaccinated with B. abortus Strain 19 (S19). Sensitivity (Se), specificity (Sp) and the capability to discriminate vaccinated cattle (ADV) were determined. Using PS antigen, Se was 100% and the Sp was 97.7%, while the highest Sp was obtained by using the O-chain (99.2%). For the NH antigen, Se was 94.7% and the Sp was 90.0%. The ADV of the three antigens was approximately 85%. Statistical analysis showed significant differences between O-chain/PS and O-chain/NH antigens. The agreement among antigens determined by kappa coefficient was 0.899 for O-chain/PS, 0.845 for O-chain/NH and 0.795 for PS/NH.
Infection and immunity, 1992
Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. ab...
PubMed, 1989
Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.
Veterinary Microbiology, 1991
Belzer, C.A., Tabatabai, L.B. and Deyoe, B.L., 1991. Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus. Vet. Microbiol., 27: 79-90. Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31 000 to 45 000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66 000 to 71 000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhoodvaccinated cattle did not react. INTRODUCTION Standard serologic tests for diagnosis of bovine brucellosis have been in use since 1940 (Manthei et al., 1956), but the most difficult task has been in distinguishing antibodies of infected from those of vaccinated animals. To date, most diagnostic tests for Brucella abortus rely on detecting a humoral immune response, although the most definitive diagnostic test is bacterial ~To whom correspondence should be addressed.
Brazilian Journal of Veterinary Research and Animal Science, 1999
Three soluble antigens were compared by radial immunodiffusion (RID) and agar gel immunodiffusion (AGID) tests: a native haptene (NH) from Brucella melitensis 16M, and a polysaccharide (PS) from B. abortus 1119-3, both obtained by non-hydrolytic methods, and the (O-Chain) polysaccharide extracted also from B. abortus 1119-3 but using an hydrolytic method. Three groups ofbovine scra were tested: a) Naturally infected (n = 76); b) Non-infected (n = 130) and c) S-19 vaccinated (n = 61); the sensitivity (Se), the specificity (Sp) and the ability to differentiate vaccinated (ADV) were determined in each group a, b and c respectively. The highest Se in lhe RID test (84.3%) was achieved by NH; while the three antigens gave 100% Sp. The O-Chain showed 100% ADV in this test. In the AGID test PS antigen showed the best Se (86.6%), and ali antigens showed 100% of Sp and ADV Finally, for its production qualities and efficiency the antigens PS and NH represen~a promising alternative for complementary diagnosis of brucellosis.
Veterinary World, 2020
Background and Aim: Brucellosis remains one of the most common zoonoses. The current anti-brucellosis measures are largely deemed ineffective due to a lack of specificity of conventional serological tests. This study evaluated the use of Brucella outer membrane protein (Omp)19 for serodiagnostic testing. Materials and Methods: The antigenicity of recombinant Brucella Omp19, Omp25, and Omp31 was examined in serum samples from mice and rabbits immunized with Omp19 or Brucella abortus 19 whole cell (WC) and 12 and 152 cows experimentally or naturally infected with brucellosis, respectively. Serum samples were collected from 151 cows that were vaccinated with B. abortus 19 and 12 unvaccinated heifers that were maintained on a brucellosis-free farm. Results: Immunization with Omp19 resulted in antibody production in mice after a single injection without the use of adjuvant. Serum antibodies obtained from rabbits immunized with inactivated B. abortus strain 19 WC targeted Omps by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibodies targeting Omp19 were identified in all B. abortus strain 544 experimentally infected cows at day 14 post-inoculation (p.i.); Omp25 was detected by ELISA at day 28 p.i., while an ELISA targeting Omp31 was negative for 25% of cows at this time point. Omp19 and Omp25 were readily detected by sera from cows from a new epizootic focus. Antibodies recognizing Omps were also detected in >50% of the animals maintained in a brucellosis-free herd at 10 months after vaccination. Conclusion: Brucella Omp19 in combination with Omp25 and Omp31 may be utilized as target antigens in an ELISA designed for serological testing of unvaccinated cattle.
Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus
Journal of clinical microbiology, 1984
Six soluble antigens prepared from Brucella abortus were compared with a salt-extractable protein (CSP) antigen in an enzyme-linked immunosorbent assay for the detection of antibody to B. abortus in cattle sera. Of seven preparations tested, antigens from B. abortus soluble antigen (prepared from an autoclaved cell suspension) and CSP were stable on frozen storage. Enzyme-linked immunosorbent assay with CSP antigen under optimal conditions was from 100- to 700-fold more sensitive than the standard agglutination, card, Rivanol precipitation-plate agglutination, and the complement fixation tests in detecting immunoglobulin G antibody. From a practical point of view, however, using the most stringent criteria for determining an "upper negative" value, the enzyme-linked immunosorbent assay with CSP was at least 12-fold more sensitive than the standard agglutination test and any of the other serological tests. Furthermore, the enzyme-linked immunosorbent assay with CSP was spec...
Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle
Veterinary World
Background and Aim: An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA). Materials and Methods: The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with ...