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Sample records for abortus inmunidad vacunas

  1. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus...

  2. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus...

  3. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus...

  4. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus...

  5. 9 CFR 113.65 - Brucella Abortus Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Brucella Abortus Vaccine. 113.65... Bacterial Vaccines § 113.65 Brucella Abortus Vaccine. Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus...

  6. Brucella abortus Infection Acquired in Microbiology Laboratories

    PubMed Central

    Fiori, Pier Luigi; Mastrandrea, Scilla; Rappelli, Paola; Cappuccinelli, Piero

    2000-01-01

    We report an outbreak of laboratory-acquired Brucella abortus infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated. PMID:10790142

  7. Bactericidal Effect of Silver Nanoparticles on Intramacrophage Brucella abortus 544

    PubMed Central

    Alizadeh, Hamed; Salouti, Mojtaba; Shapouri, Reza

    2014-01-01

    Background: Brucellosis is an infectious disease that is caused by Brucella spp. As Brucella spp. are intramacrophage pathogens, the treatment of this infection is very difficult. On the other hand, due to the side effects of the brucellosis treatment regime, it is necessary to find new antimicrobial agents against it. Objectives: The aim of this study was to investigate the antimicrobial effect of silver nanoparticles against Brucella abortus 544 in the intramacrophage condition. Materials and Methods: The antimicrobial effect of silver nanoparticles was determined by an agar well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against B. abortus 544 were determined by a broth macrodilution method. The effect of time on the antimicrobial activity of silver nanoparticles was analyzed. The effect of silver nanoparticles on the intramacrophage survival of B. abortus 544 was studied on mice peritoneal macrophages. Results: The well diffusion agar study showed that silver nanoparticles have an antimicrobial effect on B. abortus 544. The MIC and MBC of silver nanoparticles against B. abortus 544 were; 6 ppm and 8 ppm, respectively. The silver nanoparticles showed antibacterial effects within 40 minutes. The results of the macrophage culture indicated that silver nanoparticles have antibacterial activity against intramacrophage B. abortus 544, and the highest efficiency was observed at a concentration of 8-10 ppm of silver nanoparticles. Conclusions: The results showed that silver nanoparticles have an antimicrobial effect against intramacrophage B. abortus 544. PMID:25147682

  8. Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice.

    PubMed

    Lim, Jeong Ju; Kim, Dong Hyeok; Lee, Jin Ju; Kim, Dae Geun; Min, Wongi; Lee, Hu Jang; Rhee, Man Hee; Kim, Suk

    2012-09-01

    The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.

  9. Aneuploidy in abortuses following IVF and ICSI

    PubMed Central

    Frattarelli, John L.

    2009-01-01

    Purpose To compare aneuploidy rates in first trimester pregnancy losses following IVF ± ICSI. Methods A retrospective cohort analysis of karyotypes of abortuses following conventional IVF (n = 159) and ICSI (n = 196). Results 50.1% of losses were found to be cytogenetically abnormal among all patients undergoing IVF ± ICSI. A significant increase in fetal aneuploidy rate was noted with increasing maternal age (<30 years = 26.1% vs. 31 to 34 years. = 38.2% vs. 35 to 39 years. = 51.3% vs. >39 years. = 65.9%). Aneuploidy rates were similar in the ICSI vs. conventional IVF groups (52.6% vs. 47.2% [p 0.31, RR 1.11, 95% CI 0.90, 1.38]). More sex chromosome anomalies were noted in the ICSI group. Conclusions The aneuploidy rate in first trimester abortuses significantly increases with increasing maternal age. ICSI was not shown to significantly increase the aneuploidy rate. However, more sex chromosome anomalies were found among pregnancies resulting from ICSI. PMID:19224361

  10. Lung lesions in bovine fetuses aborted by Brucella abortus.

    PubMed Central

    López, A; Hitos, F; Pérez, A; Navarro-Fierro, R R

    1984-01-01

    Considering the poor facilities available for microbiological diagnosis in some countries where Brucella abortus is a frequent cause of bovine abortion, a study was conducted to determine if isolation of B. abortus from an aborted bovine fetus could be predicted from a detailed histological study of the formalized lung. Thirty-nine samples of B. abortus positive and 20 negative fetal samples were examined for the presence of 14 different pulmonary lesions. Differences in the frequency of observed lesions between the positive and negative groups, were determined by odds ratios and chi square statistic. The confidence of the prediction was calculated by means of the logistic computer model. The frequency of eight lung lesions was found to be significantly (p less than 0.05) different between the groups; nevertheless, these lesions were not specific enough to be able to incriminate B. abortus as the cause of abortion. PMID:6434166

  11. Immune response triggered by Brucella abortus following infection or vaccination.

    PubMed

    Dorneles, Elaine M S; Teixeira-Carvalho, Andréa; Araújo, Márcio S S; Sriranganathan, Nammalwar; Lage, Andrey P

    2015-07-17

    Brucella abortus live vaccines have been used successfully to control bovine brucellosis worldwide for decades. However, due to some limitations of these live vaccines, efforts are being made for the development of new safer and more effective vaccines that could also be used in other susceptible species. In this context, understanding the protective immune responses triggered by B. abortus is critical for the development of new vaccines. Such understandings will enhance our knowledge of the host/pathogen interactions and enable to develop methods to evaluate potential vaccines and innovative treatments for animals or humans. At present, almost all the knowledge regarding B. abortus specific immunological responses comes from studies in mice. Active participation of macrophages, dendritic cells, IFN-γ producing CD4(+) T-cells and cytotoxic CD8(+) T-cells are vital to overcome the infection. In this review, we discuss the characteristics of the immune responses triggered by vaccination versus infection by B. abortus, in different hosts.

  12. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  13. Brucella abortus RB51 in milk of vaccinated adult cattle.

    PubMed

    Miranda, Karina Leite; Poester, Fernando Padilla; Dorneles, Elaine Maria Seles; Resende, Thiago Magalhães; Vaz, Adil Knackfuss; Ferraz, Sandra Maria; Lage, Andrey Pereira

    2016-08-01

    The aim of this study was to evaluate the shedding of Brucella abortus in the milk of cows vaccinated with a full dose of RB51 during lactation. Eighteen cows, nine previously vaccinated with S19 as calves and nine non-vaccinated, were immunized subcutaneously with 1.3×10(10)CFU of B. abortus RB51, 30-60days after parturition. Milk samples from all animals were collected daily until day 7, and at weekly interval for the next 9 weeks after vaccination. To evaluate the shedding of B. abortus, milk samples were submitted for culture and PCR. No B. abortus was isolated from any sample tested. Only one sample, collected on first day after vaccination from a cow previously vaccinated, was faintly positive in the PCR. In conclusion, the public health hazard associated with milk consumption from cows vaccinated with RB51 in post-partum is very low, despite vaccination with the full dose and regardless of previous S19 vaccination.

  14. High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

    PubMed

    Vorimore, Fabien; Cavanna, Noémie; Vicari, Nadia; Magnino, Simone; Willems, Hermann; Rodolakis, Annie; Siarkou, Victoria I; Laroucau, Karine

    2012-09-01

    We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

  15. Identification and Characterization of Chlamydia abortus Isolates from Yaks in Qinghai, China

    PubMed Central

    Li, Zhaocai; Cao, Xiaoan; Fu, Baoquan; Chao, Yilin; Cai, Jinshan; Zhou, Jizhang

    2015-01-01

    Recently, the yak population has exhibited reproductive disorders, which are considered to be associated with Chlamydia abortus (C. abortus) in Qinghai, China. In this study, a total of 9 aborted fetuses (each from a different herd) and 126 vaginal swab samples from the 9 herds were collected and analyzed. C. abortus DNA was detected from all of the 9 aborted fetuses and 30 of the 126 vaginal swab samples (23.81%) from yak cows in the selected herds. Four C. abortus strains were isolated from embryonated egg yolk sacs inoculated with foetal organ suspensions. The isolated C. abortus strains were further identified, which showed identical restriction profiles with the C. abortus reference strain using AluI restriction enzyme in the RFLP test. Moreover, the isolated C. abortus strains and C. abortus-positive vaginal swab samples were genotyped by multiple loci variable number tandem repeat analysis and all belonged to the genotype 2 group. These findings suggested that C. abortus played a substantial role in yak abortion in Qinghai, China. PMID:26060818

  16. Brucella abortus Cell Cycle and Infection Are Coordinated.

    PubMed

    De Bolle, Xavier; Crosson, Sean; Matroule, Jean-Yves; Letesson, Jean-Jacques

    2015-12-01

    Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection.

  17. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    PubMed

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  18. Deletion of the BCSP31 gene of Brucella abortus by replacement.

    PubMed Central

    Halling, S M; Detilleux, P G; Tatum, F M; Judge, B A; Mayfield, J E

    1991-01-01

    The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain. Images PMID:1937745

  19. Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system.

    PubMed

    Campos, Priscila Carneiro; Gomes, Marco Túlio Ribeiro; Guimarães, Gabriela; Costa Franco, Miriam Maria Silva; Marim, Fernanda Martins; Oliveira, Sergio Costa

    2014-12-01

    Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field.

  20. Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge

    PubMed Central

    Nol, Pauline; Olsen, Steven C.; Rhyan, Jack C.; Sriranganathan, Nammalwar; McCollum, Matthew P.; Hennager, Steven G.; Pavuk, Alana A.; Sprino, Phillip J.; Boyle, Stephen M.; Berrier, Randall J.; Salman, Mo D.

    2016-01-01

    In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk. PMID:26904509

  1. Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge.

    PubMed

    Nol, Pauline; Olsen, Steven C; Rhyan, Jack C; Sriranganathan, Nammalwar; McCollum, Matthew P; Hennager, Steven G; Pavuk, Alana A; Sprino, Phillip J; Boyle, Stephen M; Berrier, Randall J; Salman, Mo D

    2016-01-01

    In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

  2. A dual-targeting approach to inhibit Brucella abortus replication in human cells

    PubMed Central

    Czyż, Daniel M.; Jain-Gupta, Neeta; Shuman, Howard A.; Crosson, Sean

    2016-01-01

    Brucella abortus is an intracellular bacterial pathogen and an etiological agent of the zoonotic disease known as brucellosis. Brucellosis can be challenging to treat with conventional antibiotic therapies and, in some cases, may develop into a debilitating and life-threatening chronic illness. We used multiple independent assays of in vitro metabolism and intracellular replication to screen a library of 480 known bioactive compounds for novel B. abortus anti-infectives. Eighteen non-cytotoxic compounds specifically inhibited B. abortus replication in the intracellular niche, which suggests these molecules function by targeting host cell processes. Twenty-six compounds inhibited B. abortus metabolism in axenic culture, thirteen of which are non-cytotoxic to human host cells and attenuate B. abortus replication in the intracellular niche. The most potent non-cytotoxic inhibitors of intracellular replication reduce B. abortus metabolism in axenic culture and perturb features of mammalian cellular biology including mitochondrial function and receptor tyrosine kinase signaling. The efficacy of these molecules as inhibitors of B. abortus replication in the intracellular niche suggests “dual-target” compounds that coordinately perturb host and pathogen are promising candidates for development of improved therapeutics for intracellular infections. PMID:27767061

  3. Brucella abortus Synthesizes Phosphatidylcholine from Choline Provided by the Host

    PubMed Central

    Comerci, Diego J.; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.

    2006-01-01

    The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-β-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process. PMID:16484204

  4. Chlamydia abortus in Cows Oviducts, Occasional Event or Causal Connection?

    PubMed

    Appino, S; Vincenti, L; Rota, A; Pellegrini, S; Chieppa, M N; Cadoni, V; Pregel, P

    2015-06-01

    Fifty-seven genital tracts of regularly slaughtered culled Piedmontese cows, aged 7.4 ± 4.3 years (mean ± SD), range: 2.6-15.6 years, were grossly and microscopically examined. DNA extracted from oviducts was subjected to PCR to evaluate the presence of Chlamydia spp. The 15 PCR-positive oviducts were subjected to Sanger sequencing and showed the presence of Chamydia abortus, with an identity range between 99 and 100%. Nine of the PCR-positive samples belonged to the 24 animals with a normal macroscopic appearance of the whole genital tract (percentage of positive oviducts in normal genital tracts 9/24 = 37.5%), while six belonged to the 33 genital tracts with lesions in one or more organs (percentage of positive oviducts in pathological genital tracts 6/33 = 18.1%); of these, a single animal had salpingitis. The detection of C. abortus in bovine oviducts is of particular interest because it has never been previously investigated or reported.

  5. Immunogenicity and protective effect of recombinant Brucella abortus Ndk (rNdk) against a virulent strain B. abortus 544 infection in BALB/c mice.

    PubMed

    Hop, Huynh Tan; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2015-02-01

    In this study, we particularly evaluated the protective effect of recombinant protein encoded by Brucella abortus 544 ndk (nucleoside diphosphate kinase) gene against B. abortus infection in the BALB/c mice. Cloning and expression of B. abortus Ndk was accomplished by PCR amplification into a pMAL expression system, and purification of a recombinant Ndk (rNdk). As for the determination of IgG responses, rNdk induced vigorous IgG production, especially higher in IgG2a compared to IgG1 with titers of 5.2 and 4.8, respectively, whereas titers of these in mice immunized with MBP were 2.4 of IgG2a and 2.6 of IgG1. The analysis of cytokine has revealed that rNdk can strongly induce production of IFN-γ as well as proinflammatory cytokines (TNF, MCP1 and IL-6) but not much IL-10, suggesting rNdk elicited predominantly cell-mediated immune responses. Furthermore, the spleen proliferation and bacterial burden in the spleen of rNdk immunized mice were significantly lower than those of MBP-immunized mice against virulent B. abortus challenge (P < 0.01). Conclusionly, rNdk immunization enables to elicit both of the humoral and cellular response, ultimately enhancing protection level in experimental mice, suggesting that rNdk of B. abortus might be a useful candidate for subunit vaccine for brucellosis in animals.

  6. [Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina].

    PubMed

    Hollender, Daiana; Conde, Sandra B; Salustio, Eduardo; Samartino, Luis E

    2013-01-01

    Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

  7. Biotyping and Genotyping (MLVA16) of Brucella abortus Isolated from Cattle in Brazil, 1977 to 2008

    PubMed Central

    Minharro, Sílvia; Silva Mol, Juliana P.; Dorneles, Elaine M. S.; Pauletti, Rebeca B.; Neubauer, Heinrich; Melzer, Falk; Poester, Fernando P.; Dasso, Maurício G.; Pinheiro, Elaine S.; Soares Filho, Paulo M.; Santos, Renato L.; Heinemann, Marcos B.; Lage, Andrey P.

    2013-01-01

    Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region. PMID:24324670

  8. Molecular epidemiology of Brucella abortus isolated from cattle in Brazil, 2009-2013.

    PubMed

    Oliveira, Mayra Silva; Dorneles, Elaine Maria Seles; Soares, Paulo Martins Filho; Fonseca, Antônio Augusto; Orzil, Lívia; de Souza, Patrícia Gomes; Lage, Andrey Pereira

    2017-02-01

    The aims of the present study were to genotype Brucella abortus strains isolated from cattle in Brazil between 2009 and 2013, and to analyze their distribution to support the Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose (PNCEBT) (National Brucellosis and Tuberculosis Control and Eradication Program). One hundred forty B. abortus strains isolated from cattle in Brazil between 2009 and 2013 were genotyped using a set of 18 variable number of tandem repeats (VNTR) (MLVA16+HOOF-Print 3 and 4). The multiple locus VNTR analysis (MLVA) composed by eight markers (MLVA8) revealed eight different genotypes among B. abortus strains, including five previously described and three new ones. Analysis of the MLVA16 loci revealed fifty-eight distinct genotypes, from which three were identical, thirty-eight were considered very close, and seventeen were considered distant compared to those previously described and deposited in MLVAbank. Analysis of the HOOF-Prints 3 and 4 revealed the larger number of different alleles among all VNTR assessed, exhibiting maximum resolution when associated with MLVA16 markers. This study also provides insights on the genotypes of B. abortus circulating in Brazil, which certainly contribute for the better understanding of the epidemiology and control of bovine brucellosis in the country. Moreover, our data showed a high genetic diversity among the B. abortus strains isolated between 2009 and 2013, and a close relationship among these strains and Brazilian B. abortus deposited by MLVAbank.

  9. Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

    PubMed

    Lee, Jin Ju; Lim, Jeong Ju; Kim, Dae Geun; Simborio, Hannah Leah; Kim, Dong Hyeok; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2014-09-01

    In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

  10. NLRP12 negatively regulates proinflammatory cytokine production and host defense against Brucella abortus.

    PubMed

    Silveira, Tatiana N; Gomes, Marco Túlio R; Oliveira, Luciana S; Campos, Priscila C; Machado, Gabriela G; Oliveira, Sergio C

    2017-01-01

    Brucella abortus is the causative agent of brucellosis, which causes abortion in domestic animals and undulant fever in humans. This bacterium infects and proliferates mainly in macrophages and dendritic cells, where it is recognized by pattern recognition receptors (PRRs) including Nod-like receptors (NLRs). Our group recently demonstrated the role of AIM2 and NLRP3 in Brucella recognition. Here, we investigated the participation of NLRP12 in innate immune response to B. abortus. We show that NLRP12 inhibits the early production of IL-12 by bone marrow-derived macrophages upon B. abortus infection. We also observed that NLRP12 suppresses in vitro NF-κB and MAPK signaling in response to Brucella. Moreover, we show that NLRP12 modulates caspase-1 activation and IL-1β secretion in B. abortus infected-macrophages. Furthermore, we show that mice lacking NLRP12 are more resistant in the early stages of B. abortus infection: NLRP12(-/-) infected-mice have reduced bacterial burdens in the spleens and increased production of IFN-γ and IL-1β compared with wild-type controls. In addition, NLRP12 deficiency leads to reduction in granuloma number and size in mouse livers. Altogether, our findings suggest that NLRP12 plays an important role in negatively regulating the early inflammatory responses against B. abortus.

  11. Seroprevalence and risk factors of Chlamydia abortus infection in Tibetan sheep in Gansu province, northwest China.

    PubMed

    Qin, Si-Yuan; Yin, Ming-Yang; Cong, Wei; Zhou, Dong-Hui; Zhang, Xiao-Xuan; Zhao, Quan; Zhu, Xing-Quan; Zhou, Ji-Zhang; Qian, Ai-Dong

    2014-01-01

    Chlamydia abortus, an important pathogen in a variety of animals, is associated with abortion in sheep. In the present study, 1732 blood samples, collected from Tibetan sheep between June 2013 and April 2014, were examined by the indirect hemagglutination (IHA) test, aiming to evaluate the seroprevalence and risk factors of C. abortus infection in Tibetan sheep. 323 of 1732 (18.65%) samples were seropositive for C. abortus antibodies at the cut-off of 1:16. A multivariate logistic regression analysis was used to evaluate the risk factors associated with seroprevalence, which could provide foundation to prevent and control C. abortus infection in Tibetan sheep. Gender of Tibetan sheep was left out of the final model because it is not significant in the logistic regression analysis (P > 0.05). Region, season, and age were considered as major risk factors associated with C. abortus infection in Tibetan sheep. Our study revealed a widespread and high prevalence of C. abortus infection in Tibetan sheep in Gansu province, northwest China, with higher exposure risk in different seasons and ages and distinct geographical distribution.

  12. Seroprevalence and Risk Factors of Chlamydia abortus Infection in Tibetan Sheep in Gansu Province, Northwest China

    PubMed Central

    Qin, Si-Yuan; Yin, Ming-Yang; Cong, Wei; Zhou, Dong-Hui; Zhang, Xiao-Xuan; Zhao, Quan; Zhu, Xing-Quan; Zhou, Ji-Zhang; Qian, Ai-Dong

    2014-01-01

    Chlamydia abortus, an important pathogen in a variety of animals, is associated with abortion in sheep. In the present study, 1732 blood samples, collected from Tibetan sheep between June 2013 and April 2014, were examined by the indirect hemagglutination (IHA) test, aiming to evaluate the seroprevalence and risk factors of C. abortus infection in Tibetan sheep. 323 of 1732 (18.65%) samples were seropositive for C. abortus antibodies at the cut-off of 1 : 16. A multivariate logistic regression analysis was used to evaluate the risk factors associated with seroprevalence, which could provide foundation to prevent and control C. abortus infection in Tibetan sheep. Gender of Tibetan sheep was left out of the final model because it is not significant in the logistic regression analysis (P > 0.05). Region, season, and age were considered as major risk factors associated with C. abortus infection in Tibetan sheep. Our study revealed a widespread and high prevalence of C. abortus infection in Tibetan sheep in Gansu province, northwest China, with higher exposure risk in different seasons and ages and distinct geographical distribution. PMID:25401129

  13. Risks of Brucella abortus spillover in the Greater Yellowstone area.

    PubMed

    Schumaker, B

    2013-04-01

    Recurrent spillover of Brucella abortus from wildlife reservoirs to domestic cattle in the Greater Yellowstone Area (GYA) has prevented the United States from completely eradicating bovine brucellosis. Risks to cattle are a function of the size and location of wildlife and livestock populations, the degree and nature of spatio-temporal interactions between the various hosts, the level of disease in wildlife, and the susceptibility of livestock herds. While the brucellosis prevalence in wild, free-ranging GYA bison (Bison bison) is high, current management actions have successfully limited contact between bison and cattle. Under current management practices, the risks to cattle in the GYA are predominantly from wild elk (Cervus elaphus). Intra- and inter-species transmission events, while uncommon, are nevertheless crucial for the maintenance of brucellosis in the GYA. Future management actions should focus on decreasing elk herd densities and group sizes and on understanding the behavioural and environmental drivers that result in co-mingling that makes transmission possible.

  14. Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

    PubMed

    Siarkou, Victoria I; Vorimore, Fabien; Vicari, Nadia; Magnino, Simone; Rodolakis, Annie; Pannekoek, Yvonne; Sachse, Konrad; Longbottom, David; Laroucau, Karine

    2015-01-01

    Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of

  15. Vaccination of elk (Cervus canadensis) with Brucella abortus strain RB51 overexpressing superoxide dismutase and glycosyltransferase genes does not induce adequate protection against experimental brucella abortus challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area (GYA). In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the d...

  16. Complete Genome Sequence of Brucella abortus SKN 13 Isolated from Placenta of Aborted Cattle in Gujarat, India

    PubMed Central

    Chauhan, H. C.; Chandel, B. S.; Patel, Kirit B.; Patel, A. C.; Shrimali, M. D.; Patel, S. S.; Bhagat, A. G.; Rajgor, Manish; Patel, Mitul A.; Patel, Maulik; Kala, Jitendra; Patel, Bhumika

    2016-01-01

    Brucella abortus is generally known to cause brucellosis in cattle and buffalo. Here, we report the draft genome sequence of Brucella abortus SKN 13, isolated from aborted cattle placenta in the area of Gujarat, India, providing precious resources for comparative genomic analyses of Brucella field strains. PMID:27789633

  17. Cytokine responses in camels (Camelus bactrianus) vaccinated with Brucella abortus strain 19 vaccine.

    PubMed

    Odbileg, Raadan; Purevtseren, Byambaa; Gantsetseg, Dorj; Boldbaatar, Bazartseren; Buyannemekh, Tumurjav; Galmandakh, Zagd; Erdenebaatar, Janchivdorj; Konnai, Satoru; Onuma, Misao; Ohashi, Kazuhiko

    2008-02-01

    In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1alpha, IL-1beta, TNFalpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Th1 cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.

  18. A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection

    PubMed Central

    Spera, Juan Manuel; Ugalde, Juan Esteban; Mucci, Juan; Comerci, Diego J.; Ugalde, Rodolfo Augusto

    2006-01-01

    Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella. PMID:17053080

  19. Therapeutic Chlamydophila abortus and C. pecorum Vaccination Transiently Reduces Bovine Mastitis Associated with Chlamydophila Infection▿

    PubMed Central

    Biesenkamp-Uhe, Carolin; Li, Yihang; Hehnen, Hans-Robert; Sachse, Konrad; Kaltenboeck, Bernhard

    2007-01-01

    Infections with Chlamydophila abortus and C. pecorum are highly prevalent in cattle and have been associated with bovine mastitis. A prospective cohort study was conducted with a herd of 140 Holstein dairy cows to investigate the influence of Chlamydophila infection on subclinical inflammation of the bovine mammary gland as characterized by somatic cell numbers in milk. PCR detection of C. abortus and low serum antibody levels against Chlamydophila spp. were significantly associated with subclinical mastitis. To examine the effect of the infection by response modification, immune perturbation was done by two subcutaneous administrations of an experimental vaccine preparation of inactivated C. abortus and C. pecorum elementary bodies. Vaccination against Chlamydophila highly significantly decreased milk somatic cell numbers, thus reducing bovine mastitis, and increased antibody levels against Chlamydophila but did not eliminate shedding of C. abortus in milk as detected by PCR. The protective effect peaked at 11 weeks after vaccination and lasted for a total of 14 weeks. Vaccination with the Chlamydophila vaccine, a mock vaccine, or a combination vaccine against bovine viral diseases highly significantly increased C. abortus shedding in milk for 1 week, presumably mediated by the vaccine adjuvant. In summary, this study shows an etiological involvement of the widespread Chlamydophila infections in bovine mastitis, a herd disease of critical importance for the dairy industry. Furthermore, this investigation shows the potential for temporary improvement of chlamydial disease by therapeutic vaccination. Chlamydophila vaccination of cattle might serve as a testing ground for vaccines against human chlamydial infections. PMID:17118976

  20. N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

    PubMed Central

    Mora-Cartín, Ricardo; Chacón-Díaz, Carlos; Gutiérrez-Jiménez, Cristina; Gurdián-Murillo, Stephany; Lomonte, Bruno; Chaves-Olarte, Esteban

    2016-01-01

    Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus. In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus. Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution. PMID:27001541

  1. Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis.

    PubMed

    Kim, Ji-Yeon; Sung, So-Ra; Lee, Kichan; Lee, Hyang-Keun; Kang, Sung-Il; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Her, Moon

    2014-08-15

    The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3-5.6, 4-7 and 6-11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera - B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity.

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

    PubMed

    Wei, Pan; Lu, Qiang; Cui, Guimei; Guan, Zhenhong; Yang, Li; Sun, Changjiang; Sun, Wanchun; Peng, Qisheng

    2015-02-15

    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.

  3. Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response.

    PubMed

    Kurar, E; Splitter, G A

    1997-12-01

    Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. As a test of this new methodology, the immune response to the in vivo-expressed Brucella abortus ribosomal L7/12 gene in the muscle cells of mice was examined. To accomplish this goal the eukaryotic expression systems pcDNA3 and p6 were used. Single intramuscular injection of the L7/L12 gene driven by the human cytomegalovirus (CMV) promoter (pcDNA3) or bovine MHC 1 promoter (p6) resulted in intracellular expression of the B. abortus L7/L12 immunodominant protein encoded by this gene. This application facilitated directed antigen presentation to the immune system and established specific antibody and T-cell responses compared with vector only (pcDNA3) negative controls and B. abortus S19 injected positive controls. Although pcDNA3-encoded L7/L12 gene-inoculated mice possessed significant protection, p6-L7/L12 did not engender significant protection against B. abortus S2308 infection compared to positive control mice. These data suggest a promising antigen-specific response, and L7/L12 nucleic acid vaccination may be an initial step in the development of genetically engineered candidate vaccines against brucellosis. This study for the first time focuses on DNA immunization of a gene from B. abortus.

  4. Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

    2015-06-01

    A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field.

  5. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants.

    PubMed

    Mol, Juliana P S; Pires, Simone F; Chapeaurouge, Alexander D; Perales, Jonas; Santos, Renato L; Andrade, Hélida M; Lage, Andrey P

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.

  6. In silico functional elucidation of uncharacterized proteins of Chlamydia abortus strain LLG

    PubMed Central

    Singh, Gagandeep; Sharma, Dixit; Singh, Vikram; Rani, Jyoti; Marotta, Francessco; Kumar, Manoj; Mal, Gorakh; Singh, Birbal

    2017-01-01

    Aim: This study reports structural modeling, molecular dynamics profiling of hypothetical proteins in Chlamydia abortus genome database. Methodology: The hypothetical protein sequences were extracted from C. abortus LLG Genome Database for functional elucidation using in silico methods. Results: Fifty-one proteins with their roles in defense, binding and transporting other biomolecules were unraveled. Forty-five proteins were found to be nonhomologous to proteins present in hosts infected by C. abortus. Of these, 31 proteins were related to virulence. The structural modeling of two proteins, first, WP_006344020.1 (phosphorylase) and second, WP_006344325.1 (chlamydial protease/proteasome-like activity factor) were accomplished. The conserved active sites necessary for the catalytic function were analyzed. Conclusion: The finally concluded proteins are envisioned as possible targets for developing drugs to curtail chlamydial infections, however, and should be validated by molecular biological methods. PMID:28344832

  7. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

    PubMed Central

    Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

  8. Brucella abortus is Prevalent in Both Humans and Animals in Bangladesh.

    PubMed

    Rahman, A K M A; Saegerman, C; Berkvens, D; Melzer, F; Neubauer, H; Fretin, D; Abatih, E; Dhand, N; Ward, M P

    2017-01-09

    To determine the role of different Brucella (B.) spp. in Bangladesh, 62 animal samples and 500 human sera were tested. Animal samples from cattle, goats and sheep (including milk, bull semen, vaginal swabs and placentas) were cultured for Brucella spp. Three test-positive human sera and all animal samples were screened by Brucella genus-specific real-time PCR (RT-PCR), and positive samples were then tested by IS711 RT-PCR to detect B. abortus and B. melitensis DNA. Only B. abortus DNA was amplified from 13 human and six animal samples. This is the first report describing B. abortus as the aetiological agent of brucellosis in occupationally exposed humans in Bangladesh. Of note is failure to detect B. melitensis DNA, the species most often associated with human brucellosis worldwide. Further studies are required to explore the occurrence of Brucella melitensis in Bangladesh.

  9. Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus.

    PubMed

    Díaz Aparicio, E

    2013-04-01

    Brucellosis is a disease that causes severe economic losses for livestock farms worldwide. Brucella melitensis, B. abortus and B. suis, which are transmitted between animals both vertically and horizontally, cause abortion and infertility in their primary natural hosts - goats and sheep (B. melitensis), cows (B. abortus) and sows (B. suis). Brucella spp. infect not only their preferred hosts but also other domestic and wild animal species, which in turn can act as reservoirs of the disease for other animal species and humans. Brucellosis is therefore considered to be a major zoonosis transmitted by direct contact with animals and/or their secretions, or by consuming milk and dairy products.

  10. Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections.

    PubMed

    Skyberg, Jerod A; Thornburg, Theresa; Rollins, Maryclare; Huarte, Eduardo; Jutila, Mark A; Pascual, David W

    2011-01-01

    γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ(-/-) mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα(-/-), and GMCSF(-/-) mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ(-/-) mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.

  11. Colonization of mouse placentas by Brucella abortus inoculated during pregnancy.

    PubMed Central

    Bosseray, N.

    1980-01-01

    Pregnant mice were challenged at Day 3, 7, 11 or 15 of pregnancy with Brucella abortus Strain 544 and killed at Day 18 of pregnancy for the enumeration of brucella in the spleens and individual placentas. Whatever the route of challenge--i.p., i.v. or s.c. into the foot-pad (F-s.c.) no abortions or foetal deaths were observed. Placental colonization involved either all, none or only some of the placentas in the same uterus (partial placental colonization: 25% of the mice). In the latter case, the probability of colonization was the same for all sites of implantation. Placentas were independent units as regards colonization and bacterial proliferation. Placental colonization was expressed either by (1) the class of placental infection within the uterus, which might be total, partial or nil; (2) the ratio of infected to total placentas analysed per group; or (3) the mean degree of infection per group. Whatever means of expression was chosen, placental colonization increased with the dose of challenge in parallel with splenic infection in the mouse. The challenge doses required at Day 7 to infect 50% of the placentas differed according to the route (i.p. = 54; i.v. = 5.6 x 10(2) and F-s.c. = 3.6 x 10(4) brucella). Placentas were more frequently and more intensively colonized when the challenge was performed at Days 7 and 11 than at Days 3 and 15 at pregnancy. Mice immunized with H.38 B. melitensis killed vaccine 36 days before pregnancy were found to be protected against an i.p. challenge with 2 x 10(5) brucella at Day 7 of pregnancy. PMID:6775668

  12. Brucella abortus Exposure during an Orthopedic Surgical Procedure—New Mexico, 2010

    PubMed Central

    Nichols, Megin; Thompson, Deborah; Carothers, Joshua T.; Klauber, Judy; Stoddard, Robyn A.; Guerra, Marta A.; Benoit, Tina J.; Traxler, Rita M.

    2015-01-01

    We describe a periprosthetic Brucella abortus infection in a case-patient undergoing hip replacement revision surgery, and the subsequent investigation of laboratory and surgical staff exposures. Although exposures are rare, it is important to have infection prevention recommendations for surgical procedures among patients with suspected or unidentified Brucella spp. infection. PMID:25026630

  13. Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus.

    PubMed

    Buendía, A J; Nicolás, L; Ortega, N; Gallego, M C; Martinez, C M; Sanchez, J; Caro, M R; Navarro, J A; Salinas, J

    2007-01-15

    Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8(+) T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines.

  14. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro.

    PubMed Central

    Detilleux, P G; Deyoe, B L; Cheville, N F

    1990-01-01

    In pregnant ruminants, Brucella abortus localizes and replicates within the rough endoplasmic reticulum of trophoblastic epithelial cells. In this study, Vero cells were exposed to B. abortus to investigate its internalization and intracellular growth in nonphagocytic cells. A new double-fluorescence staining procedure to discriminate between extracellular and intracellular bacteria was developed. Studies with the double-fluorescence staining procedure and quantitative bacteriologic culture of disrupted host cells showed that various B. abortus strains replicated within Vero cells, including smooth virulent (strains 2308S and 544), smooth attenuated (strain 19), and rough (strains 45/20 and 2308R) strains. Rough brucellae were more adherent and entered a greater number of Vero cells. Intracellular replication occurred in a larger percentage of cells with smooth virulent (2308S and 544) strains than with smooth attenuated (19) or rough (45/20 and 2308R) strains. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth (2308S) and rough (45/20) brucellae were consistently found within cisternae of the rough endoplasmic reticulum and nuclear envelope. The results suggest that transfer to the rough endoplasmic reticulum is the limiting step in the infection of nonphagocytic cells by B. abortus. Images PMID:2114362

  15. Abortion and premature birth in cattle following vaccination with Brucella abortus strain RB51.

    PubMed

    Fluegel Dougherty, Amanda M; Cornish, Todd E; O'Toole, Donal; Boerger-Fields, Amy M; Henderson, Owen L; Mills, Ken W

    2013-09-01

    Brucella abortus RB51 is the vaccine strain currently licensed for immunizing cattle against brucellosis in the United States. Most cattle are vaccinated as heifer calves at 4-12 months of age. Adult cattle may be vaccinated in selected high-risk situations. Two herds of pregnant adult cattle in the brucellosis-endemic area of Wyoming were vaccinated with a standard label dose (1.0-3.4 × 10(10) organisms) of RB51. Reproductive losses in the vaccinated herds were 5.3% (herd A) and 0.6% (herd B) and included abortions, stillbirths, premature calves, and unbred cows (presumed early abortion). Brucella abortus was cultured from multiple tissues of aborted and premature calves (7/9), and from placenta. Isolates were identified as B. abortus strain RB51 by standard strain typing procedures and a species-specific polymerase chain reaction. Bronchopneumonia with intralesional bacteria and placentitis were observed microscopically. There was no evidence of involvement of other infectious or toxic causes of abortion. Producers, veterinarians, and laboratory staff should be alert to the risk of abortion when pregnant cattle are vaccinated with RB51, to potential human exposure, and to the importance of distinguishing field from vaccinal strains of B. abortus.

  16. Characterization and protective property of Brucella abortus cydC and looP mutants.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

    2014-11-01

    Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.

  17. On the link between cell cycle and infection of the Alphaproteobacterium Brucella abortus

    PubMed Central

    Deghelt, Michaël; Letesson, Jean-Jacques; De Bolle, Xavier

    2014-01-01

    Bacteria of the Brucella genus are responsible for brucellosis, a worldwide zoonosis. These bacteria are known to have a peculiar intracellular trafficking, with a first long and non-proliferative endosomal stage and a second proliferation stage, often associated with its localization of the bacteria in the endoplasmic reticulum (ER). However, the status of the bacterial cell cycle during the non-proliferative phase was still unknown. In a recent study [Nat. Communic. 5:4366], we followed the cell cycle of B. abortus in culture and inside the host cells. In culture, B. abortus initiates the replication of its large chromosome before the small chromosome. The origin and terminator regions of these two chromosomes display distinct localization and dynamics within B. abortus. In HeLa cells and RAW264.7 macrophages, the bacteria in G1 (i.e. before the initiation of chromosomes replication) are preferentially found during the endosomal stage of the infection. During this period, growth is also arrested. The cell cycle arrest and resume during the B. abortus trafficking in host cell suggest that like the model Alphaproteobacterium Caulobacter crescentus, these bacteria are able to block their cell cycle at the G1 phase when starvation is sensed. PMID:28357212

  18. Experimental Infection of Richardson's Ground Squirrels (Spermophilus richardsonii) with Attenuated and Virulent Strains of Brucella abortus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Exposure of non-target species to wildlife vaccines is an important concern when evaluating a candidate vaccine for use in the field. A previous investigation of the safety of Brucella abortus strain RB51 (sRB51) in various non-target species suggested that Richardson’s ground squirrels (Spermophil...

  19. Genetic characterization of Brucella melitensis and Brucella abortus geographical clusters in Italy.

    PubMed

    De Massis, Fabrizio; Garofolo, Giuliano; Cammà, Cesare; Ippoliti, Carla; Candeloro, Luca; Ancora, Massimo; Calistri, Paolo

    2015-01-01

    The genetic diversity of Brucella melitensis and Brucella abortus strains isolated in 199 cattle and sheep from 156 brucellosis outbreaks which occurred in 8 regions of Southern Italy in 2011, was determined using a Multiple-Locus Variable Number of Tandem Repeats Analysis approach. The existence of possible genetic clusters was verified through a hierarchical cluster analysis based on 'single link', which is closely related to the minimum spanning tree. The Hamming weighted distance matrix was adopted in the analysis. All calculations were performed using R and the additional libraries phangorn and Cluster. For a number of clusters, ranging from 2 to 15, the average silhouette width was calculated. The number of clusters adopted was identified according to the maximum average silhouette width. For B. abortus and B. melitensis, 6 and 11 genetic clusters were identified, respectively. Three out of 6 B. abortus clusters included the 96.7% of all B. abortus isolates. Clusters were clearly geographically separated, and this highlighted the known epidemiological links among them. Brucella melitensis genotypes resulted more heterogeneous; the 3 more representative genetic clusters included 79.7% of all B. melitensis isolates. A clear geographical clusterization of genotypes is recognizable only for 1 cluster, whereas the others are more widespread across Southern Italy. The genetic characterization of Brucella strains isolated from animals may be a useful tool to better understand the epidemiology and dissemination patterns of this pathogen through host populations.

  20. Identification of Immunologically Relevant Proteins of Chlamydophila abortus Using Sera from Experimentally Infected Pregnant Ewes▿ †

    PubMed Central

    Marques, P. X.; Souda, Puneet; O'Donovan, J.; Gutierrez, J.; Gutierrez, E. J.; Worrall, S.; McElroy, M.; Proctor, A.; Brady, C.; Sammin, D.; Basset, H. F.; Whitelegge, Julian P.; Markey, B. E.; Nally, J. E.

    2010-01-01

    Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 × 106 inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein. PMID:20554807

  1. Immune responses of bison and efficacy after booster vaccination with Brucella abortus strain RB51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-one bison heifers were randomly assigned to saline (control; n=7) or single vaccination (n=24) with 1010 CFU of B. abortus strain RB51 (RB51). Some vaccinated bison were randomly selected for booster vaccination with 10**10 CFU of RB51 at 11 months after initial vaccination (n=16). When comp...

  2. In Vitro Antibacterial Effects of Five Volatile Oil Extracts Against Intramacrophage Brucella Abortus 544

    PubMed Central

    Al-Mariri, Ayman; Saour, George; Hamou, Razan

    2012-01-01

    Background: Brucella abortus is a gram-negative facultative intracellular bacterium that can cause a highly contagious disease in sheep, goats, cattle and one-humped camels. It is responsible for one of the most important zoonosis in human. The aim of this study was to evaluate the role of Mentha piperita, Origanum majorana, Citrus lemon, Cinnamomum verum and Myristica fragrans essential volatile oil extracts on human macrophages infected by B. abortus 544. Methods: Essential volatile oil extracts from M. piperita, O. majorana, C. lemon, C. verum and M. fragrans were extracted. Human macrophages were cultured at a density of 2×105 cells per well in sterile 96-well microtiter plates, and infected with B. abortus 544 at a ratio of 1:100 bacteria/cell. Then essential volatile oil extracts were added at a concentration of 1%. At specified times; cells were washed, lysed with 0.1% Triton, and plated on 2YT agar to determine the number of intracellular bacteria. Results: Cinnamomum verum volatile oil at a concentration of 1% had the highest antibacterial activity against B. abortus 544 inside human macrophages. Its inhibitory effect observed from 24 h and continued till 144 h after the infection. Moreover, C. verum (0.1%) in combination with 1% concentration of M. piperita, O. majorana, C. lemon or M. fragrans volatile oil extracts produced a synergistic inhibitory effect against B. abortus 544. Conclusion: The results indicate that, among the five selected oil extracts, C. verum volatile oil applied either separately or in combination with other oil extracts had the most effective antimicrobial activity against Brucella. PMID:23115441

  3. Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

    PubMed

    Garin-Bastuji, Bruno; Mick, Virginie; Le Carrou, Gilles; Allix, Sebastien; Perrett, Lorraine L; Dawson, Claire E; Groussaud, Pauline; Stubberfield, Emma J; Koylass, Mark; Whatmore, Adrian M

    2014-03-01

    Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

  4. Examination of Taxonomic Uncertainties Surrounding Brucella abortus bv. 7 by Phenotypic and Molecular Approaches

    PubMed Central

    Garin-Bastuji, Bruno; Le Carrou, Gilles; Allix, Sebastien; Perrett, Lorraine L.; Dawson, Claire E.; Groussaud, Pauline; Stubberfield, Emma J.; Koylass, Mark; Whatmore, Adrian M.

    2014-01-01

    Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position. PMID:24362435

  5. Genetic diversity of Brucella abortus and Brucella melitensis in Kazakhstan using MLVA-16.

    PubMed

    Shevtsov, Alexandr; Ramanculov, Erlan; Shevtsova, Elena; Kairzhanova, Alma; Tarlykov, Pavel; Filipenko, Maxim; Dymova, Maya; Abisheva, Gulzada; Jailbekova, Aygul; Kamalova, Dinara; Chsherbakov, Andrei; Tulegenov, Samat; Akhmetova, Assel; Sytnik, Igor; Karibaev, Talgat; Mukanov, Kasim

    2015-08-01

    Brucellosis is an endemic disease in Central Asia characterized by high infection rates in humans and animals. Currently, little is known about the genetic diversity of Brucella spp. circulating in the region, despite the high prevalence of brucellosis. This study aimed to analyze the genetic diversity of Brucella melitensis and Brucella abortus strains circulating in the Republic of Kazakhstan. We genotyped 128 B. melitensis and 124 B. abortus strains collected in regions with the highest prevalence of brucellosis. Genotyping was performed using multi-locus variable-number tandem-repeat analysis (MLVA). Analysis of a subset of 8 loci (MLVA-8) of 128 B. melitensis strains identified genotypes 42 (n=108), 43 (n=2), and 63 (n=19) related to the 'East Mediterranean' group. An MLVA-16 assay sorted 128 B. melitensis strains into 25 different genotypes. Excluding one variable locus, MLVA-15 of B. melitensis was distinct from strains originating in the Mediterranean region; however, 77% of them were identical to strains isolated in China. A minimum spanning tree for B. melitensis using MLVA-15 analysis clustered the local strains together with strains previously collected in China. MLVA-8 analysis of 124 B. abortus strains identified them as genotype 36, suggesting Eurasian distribution of this lineage. Complete MLVA-16 assay analysis clustered the strains into five genotypes, revealing little diversity of B. abortus when compared on the global scale. A minimum spanning tree for B. abortus obtained using MLVA-15 analysis clustered the 2 most prevalent genotypes (n=117) together with strains previously collected in China. Thus, MLVA analysis was used to characterize 252 strains of Brucella collected in Kazakhstan. The analysis revealed genetic homogeneity among the strains. Interestingly, identical MLVA-15 profiles were found in seemingly unrelated outbreaks in China, Turkey, and Kazakhstan. Further analysis is needed for better understanding of the epidemiology of

  6. Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization.

    PubMed

    Rolán, Hortensia García; Tsolis, Renée M

    2007-06-01

    The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.

  7. Intranasal Infection with Chlamydia abortus Induces Dose-Dependent Latency and Abortion in Sheep

    PubMed Central

    Longbottom, David; Livingstone, Morag; Maley, Stephen; van der Zon, Arjan; Rocchi, Mara; Wilson, Kim; Wheelhouse, Nicholas; Dagleish, Mark; Aitchison, Kevin; Wattegedera, Sean; Nath, Mintu; Entrican, Gary; Buxton, David

    2013-01-01

    Background Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus. Methodology/Principal Findings Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×103, 5×105 and 5×107 inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×106 IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50–67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium. Conclusions/Significance The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for

  8. Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and he...

  9. The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

    PubMed Central

    Stemshorn, B; Nielsen, K

    1977-01-01

    Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:405088

  10. Molecular typing of isolates obtained from aborted foetuses in Brucella-free Holstein dairy cattle herd after immunisation with Brucella abortus RB51 vaccine in Egypt.

    PubMed

    Wareth, Gamal; Melzer, Falk; Böttcher, Denny; El-Diasty, Mohamed; El-Beskawy, Mohamed; Rasheed, Nesma; Schmoock, Gernot; Roesler, Uwe; Sprague, Lisa D; Neubauer, Heinrich

    2016-12-01

    Bovine brucellosis is endemic in Egypt in spite of application of surveillance and control measures. An increase of abortions was reported in a Holstein dairy cattle herd with 600 animals in Damietta governorate in Egypt after immunisation with Brucella (B.) abortus RB51 vaccine. Twenty one (10.6%) of 197 vaccinated cows aborted after 3 months. All aborted cows had been tested seronegative for brucellosis in the past 3 years. B. abortus was isolated from four foetuses. Conventional biochemical and bacteriological identification and polymerase chain reaction (PCR) confirmed two B. abortus biovar (bv.) 1 smooth and two B. abortus rough strains. None of the B. abortus isolates were identified as RB51. Genotyping analysis by multiple locus of variable number tandem repeats analysis based on 16 markers (MLVA-16) revealed two different profiles with low genetic diversity. B. abortus bv1 was introduced in the herd and caused abortions.

  11. Naturally occurring lesions of the uterine tube in sheep and serologic evidence of exposure to Chlamydophila abortus.

    PubMed Central

    Tomlinson, L; Barker, I K; Foster, R A; McEwen, S A; Menzies, P I; Shewen, P E

    2000-01-01

    The uterine tubes from 405 ewes, collected at an abattoir, were assessed grossly and microscopically for abnormalities that correlated with serological evidence of exposure to Chlamydophila abortus. Gross lesions were found in 41 ewes and 86 had microscopic lesions. Enzyme immunoassay (EIA) of serum was used as an indication of exposure of individual ewes to C. abortus; 52 were found to be positive. Chi-squared analysis indicated no association between EIA-positive animals and lesions of the uterine tube. PMID:11041501

  12. Effects of partial deletion of the wzm and wzt genes on lipopolysaccharide synthesis and virulence of Brucella abortus S19.

    PubMed

    Wang, Xiuran; Wang, Lin; Lu, Tiancheng; Yang, Yanling; Chen, Si; Zhang, Rui; Lang, Xulong; Yan, Guangmou; Qian, Jing; Wang, Xiaoxu; Meng, Lingyi; Wang, Xinglong

    2014-06-01

    Brucellosis is a worldwide human and animal infectious disease, and the effective methods of its control are immunisation of animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines with virulence in the control of cattle Brucellosis. In the present study, allelic exchange plasmids of wzm and wzt genes and partial knockout mutants of wzm and wzt were constructed to evaluate the resulting difference in virulence of B. abortus S19. PCR analysis revealed that the target genes were knocked out. The mutants were rough mutants and they could be differentiated from natural infection by the Rose Bengal plate and standard agglutination tests. The molecular weights of lipopolysaccharides of the Δwzm and Δwzt mutants were clustered between 25 and 40 kDa, and 30 and 35 kDa separately, and were markedly different from those in B. abortus S19. The virulence of B. abortus Δwzm and Δwzt was decreased compared with that of B. abortus S19 in mice. All these results identified that there were several differences between the wzm and wzt genes on lipopolysaccharide synthesis and on the virulence of B. abortus.

  13. Genotyping of Brucella melitensis and Brucella abortus strains currently circulating in Xinjiang, China.

    PubMed

    Sun, Ming-Jun; Di, Dong-Dong; Li, Yan; Zhang, Zhi-Cheng; Yan, Hao; Tian, Li-Li; Jing, Zhi-Gang; Li, Jin-Ping; Jiang, Hai; Fan, Wei-Xing

    2016-10-01

    Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n=38, 76%) and 63 (n=11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n=7, 77.8%), 86 (n=1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n=49) were identified as sequence type ST8, and most B. abortus strains (n=8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.

  14. Prime-booster vaccination of cattle with an influenza viral vector Brucella abortus vaccine induces a long-term protective immune response against Brucella abortus infection.

    PubMed

    Tabynov, Kaissar; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Kydyrbayev, Zhailaubay; Kozhamkulov, Yerken; Inkarbekov, Dulat; Sansyzbay, Abylai

    2016-01-20

    This study analyzed the duration of the antigen-specific humoral and T-cell immune responses and protectiveness of a recently-developed influenza viral vector Brucella abortus (Flu-BA) vaccine expressing Brucella proteins Omp16 and L7/L12 and containing the adjuvant Montadine Gel01 in cattle. At 1 month post-booster vaccination (BV), both humoral (up to 3 months post-BV; GMT IgG ELISA titer 214±55 to 857±136, with a prevalence of IgG2a over IgG1 isotype antibodies) and T-cell immune responses were observed in vaccinated heifers (n=35) compared to control animals (n=35, injected with adjuvant/PBS only). A pronounced T-cell immune response was induced and maintained for 12 months post-BV, as indicated by the lymphocyte stimulation index (2.7±0.4 to 10.1±0.9 cpm) and production of IFN-γ (13.7±1.7 to 40.0±3.0 ng/ml) at 3, 6, 9, and 12 months post-BV. Prime-boost vaccination provided significant protection against B. abortus infection at 3, 6, 9 and 12 months (study duration) post-BV (7 heifers per time point; alpha=0.03-0.01 vs. control group). Between 57.1 and 71.4% of vaccinated animals showed no signs of B. abortus infection (or Brucella isolation) at 3, 6, 9 and 12 months post-BV; the severity of infection, as indicated by the index of infection (P=0.0003 to <0.0001) and rates of Brucella colonization (P=0.03 to <0.0001), was significantly lower for vaccinated diseased animals than appropriate control animals. Good protection from B. abortus infection was also observed among pregnant vaccinated heifers (alpha=0.03), as well as their fetuses and calves (alpha=0.01), for 12 months post-BV. Additionally, 71.4% of vaccinated heifers calved successfully whereas all pregnant control animals aborted (alpha=0.01). Prime-boost vaccination of cattle with Flu-BA induces an antigen-specific humoral and pronounced T cell immune response and most importantly provides good protectiveness, even in pregnant heifers, for at least 12 months post-BV.

  15. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    PubMed Central

    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  16. Chlamydophila abortus infection in the mouse: a useful model of the ovine disease.

    PubMed

    Caro, M R; Buendía, A J; Del Rio, L; Ortega, N; Gallego, M C; Cuello, F; Navarro, J A; Sanchez, J; Salinas, J

    2009-03-16

    Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.

  17. A case of unusual septic knee arthritis with Brucella abortus after arthroscopic meniscus surgery.

    PubMed

    Lee, Keun Hwa; Kang, Hyunseong; Kim, Taejung; Choi, Sungwook

    2016-01-01

    We present a 51-year-old male patient with Brucella abortus septic arthritis in the right knee following arthroscopic meniscus surgery. He had eaten a traditional dish of raw minced cattle conceptus (bovine fetus) that was prepared after the cow was slaughtered. Despite treatment with empirical antibiotics and debridement of the postoperative surgical wound, the infection persisted without improvement. Polymerase chain reaction sequencing identified Brucella abortus from tissue samples obtained from the patient. After confirmation of the diagnosis of brucellar infection, antibiotics were replaced with doxycycline and rifampin, which were used for 4 months. In patients with a non-specific arthralgia who eat raw meat or live close to animals, it is important to consider the possibility of septic arthritis due to infection with Brucella spp.

  18. Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes.

    PubMed

    Muendo, Esther N; Mbatha, Peter M; Macharia, Joseph; Abdoel, Theresia H; Janszen, Paul V; Pastoor, Rob; Smits, Henk L

    2012-01-01

    Brucella melitensis biovar 1 was isolated from bovine milk samples from a herd in central Kenya, and Brucella abortus biovar 3 was isolated from aborted fetus materials and vaginal discharge fluids from cattle in central and eastern provinces of Kenya. All infections including those with B. melitensis were in cattle with reproductive problems kept in mixed herds indicating that cross infection occurs from small ruminants. Multiple-locus variable-number tandem repeat analysis genotyping revealed a close molecular homology of the B. melitensis isolates with an isolate from Israel and a close homology of the B. abortus isolates with an isolate from Uganda indicating that these genotypes have a wide geographic distribution. Infection of cattle with B. melitensis may complicate the control of brucellosis in this country.

  19. Immunogenic response induced by wzm and wzt gene deletion mutants from Brucella abortus S19.

    PubMed

    Wang, Xiu-Ran; Yan, Guang-Mou; Zhang, Rui; Lang, Xu-Long; Yang, Yan-Ling; Li, Xiao-Yan; Chen, Si; Qian, Jing; Wang, Xing-Long

    2014-02-01

    Brucellosis is an infectious disease affecting humans and animals worldwide. Effective methods of control include inducing immunity in animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines for control of cattle brucellosis, as it has low virulence. In this paper, allelic exchange plasmids of wzm and wzt genes were constructed and partially knocked out to evaluate the effects on the induction of immunity to Brucella abortus S19 mutants. Cytokine secretion in vitro, INF-γ induction in vivo and antibody dynamics were evaluated. These data suggested that the immunity-eliciting ability of the wzm and wzt gene deletion mutants was similar, although reduced compared with the S19 strain. The results demonstrated that the wzt gene may be more important in the regulation of the induction of immunity than the wzm gene.

  20. Seroprevalence of antibodies to Chlamydophila abortus in Ovine in the State of Alagoas, Brazil

    PubMed Central

    Pinheiro Junior, José Wilton; Mota, Rinaldo Aparecido; Piatti, Rosa Maria; Oliveira, Andréa Alice da Fonseca; da Silva, Aline Melo; de Oliveira Abreu, Sílvio Romero; Anderlini, Giulliano Aires; Valença, Rômulo Menna Barreto

    2010-01-01

    The goal of this study was to perform a seroepidemiological investigation and to identify risk factors associated with infection of Chlamydophila abortus of sheep herds in the Brazilian state of Alagoas. The study was conducted with samples of 274 ewes with ages equal to or higher than 24 months in 25 herds and in 23 towns located in three regions of the state (Sertão, Agreste and Eastern Alagoas). Anti-C. abortus antibodies were detected using the microcomplement fixation test. The risk factors, were determined based on questionnaires consisting of objective questions, about the farmer and general characteristics of the herd like size, sanitary situation and reproductive management. Among 274 sera samples analyzed for C. abortus, 59 (21.5%) were positive with titers ≥32, 187 (68.3%) negative and 28 (10.2%) suspect with titers ≥16. In the 23 towns studied, 20 had positive animals. Among herds 21 (77.7%) of had positive animals. The only variable which appeared to be significant in the multivariate analysis was the region, and Sertão was the most significant (p<0.001; OR=3.48; T.I. 1.79 – 6.76). Results indicate that infection by Chlamydophila abortus is widespread on sheep farms in the State of Alagoas. Others studies, however, have to be conducted to isolate the agent in order to confirm the role of the bacteria is reproductive disturbances in sheeps. In addition to that, control and prophylactic measures along with health promoting programs have to be encouraged on the studied farms so that infection reates are reduced. PMID:24031504

  1. Transposon-Derived Brucella abortus Rough Mutants Are Attenuated and Exhibit Reduced Intracellular Survival

    PubMed Central

    Allen, Chris A.; Adams, L. Garry; Ficht, Thomas A.

    1998-01-01

    The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential

  2. Epidemiology of Brucellosis and Genetic Diversity of Brucella abortus in Kazakhstan

    PubMed Central

    Shevtsova, Elena; Shevtsov, Alexandr; Mukanov, Kasim; Filipenko, Maxim; Kamalova, Dinara; Sytnik, Igor; Syzdykov, Marat; Kuznetsov, Andrey; Akhmetova, Assel; Zharova, Mira; Karibaev, Talgat; Tarlykov, Pavel; Ramanculov, Erlan

    2016-01-01

    Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan. PMID

  3. A rapid cycleave PCR method for distinguishing the vaccine strain Brucella abortus A19 in China.

    PubMed

    Nan, Wenlong; Zhang, Yueyong; Tan, Pengfei; Xu, Zouliang; Chen, Yuqi; Mao, Kairong; Chen, Yiping

    2016-05-01

    Brucellosis is a widespread zoonotic disease caused by Brucella spp. Immunization with attenuated vaccines has proved to be an effective method of prevention; however, it may also interfere with diagnosis. Brucella abortus strain A19, which is homologous to B. abortus strain S19, is widely used for the prevention of bovine brucellosis in China. For effective monitoring of the control of brucellosis, it is essential to distinguish A19 from field strains. Single-nucleotide polymorphism-based assays offer a new approach to such discrimination studies. In the current study, we developed a cycleave PCR assay that successfully distinguished attenuated vaccine strains A19 and S19 from 22 strains of B. abortus and 57 strains of 5 other Brucella species. The assay gave a negative reaction with 4 non-Brucella species. The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 7.6 fg for the A19 strain and 220 fg for the single non-A19/non-S19 Brucella strain tested (B. abortus 104M). The assay was also reproducible (intra- and interassay coefficients of variation: 0.003-0.01 and 0.004-0.025, respectively). The cycleave assay gave an A19/S19-specific reaction in 3 out of 125 field serum samples, with the same 3 samples being positive in an alternative A19/S19-specific molecular assay. The cycleave assay gave a total of 102 Brucella-specific reactions (3 being the A19/S19-specific reactions), whereas an alternative Brucella-specific assay gave 92 positive reactions (all also positive in the cycleave assay). Therefore, this assay represents a simple, rapid, sensitive, and specific tool for use in brucellosis control.

  4. Effects of gamma radiation and azathioprine on Brucella abortus infection in BALB/c mice

    SciTech Connect

    Elzer, P.H.; Rowe, G.E.; Enright, F.M.; Winter, A.J. )

    1991-06-01

    Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 {times} 10(4) virulent Brucella abortus, caused significant (P less than 0.01) reductions in bacterial numbers in comparison with numbers in unirradiated controls. Numbers of brucellae in the spleen were significantly lower by 5 days after inoculation and decreased thereafter, so that at 2 and 3 weeks after inoculation, there were up to 1,000-fold fewer organisms in the spleen of irradiated mice. The number of brucellae in the spleen increased in irradiated mice thereafter. The course of events in the liver was similar, but developed more slowly, and peak differences in bacterial numbers were about 1 log less. These phenomena were not attributable to differences in implantation of brucellae in the liver or spleen, nor to an abnormal distribution of organisms in other organs of irradiated mice. Irradiation of mice during the plateau phase of infection also resulted in significant (P less than 0.05) reductions in bacterial counts in the spleen during the succeeding 4 weeks. Macrophage activation in the spleen, measured by a Listeria monocytogenes-killing assay, was significantly (P less than 0.01) increased by irradiation alone at 1 week after inoculation and at that time was significantly (P less than 0.01) greater in B abortus-infected, irradiated mice than in B abortus-infected controls. Histologic, cytologic, and immunologic studies revealed that the decrease in numbers of organisms between 1 and 2 weeks after inoculation in irradiated mice occurred at a time when their immune response to B abortus was suppressed and when numbers of neutrophils and monocytes infiltrating the spleen were significantly (P less than 0.01) diminished.

  5. Evaluation of transmission of Brucella abortus strain 19 in bison by intravaginal, intrauterine, and intraconjunctival inoculation.

    PubMed

    Uhrig, Samantha R; Nol, Pauline; McCollum, Matt; Salman, Mo; Rhyan, Jack C

    2013-07-01

    Bovine brucellosis, caused by the bacterium Brucella abortus, is endemic in bison (Bison bison) and elk (Cervus elaphus nelsoni) populations in the area of Yellowstone National Park, USA. Two strategies have been proposed to reduce the risk of transmission of disease in bison: remote vaccination with the vaccine RB51, and the use of immunocontraception of bison to decrease shedding of organisms from infected females. The frequent occurrence of venereal transmission in bison would complicate either of these strategies, requiring vaccination of males as well as females, and rendering immunocontraception less effective in reducing transmission of B. abortus. To address the question of venereal transmission, we inoculated each of 18 bison cows with 4.5 × 10(8) colony-forming units of B. abortus strain 19, as a surrogate of field strain, by three routes: intraconjunctival (IC), intravaginal (VI), and intracervical/intrauterine (AI). Bison semen was mixed with strain 19 inoculum for the latter route. Bison were monitored by serology and culture for 12 wk, at which time they were euthanized and specimens collected for culture. All IC-inoculated animals seroconverted on multiple tests and one was culture positive at 12 wk postexposure. Seven of eight VI bison developed suspect or positive serologic tests and four were positive at one or more time points. Weak transient serologic responses (suspect) were seen in four of five AI bison. Results showed that IC inoculation with strain 19 was a suitable surrogate for field strain to demonstrate exposure to the B. abortus. The seroconversion of four of eight VI bison indicated exposure of the immune system to the agent and the need for further studies on venereal transmission in bison.

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

    PubMed

    Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G; Gorvel, Jean-Pierre; Moreno, Edgardo

    2015-05-01

    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.

  7. Comparison of Biological and Immunological Characterization of Lipopolysaccharides From Brucella abortus RB51 and S19

    PubMed Central

    Kianmehr, Zahra; Kaboudanian Ardestani, Sussan; Soleimanjahi, Hoorieh; Fotouhi, Fatemeh; Alamian, Saeed; Ahmadian, Shahin

    2015-01-01

    Background: Brucella abortus RB51 is a rough stable mutant strain, which has been widely used as a live vaccine for prevention of brucellosis in cattle instead of B. abortus strain S19. B. abortus lipopolysaccharide (LPS) has unique properties in comparison to other bacterial LPS. Objectives: In the current study, two types of LPS, smooth (S-LPS) and rough (R-LPS) were purified from B. abortus S19 and RB51, respectively. The aim of this study was to evaluate biological and immunological properties of purified LPS as an immunogenical determinant. Materials and Methods: Primarily, S19 and RB51 LPS were extracted and purified by two different modifications of the phenol water method. The final purity of LPS was determined by chemical analysis (2-keto-3-deoxyoctonate (KDO), glycan, phosphate and protein content) and different staining methods, following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). C57BL/6 mice were immunized subcutaneously three times at biweekly intervals with the same amount of purified LPSs. The humoral immunity was evaluated by measuring specific IgG levels and also different cytokine levels, such as IFN-γ, TNF-α, IL-4 and IL-10, were determined for assessing T-cell immune response. Results: Biochemical analysis data and SDS-PAGE profile showed that the chemical nature of S19 LPS is different from RB51 LPS. Both S and R-LPS induce an immune response. T-cell immune response induced by both S and R-LPS had almost the same pattern whereas S19 LPS elicited humoral immunity, which was higher than RB51 LPS. Conclusions: Purified LPS can be considered as a safe adjuvant and can be used as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. PMID:26862376

  8. Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

    PubMed

    Velásquez, Lis N; Milillo, M Ayelén; Delpino, M Victoria; Trotta, Aldana; Fernández, Pablo; Pozner, Roberto G; Lang, Roland; Balboa, Luciana; Giambartolomei, Guillermo H; Barrionuevo, Paula

    2017-03-01

    Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4(+) T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.

  9. Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

    PubMed Central

    2009-01-01

    Background Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals. Results Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas) were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta) and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta) were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens. Conclusion We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and the pathogenetic

  10. Purification and properties of Cu-Zn superoxide dismutase extracted from Brucella abortus strain 19

    SciTech Connect

    Tabatabai, L.B. )

    1991-03-11

    Recent work showed that a recombinant 20 kDa protein from Brucella abortus expressed in E. coli is a Cu-Zn superoxide dismutase (SOD). Western blot and ELISA results indicated that cattle with brucellosis have antibody to SOD. Here the authors report the purification and properties of the native B. abortus Cu-Zn SOD. SOD was extracted from methanol-killed Brucella abortus strain 19 with 0.1 M sodium citrate-1.0 M sodium chloride solution. The extract was dialyzed and protein precipitated by ammonium sulfate at 70-100% saturation was collected. The SOD was purified by HPLC anion exchange chromatography. SOD activity was assayed with a coupled enzyme assay using xanthine oxidase-cytochrome C reduction assay. The authors determined that the Brucella SOD is present in two molecular forms both inhibitable with KCN with Ki's of 0.32 mM and 4.98 mM, respectively. No other form of SOD was identified in the extract. Polyclonal antibody to SOD and polyclonal antibody to SOD synthetic peptide residues 134-143 inhibited SOD activity by 50% and 13%, respectively. Both SOD and the synthetic peptide inhibited binding of anti-SOD antibody to SOD by 60% and 20%, respectively. Based on these results the SOD and its amphipathic peptide will be considered as candidates for the design of synthetic multiple peptide vaccines and diagnostic reagents for bovine brucellosis.

  11. Intracellularly induced cyclophilins play an important role in stress adaptation and virulence of Brucella abortus.

    PubMed

    Roset, Mara S; García Fernández, Lucía; DelVecchio, Vito G; Briones, Gabriel

    2013-02-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.

  12. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks

    PubMed Central

    Villagra-Blanco, R.; Dolz, G.; Montero-Caballero, D.; Romero-Zúñiga, J.J.

    2015-01-01

    A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA). Antibodies were detected in 19 (5.29%) sheep from 12 (80%) flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific) determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two risk factors associated with seropositivity; introducing animals (males and females), embryos, or semen from other farms or from abroad without any sanitary certification, and flocks not having quarantine areas or separated boxes for diseased animals. No clinical signs of disease were observed in positive seroreactors. C. abortus seems to be present in Costa Rica in a very low prevalence in sheep flocks. Further studies, to isolate the bacteria are required. Finally, implementation of control measures to prevent the spread of C. abortus is recommended. PMID:26623377

  13. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks.

    PubMed

    Villagra-Blanco, R; Dolz, G; Montero-Caballero, D; Romero-Zúñiga, J J

    2015-01-01

    A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA). Antibodies were detected in 19 (5.29%) sheep from 12 (80%) flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific) determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two risk factors associated with seropositivity; introducing animals (males and females), embryos, or semen from other farms or from abroad without any sanitary certification, and flocks not having quarantine areas or separated boxes for diseased animals. No clinical signs of disease were observed in positive seroreactors. C. abortus seems to be present in Costa Rica in a very low prevalence in sheep flocks. Further studies, to isolate the bacteria are required. Finally, implementation of control measures to prevent the spread of C. abortus is recommended.

  14. The role of innate immune signals in immunity to Brucella abortus.

    PubMed

    Gomes, Marco Túlio R; Campos, Priscila C; de Almeida, Leonardo A; Oliveira, Fernanda S; Costa, Miriam Maria S; Marim, Fernanda M; Pereira, Guilherme S M; Oliveira, Sergio C

    2012-01-01

    Innate immunity serves as the first line of defense against infectious agents such as intracellular bacteria. The innate immune platform includes Toll-like receptors (TLRs), retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, nucleotide-binding and oligomerization domain-like receptors, adaptors, kinases and other signaling molecules that are required to elicit effective responses against different pathogens. Our research group has been using the Gram-negative bacteria Brucella abortus as a model of pathogen. We have demonstrated that B. abortus triggers MAPK and NF-κB signaling pathways in macrophages in a MyD88 and IRAK-4-dependent manner. Furthermore, we claimed that so far TLR9 is the most important single TLR during Brucella infection. The identification of host receptors that recognize pathogen-derived nucleic acids has revealed an essential role for nucleic acid sensing in the triggering of immunity to intracellular pathogens. Besides TLRs, herein we describe recent advances in NOD1, NOD2, and type I IFN receptors in innate immune pathways during B. abortus infection.

  15. Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle.

    PubMed

    Davis, D S; Templeton, J W; Ficht, T A; Williams, J D; Kopec, J D; Adams, L G

    1990-07-01

    Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 x 10(7) colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 x 10(7) CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of

  16. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

    PubMed Central

    Bernardo Reyes, Alisha Wehdnesday; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee

    2016-01-01

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis. PMID:26726017

  17. Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico.

    PubMed

    Campos-Hernández, Eleuterio; Vázquez-Chagoyán, Juan Carlos; Salem, Abdelfattah Z M; Saltijeral-Oaxaca, Jorge Antonio; Escalante-Ochoa, Cristina; López-Heydeck, Sandra M; de Oca-Jiménez, Roberto Montes

    2014-08-01

    The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzyme-linked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87% (n = 246). Seropositive animals were found in six out of nine (66.6%) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51%. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico.

  18. Evaluation and comparison of different blood culture techniques for bacteriological isolation of Salmonella typhi and Brucella abortus.

    PubMed Central

    Gaviria-Ruiz, M M; Cardona-Castro, N M

    1995-01-01

    An experimental study was carried out to evaluate and compare various noncommercial methods of blood culture for the isolation of Salmonella typhi and Brucella abortus from fresh human blood samples that had been artificially inoculated with 1 to 50 microorganisms per ml of blood. The methods compared included the Ruiz-Castañeda blood culture, broth blood culture, leukocyte lysis and direct plating on agar (WBL-P), leukocyte lysis and filtration, Ficoll-Hypaque centrifugation and filtration, Ficoll-Hypaque centrifugation, and Ficoll-Hypaque centrifugation and leukocyte lysis methods. Results with the WBL-P technique showed that S. typhi was isolated in 18 h, and its recovery rate was 36.6% (calculated from the number of CFU recovered per milliliter versus the number inoculated). B. abortus was isolated in 48 h by the same technique, and its recovery rate was 48.8%. The isolation times for the other blood culture techniques were between 36 and 44 h for S. typhi and 66 h for B. abortus. The techniques which relied on filtering systems for the recovery of S. typhi and B. abortus performed poorly. The WBL-P technique for the isolation of S. typhi and B. abortus is faster than the other methods tested. PMID:7790452

  19. Evaluation of bison (Bison bison) semen from Yellowstone National Park, Montana, USA, bulls for Brucella abortus shedding.

    PubMed

    Frey, Rebecca K; Clarke, P Ryan; McCollum, Matt P; Nol, Pauline; Johnson, Kammy R; Thompson, Brent D; Ramsey, Jennifer M; Anderson, Neil J; Rhyan, Jack C

    2013-07-01

    To determine if bison (Bison bison) bulls from Yellowstone National Park (YNP), Montana, USA, shed an infective dose of Brucella abortus in semen, 50 YNP bulls were captured on public lands in Montana during the winter and early spring (April-May) of 2010 and 2011. The bulls were immobilized, and blood and semen samples were collected for serology and Brucella culture. Thirty-five bulls (70%) were antibody-positive, and B. abortus was cultured from semen in three (9%) of the 35 antibody-positive or suspect bulls, though not at concentrations considered an infective dose. Eight bulls (six antibody-positive, two negative) had palpable lesions of the testes, epididymides, or seminal vesicles consistent with B. abortus infection. Breeding soundness exams and semen analysis suggested that antibody-positive bulls were more likely to have nonviable ejaculate (8/35; 23%) than bulls without detectable antibody (2/15; 13%).

  20. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

    PubMed

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-04-30

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  1. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle

    PubMed Central

    Marchesini, María I.; Morrone Seijo, Susana M.; Guaimas, Francisco F.; Comerci, Diego J.

    2016-01-01

    Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus. PMID:27900285

  2. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    PubMed

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.

  3. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

    PubMed Central

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D.; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-01-01

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies. PMID:27144565

  4. Ability of Brucella abortus rough vaccine strains to elicit DC and innate immunity in lung using a murine respiratory model.

    PubMed

    Surendran, Naveen; Zimmerman, Kurt; Seleem, Mohamed N; Sriranganathan, Nammalwar; Boyle, Stephen M; Hiltbold, Elizabeth M; Lawler, Heather; Heid, Bettina; Witonsky, Sharon G

    2010-10-08

    Brucella abortus strains RB51 and RB51SOD are live attenuated vaccine strains which protect mice against virulent B. abortus strain 2308 intraperitoneal challenge. By comparison, limited information is available on how Brucella vaccines stimulate pulmonary immunity against respiratory infection, another route of exposure in humans. Therefore, in this study, we assessed the ability of intranasally delivered vaccine strains RB51 and RB51SOD to induce innate immunity. Based on parameters assessed, rough strain RB51 induces a better innate immune response in lung versus strain RB51SOD. Additional studies to further delineate strain RB51's ability to stimulate DC and adaptive immunity are warranted.

  5. Proinflammatory Response of Human Trophoblastic Cells to Brucella abortus Infection and upon Interactions with Infected Phagocytes.

    PubMed

    Fernández, Andrea G; Ferrero, Mariana C; Hielpos, M Soledad; Fossati, Carlos A; Baldi, Pablo C

    2016-02-01

    Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis.

  6. Different resistance patterns of reference and field strains of Brucella abortus.

    PubMed

    Miranda, Karina L; Dorneles, Elaine M S; Poester, Fernando P; Martins Filho, Paulo S; Pauletti, Rebeca B; Lage, Andrey P

    2015-03-01

    The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO 2 . Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 ( B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75-0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

  7. MLVA16 Typing of Portuguese Human and Animal Brucella melitensis and Brucella abortus Isolates

    PubMed Central

    Ferreira, Ana Cristina; Chambel, Lélia; Tenreiro, Tania; Cardoso, Regina; Flor, Lídia; Dias, Isabel Travassos; Pacheco, Teresa; Garin-Bastuji, Bruno; Le Flèche, Philippe; Vergnaud, Gilles; Tenreiro, Rogério; de Sá, Maria Inácia Corrêa

    2012-01-01

    To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16Orsay assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16Orsay showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the “Americas” (17%) and “East Mediterranean” (83%) groups. No isolate belonged to the “West Mediterranean” group. Eighty-five percent of the human isolates (39 in 46) fit in the “East-Mediterranean” group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16Orsay provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries. PMID:22905141

  8. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells

    PubMed Central

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José

    2015-01-01

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway. PMID:26667834

  9. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells.

    PubMed

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José; Delpino, María Victoria

    2015-12-14

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

  10. TLR7 and TLR3 Sense Brucella abortus RNA to Induce Proinflammatory Cytokine Production but They Are Dispensable for Host Control of Infection

    PubMed Central

    Campos, Priscila C.; Gomes, Marco Túlio R.; Guimarães, Erika S.; Guimarães, Gabriela; Oliveira, Sergio C.

    2017-01-01

    Brucella abortus is a Gram-negative, facultative intracellular bacterium that causes brucellosis, a worldwide zoonotic disease leading to undulant fever in humans and abortion in cattle. The immune response against this bacterium relies on the recognition of microbial pathogen-associated molecular patterns, such as lipoproteins, lipopolysaccharides, and DNA; however, the immunostimulatory potential of B. abortus RNA remains to be elucidated. Here, we show that dendritic cells (DCs) produce significant amounts of IL-12, IL-6, and IP-10/CXCL10, when stimulated with purified B. abortus RNA. IL-12 secretion by DCs stimulated with RNA depends on TLR7 while IL-6 depends on TLR7 and partially on TLR3. Further, only TLR7 plays a role in IL-12 production induced by B. abortus infection. Moreover, cytokine production in DCs infected with B. abortus or stimulated with bacterial RNA was reduced upon pretreatment with MAPK/NF-κB inhibitors. By confocal microscopy, we demonstrated that TLR7 is colocalized with B. abortus in LAMP-1+ Brucella-containing vacuoles. Additionally, type I IFN expression and IP-10/CXCL10 secretion in DCs stimulated with bacterial RNA were dependent on TLR3 and TLR7. Our results suggest that TLR3 and TLR7 are not required to control Brucella infection in vivo, but they play an important role on sensing B. abortus RNA in vitro. PMID:28167945

  11. Host-pathogen interactions in specific pathogen-free chickens following aerogenous infection with Chlamydia psittaci and Chlamydia abortus.

    PubMed

    Kalmar, Isabelle; Berndt, Angela; Yin, Lizi; Chiers, Koen; Sachse, Konrad; Vanrompay, Daisy

    2015-03-15

    Although Chlamydia (C.) psittaci infections are recognized as an important factor causing economic losses and impairing animal welfare in poultry production, the specific mechanisms leading to severe clinical outcomes are poorly understood. In the present study, we comparatively investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in the course of experimental aerogeneous infection with C. psittaci and the closely related C. abortus, respectively, in specific pathogen-free chicks. Clinical signs appeared sooner and were more severe in the C. psittaci-infected group. Compared to C. abortus infection, more intense systemic dissemination of C. psittaci correlated with higher and faster infiltration of immune cells, as well as more macroscopic lesions and epithelial pathology, such as hyperplasia and erosion. In thoracic air sac tissue, mRNA expression of immunologically relevant factors, such as IFN-γ, IL-1β, IL-6, IL-17, IL-22, LITAF and iNOS was significantly stronger up-regulated in C. psittaci- than in C. abortus-infected birds between 3 and 14 days post-infection. Likewise, transcription rates of the chlamydial genes groEL, cpaf and ftsW were consistently higher in C. psittaci during the acute phase. These findings illustrate that the stronger replication of C. psittaci in its natural host also evoked a more intense immune response than in the case of C. abortus infection.

  12. Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

    PubMed

    Doull, L; Wattegedera, S R; Longbottom, D; Mwangi, D; Nath, M; Glass, E J; Entrican, G

    2015-11-15

    Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1β or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.

  13. Efficacy of dart or booster vaccination with strain RB51 in protecting bison against experimental Brucella abortus challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vaccination is an effective tool for reducing the prevalence of brucellosis in natural hosts. In this study, we characterized the efficacy of the Brucella abortus strain RB51 (RB51) vaccine in bison when delivered by single intramuscular vaccination (Hand RB51), single pneumatic dart delivery (Dart ...

  14. Genome Sequences of Human and Livestock Isolates of Brucella melitensis and Brucella abortus from the Country of Georgia

    PubMed Central

    Sidamonidze, Ketevan; Hang, Jun; Yang, Yu; Dzavashvili, George; Zhgenti, Ekaterine; Trapaidze, Nino; Imnadze, Paata

    2017-01-01

    ABSTRACT Brucellosis, which is among the most widespread global zoonotic diseases, is endemic in the nation of Georgia and causes substantial human morbidity and economic loss. Here, we report whole-genome sequences of three Brucella melitensis and seven Brucella abortus isolates from cattle, sheep, and humans that represent genetic groups discovered in Georgia. PMID:28183751

  15. Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

    PubMed

    Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2017-03-28

    Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus-infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus-containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

  16. A Live Vaccine from Brucella abortus Strain 82 for Control of Cattle Brucellosis in the Russian Federation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During the first half of the 20th century, widespread regulatory efforts to control cattle brucellosis (Brucella abortus) in the Union of Soviet Socialist Republics were essentially nonexistent, and control was limited to selective test and slaughter of serologic agglutination reactors. By the 1950...

  17. Comparison of abortion and infection after experimental challenge of pregnant bison and cattle with Brucella abortus strain 2308

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comparative study was conducted using data from naive bison (n=45) and cattle (n=46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered. The incidence of abortion, fetal infection, uterine or mammary infection, or infec...

  18. Dextran sulfate sodium upregulates MAPK signaling for the uptake and subsequent intracellular survival of Brucella abortus in murine macrophages.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Arayan, Lauren Togonon; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2016-02-01

    Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection.

  19. Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization.

    PubMed

    Iannino, Florencia; Herrmann, Claudia K; Roset, Mara S; Briones, Gabriel

    2015-05-05

    Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with BabΔpgm carrier strain (t=12 days). As expected, mice immunized with BabΔpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.

  20. Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice.

    PubMed

    Adone, Rosanna; Francia, Massimiliano; Pistoia, Claudia; Petrucci, Paola; Pesciaroli, Michele; Pasquali, Paolo

    2012-06-08

    It has been demonstrated that antibodies specific for O-PS antigen of Brucella smooth strains are involved in the protective immunity of brucellosis. Since the rough strain Brucella melitensis B115 was able to protect mice against wild Brucella strains brucellosis despite the lack of anti-OPS antibodies, in this study we evaluated the biological significance of antibodies induced by this strain, directed to antigens other than O-PS, passively tranferred to untreated mice prior to infection with Brucella abortus 2308 and B. melitensis 16M virulent strains. The protective ability of specific antisera collected from mice vaccinated with B. melitensis B115, B. abortus RB51 and B. abortus S19 strains was compared. The results indicated that antibodies induced by B115 were able to confer a satisfactory protection, especially against B. abortus 2308, similar to that conferred by the antiserum S19, while the RB51 antiserum was ineffective. These findings suggest that antibodies induced by B115 could act as opsonins as well as antibodies anti-O-PS, thus triggering more efficient internalization and degradation of bacteria within phagocytes. This is the first study assessing the efficacy of antibodies directed to antigens other than O-PS in the course of brucellosis infection.

  1. Abortive Potency of Chlamydophila abortus in Pregnant Mice Is Not Directly Correlated with Placental and Fetal Colonization Levels

    PubMed Central

    Bouakane, Amel; Benchaîeb, Ilhem; Rodolakis, Annie

    2003-01-01

    Abortion, placental and fetal colonization, and levels of gamma interferon were analyzed for four Chlamydophila abortus strains presenting antigenic variations in a mouse model. Expression of virulence of these strains varied and indicated that abortion was not directly related to the number of bacteria in the placenta, and thus, other factors may have an important role in activating the abortion process. PMID:14638821

  2. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

  3. Intratracheal infection as an efficient route for testing vaccines against Chlamydia abortus in sheep.

    PubMed

    Álvarez, D; Salinas, J; Buendía, A J; Ortega, N; del Río, L; Sánchez, J; Navarro, J A; Gallego, M C; Murcia-Belmonte, A; Cuello, F; Caro, M R

    2015-09-01

    Pregnant ewes have been widely used to test vaccines against Chlamydia abortus. However, this model entails many disadvantages such as high economic costs and long periods of pregnancy. The murine model is very useful for specific studies but cannot replace the natural host for the later stages of vaccine evaluation. Therefore, a non-pregnant model of the natural host might be useful for a vaccine trial to select the best vaccine candidates prior to use of the pregnant model. With this aim, two routes of infection were assessed in young non-pregnant sheep, namely, intranasal (IN) and intratracheal (IT). In addition, groups of non-vaccinated sheep and sheep immunised with an inactivated vaccine were established to investigate the suitability of the model for testing vaccines. After the experimental infection, isolation of the microorganism in several organs, with pathological and immunohistochemical analyses, antibody production assessment and investigation by PCR of the presence of chlamydia in the vagina or rectum were carried out. Experimental IT inoculation of C. abortus induced pneumonia in sheep during the first few days post-infection, confirming the suitability of the IT route for testing vaccines in the natural host. The course of infection and the resulting pathological signs were less severe in vaccinated sheep compared with non-vaccinated animals, demonstrating the success of vaccination. IN infection did not produce evident lesions or demonstrate the presence of chlamydial antigen in the lungs and cannot be considered an appropriate model for testing vaccines.

  4. Comparison of Chemical Components of Cell Walls of Brucella abortus Strains of Low and High Virulence.

    PubMed

    Kellerman, G D; Foster, J W; Badakhsh, F F

    1970-09-01

    Amino acid, carbohydrate, and lipid components of cell walls of Brucella abortus strain 19A (low virulence) and strain 2308 (high virulence) were compared by thin layer chromatography (TLC) and by use of an amino acid analyzer. A total of 15 amino acids were detected by both chromatographic methods. Each amino acid was present in greater amounts in strain 2308 than in strain 19A when equal amounts of hydrolysates of cell wall and endotoxin-containing preparations were analyzed. A component with the same R(F) value as ethanolamine was present in strain 2308 cell wall hydrolysates but was not revealed by TLC of strain 19A cell wall hydrolysates. This component was not detected with the amino acid analyzer. TLC of cell walls tagged with 2,4-dinitrofluorobenzene prior to hydrolysis showed that phenylalanine was a terminal amino acid in cell walls of B. abortus strains 19A and 2308, B. suis strain 1776, and B. melitensis strain 2500. Carbohydrates detected in cell walls of strains 19A and 2308 by TLC were tentatively identified as glucose, mannose, rhamnose, and galactose. Colorimetric tests were also positive for 2-keto-3-deoxy-octulosonic acid, heptose, and dideoxyhexose. At least seven lipid components were detected by TLC of ether extracts of cell walls of strains 19A and 2308. It is suggested that one or more lipids is important in maintaining cell wall structure, because isolated cell walls rapidly became fragmented after exposure to ether.

  5. Identification of a protective protein from stationary-phase exoproteome of Brucella abortus.

    PubMed

    Jain, Shikha; Kumar, Subodh; Dohre, Sudhir; Afley, Prachiti; Sengupta, Nabonita; Alam, Syed I

    2014-02-01

    Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. To search the putative vaccine candidates, we studied the exoproteome of Brucella abortus NCTC 10093 using 2-DE-MS approach. Twenty-six proteins were identified using MALDI-TOF/TOF tandem mass spectrometry. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Most proteins (6, 23%) were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (4, 15%). Outer membrane protein 25, Omp2b porin and one hypothetical protein were predicted as outer membrane proteins. In addition, Omp28, Omp31 and one ribosomal protein (L9) were also identified. The ribosomal protein L9 was produced as a recombinant protein and was studied in mouse model for vaccine potential. It was found to be immunogenic in terms of generating serum antibody response and release of IFN-γ from mice spleen cells. Recombinant L9-immunized mice were protected against challenge with virulent B. abortus strain 544, suggesting usefulness of ribosomal protein L9 as a good vaccine candidate against brucellosis.

  6. An evaluation of ELISA using recombinant Brucella abortus bacterioferritin (Bfr) for bovine brucellosis.

    PubMed

    Hop, Huynh Tan; Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-04-01

    To date, detection of antibodies against the lipopolysaccharide portion is the backbone of most serodiagnostic methods for brucellosis screening. However this pose a risk for false positive reactions related to other pathogens especially that of Yersinia enterocolitica O:9 which has the most prominent cross reactivity with Brucella spp. In this study, cloning and expression of Brucella abortus bacterioferritin (Bfr) was accomplished by PCR amplification into an expression vector system, and purification of a recombinant B. abortus Bfr (rBfr). The immunogenicity of rBfr was confirmed by Western blot with Brucella-positive bovine serum. To determine whether rBfr has a potential benefit for use in the serodiagnosis of bovine brucellosis, rBfr-based ELISA was performed. Interestingly, rBfr was able to detect anti-Brucella antibodies in positive sera in a dependent manner of TAT values but did not show an immunoreaction with negative samples. Particularly, average OD492 values at the lowest, medium and highest TAT titer levels were 1.4, 2.2 and 2.6-fold increase compared with the cutoff value, respectively. The accuracy, specificity and sensitivity of rBfr showed 89.09%, 93.6% and 85.33%, respectively. These findings suggest that rBfr might be a good candidate for serological diagnosis development of bovine brucellosis.

  7. Myeloid decidual dendritic cells and immunoregulation of pregnancy: defective responsiveness to Coxiella burnetii and Brucella abortus

    PubMed Central

    Gorvel, Laurent; Ben Amara, Amira; Ka, Mignane B.; Textoris, Julien; Gorvel, Jean-Pierre; Mege, Jean-Louis

    2014-01-01

    Dendritic cells (DCs) are a component of the placental immune system, but their role in pregnancy is still poorly understood. Decidual DCs (dDCs) were selected from at-term pregnancy on the basis of CD14 and CD11c expression. A phenotypic analysis revealed that dDCs are characterized by the expression of monocyte-derived DC (moDCs) markers and specific markers such as HLA-G and its ligand ILT4. As demonstrated by whole-genome microarray, dDCs expressed a specific gene program markedly distinct from that of moDCs; it included estrogen- and progesterone-regulated genes and genes encoding immunoregulatory cytokines, which is consistent with the context of foeto-maternal tolerance. A functional analysis of dDCs showed that they were unable to mature in response to bacterial ligands such as lipopolysaccharide or peptidoglycan, as assessed by the expression of HLA-DR, CD80, CD83, and CD86. When dDCs were incubated with bacteria known for their placenta tropism, Coxiella burnetii and Brucella abortus, they were also unable to mature and to produce inflammatory cytokines. It is likely that the defective maturation of dDCs and their inability to produce inflammatory cytokines is related to the spontaneous release of IL-10 by these cells. Taken together, these results suggest that dDCs exhibit an immunoregulatory program, which may favor the pathogenicity of C. burnetii or B. abortus. PMID:25566514

  8. WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

    PubMed Central

    Herrou, Julien; Czyż, Daniel M.; Willett, Jonathan W.; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang

    2016-01-01

    ABSTRACT The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. IMPORTANCE Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of

  9. A Multiplex Approach to Molecular Detection of Brucella abortus and/or Mycobacterium bovis Infection in Cattle

    PubMed Central

    Sreevatsan, Srinand; Bookout, Jack B.; Ringpis, Fidel; Perumaalla, Veera S.; Ficht, Thomas A.; Adams, L. Garry; Hagius, Sue D.; Elzer, Philip H.; Bricker, Betsy J.; Kumar, Girish K.; Rajasekhar, M.; Isloor, Srikrishna; Barathur, Raj R.

    2000-01-01

    A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a “gold standard,” was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease

  10. Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases

    PubMed Central

    2013-01-01

    Background Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. Methods In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. Results B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing

  11. Seroprevalence and molecular characterization of Chlamydia abortus in frozen fetal and placental tissues of aborting ewes in northeastern Algeria.

    PubMed

    Hireche, Sana; Ababneh, Mustafa Mohammed Kheir; Bouaziz, Omar; Boussena, Sabrina

    2016-02-01

    Enzootic abortion of ewes is one of the most serious health problems in sheep flocks worldwide. It has a significant economic impact because abortion, decrease in milk production and weak lambs. Besides, the bacteria is zoonotic. A cross-sectional study was conducted to determine the seroprevalence and risk factors associated with Chlamydia abortus infection in 552 ewes in Constantine using a C. abortus-specific indirect ELISA kit. Chlamydial DNA was investigated in ten ovine fetuses and eight placentas using PCR- restriction fragment length polymorphism (RFLP) and DNA sequencing. The study concluded that 7.2 % of ewes were seropositive and 33.3 % of sheep flocks had at least one seropositive ewe. Adjacent farmworker visits (OR = 7.667, 95 % CI (OR) = 2.307; 27.203) was defined as a risk factor. Deliveries of weak lambs (OR = 2.920, 95 % CI (OR) = 1.022; 8.342) and septicemia in lambs (OR = 9.971, 95 % CI (OR) = 2.383; 41.713) were significantly associated with chlamydial infection. PCR-RFLP analysis revealed positive signals to C. abortus in six fetuses and four placentas. Sequencing of the omp2 gene revealed that the Algerian strain is 96 % similar with C. abortus FAS strain. C. abortus plays a major role in abortion in northeastern Algeria. Appropriate control measures must be implemented to reduce economic losses and to avoid human contamination.

  12. Studies on recombinant glucokinase (r-glk) protein of Brucella abortus as a candidate vaccine molecule for brucellosis.

    PubMed

    Vrushabhendrappa; Singh, Amit Kumar; Balakrishna, Konduru; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-09-29

    Brucellosis is one of the most prevalent zoonotic diseases of worldwide distribution caused by the infection of genus Brucella. Live attenuated vaccines such as B. abortus S19, B. abortus RB51 and B. melitensis Rev1 are found most effective against brucellosis infection in animals, contriving a number of serious side effects and having chances to revert back into their active pathogenic form. In order to engineer a safe and effective vaccine candidate to be used in both animals and human, a recombinant subunit vaccine molecule comprising the truncated region of glucokinase (r-glk) gene from B. abortus S19 was cloned and expressed in Escherichia coli BL21DE3 host. Female BALB/c mice immunized with purified recombinant protein developed specific antibody titer of 1:64,000. The predominant IgG2a and IgG2b isotypes signified development of Th1 directed immune responses. In vitro cell cytotoxicity assay using anti-r-glk antibodies incubated with HeLa cells showed 81.20% and 78.5% cell viability against lethal challenge of B. abortus 544 and B. melitensis 16M, respectively. The lymphocyte proliferative assay indicated a higher splenic lymphocyte responses at 25μg/ml concentration of protein which implies the elevated development of memory immune responses. In contrast to control, the immunized group of mice intra-peritoneal (I.P.) challenged with B. abortus 544 were significantly protected with no signs of necrosis and vacuolization in their liver and spleen tissue. The elevated B-cell response associated with Th1 adopted immunity, significant in vitro cell viability as well as protection afforded in experimental animals after challenge, supplemented with histopathological analysis are suggestive of r-glk protein as a prospective candidate vaccine molecule against brucellosis.

  13. Transcriptional analysis of in vitro expression patterns of Chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle

    PubMed Central

    Wheelhouse, Nicholas; Aitchison, Kevin; Spalding, Lucy; Livingstone, Morag; Longbottom, David

    2009-01-01

    Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in the loci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peak expression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 h p.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may be indicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis. PMID:19454212

  14. Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544.

    PubMed

    Goel, Divya; Rajendran, Vinoth; Ghosh, Prahlad C; Bhatnagar, Rakesh

    2013-02-06

    Brucellosis is a disease affecting various domestic and wild life species, and is caused by a bacterium Brucella. Keeping in view the serious economic and medical consequences of brucellosis, efforts have been made to prevent the infection through the use of vaccines. Cell-mediated immune responses [CMI] involving interferon gamma and cytotoxic CD4(+) and CD8(+) T cells are required for removal of intracellular Brucella. Omp25 has been reported to be involved in virulence of Brucella melitensis, Brucella abortus and Brucella ovis. In our previous study, we have shown the protective efficacy of recombinant Omp25, when administered intradermally. In this study, the recombinant Omp25 was formulated in PC-PE liposomes and PLGA microparticles, to enhance the protective immunity generated by it. Significant protection was seen with prime and booster liposome immunization in Balb/c mice against virulent B. abortus 544 as it was comparable to B. abortus S-19 vaccine strain. However, microparticle prime and booster immunization failed to give better protection when compared to B. abortus S-19 vaccine strain. This difference can be attributed to the stimulation of cell mediated immune response in PC-PE liposome immunized mice even after challenge which converted to cytotoxicity seen in CD4(+) and CD8(+) enriched lymphocytes. However, in PLGA microparticle immunized mice, cell mediated immunity was not generated after challenge as observed by decreased cytotoxicity of CD4(+) and CD8(+) enriched lymphocytes. Our study emphasizes on the importance of liposome encapsulating Omp25 immunization in conferring protection against B. abortus 544 challenge in Balb/c mice with a single dose immunization regimen.

  15. Bacterial survival, lymph node pathology, and serological responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19.

    PubMed

    Olsen, S C; Cheville, N F; Kunkle, R A; Palmer, M V; Jensen, A E

    1997-01-01

    From August 1993 to June 1994, 3 month-old bison (Bison bison) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6), strain 19 (S19, n = 3), or with saline (n = 1) and serologic responses and persistence of vaccine strains within lymph nodes were monitored. Bison vaccinated with S19 had granulomatous lymphadenitis and greater peak numbers of B. abortus than those vaccinated with SRB51. Bison vaccinated with RB51 had similar histological lesions and B. abortus were still present in lymph nodes at 16 weeks. Although antibodies against RB51 were produced, standard tube agglutination test responses of RB51-vaccinates remained negative. The histological lesions of B. abortus infections in bison were similar to those observed in cattle, but bison did not clear SRB51 as rapidly as cattle.

  16. TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

    PubMed

    Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

    2016-05-01

    Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9.

  17. Effect of Preventive Chlamydia abortus Vaccination in Offspring Development in Sheep Challenged Experimentally

    PubMed Central

    García-Seco, Teresa; Pérez-Sancho, Marta; Salinas, Jesús; Navarro, Alejandro; Díez-Guerrier, Alberto; García, Nerea; Pozo, Pilar; Goyache, Joaquín; Domínguez, Lucas; Álvarez, Julio

    2016-01-01

    Ovine enzootic abortion, caused by Chlamydia abortus, leads to important economic losses worldwide. In addition to reproductive failures, infection may impact lamb growth during the first weeks after birth, yet this effect has not been well characterized. Vaccination can help to control the disease but variable efficacy values have been described, possibly related with factors associated with the host, the vaccine, the parameter used for efficacy determination, and the challenge conditions. In this context, we evaluated the efficacy of an inactivated standard commercial vaccine and a 1/2 diluted dose in pregnant sheep challenged with C. abortus by examining multiple indicators of vaccine effect (including incidence of reproductive failures, bacterial excretion, and evolution of weight gain of viable lambs during the first month of life). Three groups of ewes [control non-vaccinated, C (n = 18); vaccinated with standard dose, SV (n = 16); and vaccinated with 1/2 dose, DV (n = 17)], were challenged approximately 90 days post-mating and tested using direct PCR (tissue samples and vaginal swabs) and ELISA (serum) until 31 days post-reproductive outcome. There were not significant differences in the proportions of reproductive failures or bacterial shedding after birth/abortion regardless the vaccination protocol. However, a beneficial effect of vaccination on offspring growth was detected in both vaccinated groups compared with the controls, with a mean increase in weight measured at 30 days of life of 1.5 and 2.5 kg (p = 0.056) and an increase in the geometric mean of the daily gain of 8.4 and 9.7% in lambs born from DV and SV ewes compared with controls, respectively. Our results demonstrate the effect of an inactivated vaccine in the development of the offspring of C. abortus-infected ewes at a standard and a diluted dose, an interesting finding given the difficulty in achieving sufficient antigen concentration in the production of enzootic

  18. Effect of Preventive Chlamydia abortus Vaccination in Offspring Development in Sheep Challenged Experimentally.

    PubMed

    García-Seco, Teresa; Pérez-Sancho, Marta; Salinas, Jesús; Navarro, Alejandro; Díez-Guerrier, Alberto; García, Nerea; Pozo, Pilar; Goyache, Joaquín; Domínguez, Lucas; Álvarez, Julio

    2016-01-01

    Ovine enzootic abortion, caused by Chlamydia abortus, leads to important economic losses worldwide. In addition to reproductive failures, infection may impact lamb growth during the first weeks after birth, yet this effect has not been well characterized. Vaccination can help to control the disease but variable efficacy values have been described, possibly related with factors associated with the host, the vaccine, the parameter used for efficacy determination, and the challenge conditions. In this context, we evaluated the efficacy of an inactivated standard commercial vaccine and a 1/2 diluted dose in pregnant sheep challenged with C. abortus by examining multiple indicators of vaccine effect (including incidence of reproductive failures, bacterial excretion, and evolution of weight gain of viable lambs during the first month of life). Three groups of ewes [control non-vaccinated, C (n = 18); vaccinated with standard dose, SV (n = 16); and vaccinated with 1/2 dose, DV (n = 17)], were challenged approximately 90 days post-mating and tested using direct PCR (tissue samples and vaginal swabs) and ELISA (serum) until 31 days post-reproductive outcome. There were not significant differences in the proportions of reproductive failures or bacterial shedding after birth/abortion regardless the vaccination protocol. However, a beneficial effect of vaccination on offspring growth was detected in both vaccinated groups compared with the controls, with a mean increase in weight measured at 30 days of life of 1.5 and 2.5 kg (p = 0.056) and an increase in the geometric mean of the daily gain of 8.4 and 9.7% in lambs born from DV and SV ewes compared with controls, respectively. Our results demonstrate the effect of an inactivated vaccine in the development of the offspring of C. abortus-infected ewes at a standard and a diluted dose, an interesting finding given the difficulty in achieving sufficient antigen concentration in the production of enzootic

  19. A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortus for Virulence and Intracellular Multiplication

    PubMed Central

    Sieira, Rodrigo; Comerci, Diego J.; Sánchez, Daniel O.; Ugalde, Rodolfo A.

    2000-01-01

    As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment. PMID:10940027

  20. Adrenal steroids modulate the immune response during Brucella abortus infection by a mechanism that depends on the regulation of cytokine production.

    PubMed

    Gentilini, María Virginia; Velásquez, Lis Noelia; Barrionuevo, Paula; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2015-05-01

    Human brucellosis is a protean disease with a diversity of clinical signs and symptoms resulting from infection with Brucella species. Recent reports suggest a cross-regulation between adrenal steroids (cortisol and dehydroepiandrosterone [DHEA]) and the immune system. Monocytes and macrophages are the main replication niche for Brucella. Therefore, we investigated the role of adrenal hormones on the modulation of the immune response mediated by macrophages in B. abortus infection. Cortisol treatment during B. abortus infection significantly inhibits cytokine, chemokine, and MMP-9 secretion. In contrast, DHEA treatment had no effect. However, DHEA treatment increases the expression of costimulatory molecules (CD40, CD86), the adhesion molecule CD54, and major histocompatibility complex class I (MHC-I) and MHC-II expression on the surface of B. abortus-infected monocytes. It is known that B. abortus infection inhibits MHC-I and MHC-II expression induced by gamma interferon (IFN-γ) treatment. DHEA reverses B. abortus downmodulation of the MHC-I and -II expression induced by IFN-γ. Taken together, our data indicate that DHEA immune intervention may positively affect monocyte activity during B. abortus infection.

  1. Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

    PubMed

    Martínez, Diana; Thompson, Carolina; Draghi, Graciela; Canavesio, Vilma; Jacobo, Roberto; Zimmer, Patricia; Elena, Sebastián; Nicola, Ana M; de Echaide, Susana Torioni

    2014-09-17

    An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas.

  2. The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells.

    PubMed

    Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2015-06-01

    This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection.

  3. Brucella abortus S19 and RB51 vaccine immunogenicity test: Evaluation of three mice (BALB/c, Swiss and CD-1) and two challenge strains (544 and 2308).

    PubMed

    Miranda, Karina Leite; Dorneles, Elaine Maria Seles; Pauletti, Rebeca Barbosa; Poester, Fernando Padilla; Lage, Andrey Pereira

    2015-01-15

    The aim of the present study was to evaluate the use of different mouse strains (BALB/c, Swiss and CD-1) and different challenge strains (Brucella abortus 544 and 2308) in the study of B. abortus vaccine (S19 and RB51) immunogenicity test in the murine model. No significant difference in B. abortus vaccine potency assay was found with the use of B. abortus 544 or B. abortus 2308 as challenge strain. Results of variance analysis showed an interaction between treatment and mouse strain; therefore these parameters could not be compared separately. When CD-1 groups were compared, those vaccinated showed significantly lower counts than non-vaccinated ones (P<0.05), independently of the vaccine received (S19 or RB51). Similar results were observed on BALB/c groups. However, in Swiss mouse groups, S19 was more protective than RB51 (P<0.05), which showed protection when compared to the non-vaccinated group (P<0.05). In summary, data from the present study showed that CD-1, BALB/c and Swiss mice strains, as well as both challenge strains, B. abortus strains 544 and 2308, can be used in immunogenicity tests of S19 and RB51 vaccines.

  4. Monocyte-derived macrophages from Zebu (Bos taurus indicus) are more efficient to control Brucella abortus intracellular survival than macrophages from European cattle (Bos taurus taurus).

    PubMed

    Macedo, A A; Costa, E A; Silva, A P C; Paixão, T A; Santos, R L

    2013-02-15

    Brucellosis is one of the most important zoonotic diseases in the world. Considering its strict zoonotic nature, understanding of the pathogenesis and immunity of Brucella spp. in natural animal hosts is essential to prevent human infections. Natural resistance against brucellosis has been demonstrated in cattle, and it is associated with the ability of macrophages to prevent intracellular replication of Brucella abortus. Identification of breeds that are resistant to B. abortus may contribute for controlling and eradicating brucellosis in cattle. This study aimed to compare macrophages from Nelore (Bos taurus indicus) or Holstein (Bos taurus taurus) regarding their resistance to B. abortus infection. Macrophages from Nelore were significantly more efficient in controlling intracellular growth of B. abortus when compared to Holstein macrophages even under intralysosomal iron restricting conditions. Furthermore, Nelore macrophages had higher transcription levels of inducible nitric oxide synthase (iNOS) and TNF-α at 12h post-infection (hpi) and higher levels of IL-12 at 24 hpi when compared to Holstein macrophages. Conversely, Holstein macrophages had higher levels of IL-10 transcripts at 24 hpi. Macrohages from Nelore also generated more nitric oxide (NO) in response to B. abortus infection when compared to Holstein macrophages. In conclusion, cultured Nelore macrophages are more effective in controlling intracellular replication of B. abortus, suggesting that Nelore cattle is likely to have a higher degree of natural resistance to brucellosis than Holstein.

  5. Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

    PubMed

    Jain, Lata; Rawat, Mayank; Prajapati, Awadhesh; Tiwari, Ashok Kumar; Kumar, Bablu; Chaturvedi, V K; Saxena, H M; Ramakrishnan, Sarvanan; Kumar, Jatin; Kerketta, Priscilla

    2015-09-01

    The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 μg protein and 60 μg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

  6. Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

    PubMed Central

    Zaitseva, M; Golding, H; Manischewitz, J; Webb, D; Golding, B

    1996-01-01

    Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation. PMID:8757841

  7. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán

    2015-01-01

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. PMID:26459511

  8. An ecological perspective on the changing face of Brucella abortus in the western United States

    USGS Publications Warehouse

    Cross, Paul C.; Maichak, Eric J.; Brennan, Angela; Scurlock, Brandon M.; Henningsen, John C.; Luikart, Gordon

    2013-01-01

    After a hiatus during the 1990s, outbreaks of Brucella abortus in cattle are occurring more frequently in some of the western states of the United States, namely, Montana, Wyoming and Idaho. This increase is coincident with increasing brucellosis seroprevalence in elk (Cervus elaphus), which is correlated with elk density. Vaccines are a seductive solution, but their use in wildlife systems remains limited by logistical, financial, and scientific constraints. Cattle vaccination is ongoing in the region. Livestock regulations, however, tend to be based on serological tests that test for previous exposure and available vaccines do not protect against seroconversion. The authors review recent ecological studies of brucellosis, with particular emphasis on the Greater Yellowstone Area, and highlight the management options and implications of this work, including the potential utility of habitat modifications and targeted hunts, as well as scavengers and predators. Finally, the authors discuss future research directions that will help us to understand and manage brucellosis in wildlife.

  9. Evaluation of Brucella abortus strain RB51 and strain 19 in pronghorn antelope

    USGS Publications Warehouse

    Elzer, P.H.; Smith, J.; Roffe, T.; Kreeger, T.; Edwards, J.; Davis, D.

    2002-01-01

    Free-roaming elk and bison in the Greater Yellowstone Area remain the only wildlife reservoirs for Brucella abortus in the United States, and the large number of animals and a lack of holding facilities make it unreasonable to individually vaccinate each animal. Therefore, oral delivery is being proposed as a possible option to vaccinate these wild ungulates. One of the main problems associated with oral vaccination is the potential exposure of nontarget species to the vaccines. The purpose of this study was to determine the effects of two Brucella vaccines, strain 19 (S19) and the rough strain RB51 (SRB51), in pregnant pronghorn antelope. We conclude that S19 and SRB51 rarely colonize maternal and fetal tissues of pregnant pronghorn and were not associated with fetal death. Oral delivery of either vaccine at this dose appears to be nonhazardous to pregnant pronghorn.

  10. Evaluation of Brucella abortus strain RB51 and strain 19 in pronghorn antelope.

    PubMed

    Elzer, Philip H; Smith, J; Roffe, T; Kreeger, T; Edwards, J; Davis, D

    2002-10-01

    Free-roaming elk and bison in the Greater Yellowstone Area remain the only wildlife reservoirs for Brucella abortus in the United States, and the large number of animals and a lack of holding facilities make it unreasonable to individually vaccinate each animal. Therefore, oral delivery is being proposed as a possible option to vaccinate these wild ungulates. One of the main problems associated with oral vaccination is the potential exposure of nontarget species to the vaccines. The purpose of this study was to determine the effects of two Brucella vaccines, strain 19 (S19) and the rough strain RB51 (SRB51), in pregnant pronghorn antelope. We conclude that S19 and SRB51 rarely colonize maternal and fetal tissues of pregnant pronghorn and were not associated with fetal death. Oral delivery of either vaccine at this dose appears to be nonhazardous to pregnant pronghorn.

  11. First Report of Orchitis in Man Caused by Brucella abortus Biovar 1 in Ecuador

    PubMed Central

    Ron-Román, Jorge; Saegerman, Claude; Minda-Aluisa, Elizabeth; Benítez-Ortíz, Washington; Brandt, Jef; Douce, Richard

    2012-01-01

    We present a 44-year-old man from a rural community in northern Ecuador who worked on a cattle farm where he was involved with primary veterinary care, including assistance during births (or calving) and placenta retention and artificial insemination, with minimal precautions. In September of 2009, quite abruptly, he developed asthenia and hypersomnia without any apparent cause or symptoms like fever, chills, or night sweats. On November 14, 2009, he suffered from pain and edema in the right testicle that coincided with pain in the abdomen. Clinical, serological, and bacteriological investigations confirmed the first case of unilateral orchitis in man in Ecuador caused by Brucella abortus biovar 1. Because brucellosis is a neglected disease, special attention should be given to it in the training of medical and veterinary students. PMID:22826490

  12. Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

    PubMed

    Moriyón, I; López-Goñi, I

    1998-03-01

    The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

  13. First report of orchitis in man caused by Brucella abortus biovar 1 in Ecuador.

    PubMed

    Ron-Román, Jorge; Saegerman, Claude; Minda-Aluisa, Elizabeth; Benítez-Ortíz, Washington; Brandt, Jef; Douce, Richard

    2012-09-01

    We present a 44-year-old man from a rural community in northern Ecuador who worked on a cattle farm where he was involved with primary veterinary care, including assistance during births (or calving) and placenta retention and artificial insemination, with minimal precautions. In September of 2009, quite abruptly, he developed asthenia and hypersomnia without any apparent cause or symptoms like fever, chills, or night sweats. On November 14, 2009, he suffered from pain and edema in the right testicle that coincided with pain in the abdomen. Clinical, serological, and bacteriological investigations confirmed the first case of unilateral orchitis in man in Ecuador caused by Brucella abortus biovar 1. Because brucellosis is a neglected disease, special attention should be given to it in the training of medical and veterinary students.

  14. Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains

    PubMed Central

    Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Roop II, R. Martin; Comerci, Diego J.; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Caswell, Clayton C.; Baker, Kate S.; Chaves-Olarte, Esteban; Thomson, Nicholas R.; Moreno, Edgardo; Letesson, Jean J.; De Bolle, Xavier; Guzmán-Verri, Caterina

    2016-01-01

    Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella. As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain B. abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised. PMID:27746773

  15. Immunological characteristics in cattle of allergens derived from smooth Brucella abortus S99.

    PubMed

    Cunningham, B; Miler, J J; Dolan, L; McKeon, F; O'Meara, M

    1980-10-18

    Allergens prepared from a smooth strain of Brucella abortus (S99) were used in an intradermal test for the diagnosis of brucellosis in cattle. The development and testing of the allergen from the initial less purified stages is described. The removal of serological activity noted in some of the earlier preparations can be related to the elimination of molecules of high molecular weight. The final allergen is of a high order of sensitivity and specificity and does not cause production of complement fixing or agglutinating antibodies. Intradermal testing with such an allergen could be a most useful addition to present serological procedures as it could be carried out at the same time as tuberculin testing. As a routine surveillance test, particularly in low prevalence areas, it would eliminate the necessity for extensive blood sampling and in many cases detect the infected herd before serological tests would have become positive.

  16. Properties of a cell-wall-defective variant of Brucella abortus of bovine origin.

    PubMed Central

    Corbel, M. J.; Scott, A. C.; Ross, H. M.

    1980-01-01

    The properties of an atypical Brucella strain isolated from lymph node tissue of a cow slaughtered as a brucellosis reactor were examined. The organism was Gram negative and highly pleomorphic, existing as cocci, coccobacilli, rods, branched and irregular forms which stained with fluorescent antibody conjugates prepared against rough and smooth Brucella abortus strains. It produced lecithinase and required at least 15% v/v equine or bovine serum for growth. It did not need supplementary CO2 for growth, produced H2S and was inhibited by brucella dyes and partially by i-erythritol. Growth inhibition or lysis was produced by brucella-phages. The organism was not pathogenic for guinea-pigs or mice but evoked antibodies mainly to rough Brucella antigens. Images Fig. 1 Fig. 2 Fig. 3 Fig. 1 Fig. 2 Fig. 1 Fig. 1 Fig. 2 PMID:6820027

  17. Crystal structure of cyclic nucleotide-binding-like protein from Brucella abortus.

    PubMed

    He, Zheng; Gao, Yuan; Dong, Jing; Ke, Yuehua; Li, Xuemei; Chen, Zeliang; Zhang, Xuejun C

    2015-12-25

    The cyclic nucleotide-binding (CNB)-like protein (CNB-L) from Brucella abortus shares sequence homology with CNB domain-containing proteins. We determined the crystal structure of CNB-L at 2.0 Å resolution in the absence of its C-terminal helix and nucleotide. The 3D structure of CNB-L is in a two-fold symmetric form. Each protomer shows high structure similarity to that of cGMP-binding domain-containing proteins, and likely mimics their nucleotide-free conformation. A key residue, Glu17, mediates the dimerization and prevents binding of cNMP to the canonical ligand-pocket. The structurally observed dimer of CNB-L is stable in solution, and thus is likely to be biologically relevant.

  18. Brucella abortus S19 vaccine protects dairy cattle against natural infection with Brucella melitensis.

    PubMed

    van Straten, Michael; Bardenstein, Svetlana; Keningswald, Gaby; Banai, Menachem

    2016-11-21

    Brucellosis is a zoonotic disease that can cause severe illness in humans and considerable economic loss in the livestock industry. Although small ruminants are the preferential host for Brucella melitensis, this pathogen has emerged as a cause for Brucella outbreaks in cattle. S19 vaccination is implemented in many countries where B. abortus is endemic but its effectiveness against B. melitensis has not been validated. Here we show that vaccine effectiveness in preventing disease transmission between vaccinated and unvaccinated cohorts, as determined by seroconversion, was 87.2% (95% CI 69.5-94.6%). Furthermore, vaccination was associated with a reduced risk for abortion. Together, our data emphasize the role S19 vaccination could play in preventing B. melitensis outbreaks in areas where this pathogen is prevalent in small ruminant populations.

  19. Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains.

    PubMed

    Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Roop Ii, R Martin; Comerci, Diego J; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Caswell, Clayton C; Baker, Kate S; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Letesson, Jean J; De Bolle, Xavier; Guzmán-Verri, Caterina

    2016-01-01

    Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella. As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain B. abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised.

  20. An ecological perspective on Brucella abortus in the western United States.

    PubMed

    Cross, P C; Maichak, E J; Brennan, A; Scurlock, B M; Henningsen, J; Luikart, G

    2013-04-01

    After a hiatus during the 1990s, outbreaks of Brucella abortus in cattle are occurring more frequently in some of the western states of the United States, namely, Montana, Wyoming and Idaho. This increase is coincidentwith increasing brucellosis seroprevalence in elk (Cervus elaphus), which is correlated with elk density. Vaccines are a seductive solution, but their use in wildlife systems remains limited by logistical, financial, and scientific constraints. Cattle vaccination is ongoing in the region. Livestock regulations, however, tend to be based on serological tests that test for previous exposure and available vaccines do not protect against seroconversion. The authors review recent ecological studies of brucellosis, with particular emphasis on the Greater Yellowstone Area, and highlight the management options and implications of this work, including the potential utility of habitat modifications and targeted hunts, as well as scavengers and predators. Finally, the authors discuss future research directions that will help us to understand and manage brucellosis in wildlife.

  1. Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus.

    PubMed

    Worrall, S; Sammin, D J; Bassett, H F; Reid, C R; Gutierrez, J; Marques, P X; Nally, J E; O'Donovan, J; Williams, E J; Proctor, A; Markey, B K

    2011-08-01

    Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.

  2. Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia.

    PubMed

    Rekiki, Abdessalem; Sidi-Boumedine, Karim; Souriau, Armel; Jemli, Jemaa; Hammami, Salah; Rodolakis, Annie

    2002-01-01

    Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunosorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.

  3. Efficacy of single calfhood vaccination of elk with Brucella abortus strain 19

    USGS Publications Warehouse

    Roffe, T.J.; Jones, L.C.; Coffin, K.; Drew, M.L.; Sweeney, Steven J.; Hagius, S.D.; Elzer, P.H.; Davis, D.

    2004-01-01

    Brucellosis has been eradicated from cattle in the states of Wyoming, Montana, and Idaho, USA. However, free-ranging elk (Cervus elaphus) that use feedgrounds in the Greater Yellowstone Area (GYA) and bison (Bison bison) in Yellowstone and Grand Teton national parks still have high seroprevalence to the disease and have caused loss of brucellosis-free status in Wyoming. Management tools to control or eliminate the disease are limited; however, wildlife vaccination is among the methods currently used by wildlife managers in Wyoming. We conducted a controlled challenge study of single calfhood vaccination. Elk calves, caught in January and February of 1999 and 2000 and acclimated to captivity for 3 weeks, were randomly assigned to control or vaccinate groups. The vaccinate groups received Brucetta abortus vaccine strain 19 (S19) by hand-delivered intramuscular injection. Calves were raised to adulthood and bred at either 2.5 or 3.5 years of age for 2000 and 1999 captures, respectively. Eighty-nine (44 controls, 45 vaccinates) pregnant elk entered the challenge portion of the study. We challenged elk at mid-gestation with pathogenic B. abortus strain 2308 by intraconjunctival instillation. Abortion occurred in significantly more (P = 0.002) controls (42; 93%) than vaccinates (32; 71%), and vaccine protected 25% of the vaccinate group. We used Brucella culture of fetus/calf tissues to determine the efficacy of vaccination for preventing infection, and we found that the number of infected fetuses/calves did not differ between controls and vaccinates (P = 0.14). Based on these data, single calfhood vaccination with S19 has low efficacy, will likely have only little to moderate effect on Brucella prevalence in elk, and is unlikely to eradicate the disease in wildlife of the GYA.

  4. Evaluation of Brucella abortus Phosphoglucomutase (pgm) Mutant as a New Live Rough-Phenotype Vaccine

    PubMed Central

    Ugalde, Juan Esteban; Comerci, Diego José; Leguizamón, M. Susana; Ugalde, Rodolfo Augusto

    2003-01-01

    Brucella abortus S19 is the vaccine most frequently used against bovine brucellosis. Although it induces good protection levels, it cannot be administered to pregnant cattle, revaccination is not advised due to interference in the discrimination between infected and vaccinated animals during immune-screening procedures, and the vaccine is virulent for humans. Due to these reasons, there is a continuous search for new bovine vaccine candidates that may confer protection levels comparable to those conferred by S19 but without its disadvantages. A previous study characterized the phenotype associated with the phosphoglucomutase (pgm) gene disruption in Brucella abortus S2308, as well as the possible role for the smooth lipopolysaccharide (LPS) in virulence and intracellular multiplication in HeLa cells (J. E. Ugalde, C. Czibener, M. F. Feldman, and R. A. Ugalde, Infect. Immun. 68:5716-5723, 2000). In this report, we analyze the protection, proliferative response, and cytokine production induced in BALB/c mice by a Δpgm deletion strain. We show that this strain synthesizes O antigen with a size of approximately 45 kDa but is rough. This is due to the fact that the Δpgm strain is unable to assemble the O side chain in the complete LPS. Vaccination with the Δpgm strain induced protection levels comparable to those induced by S19 and generated a proliferative splenocyte response and a cytokine profile typical of a Th1 response. On the other hand, we were unable to detect a specific anti-O-antigen antibody response by using the fluorescence polarization assay. In view of these results, the possibility that the Δpgm mutant could be used as a vaccination strain is discussed. PMID:14573645

  5. The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection.

    PubMed

    Marim, Fernanda M; Franco, Miriam M Costa; Gomes, Marco Tulio R; Miraglia, Maria Cruz; Giambartolomei, Guillermo H; Oliveira, Sergio Costa

    2017-02-01

    The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response.

  6. The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection

    PubMed Central

    Gomes, Marco Tulio R.; Miraglia, Maria Cruz; Giambartolomei, Guillermo H.; Oliveira, Sergio C.

    2016-01-01

    The innate immune system is essential for detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which leads to control of infection. This protective effect is due to inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of proinflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and proinflammatory response. PMID:27405866

  7. The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

    PubMed Central

    Iriarte, Maite; Manček-Keber, Mateja; Barquero-Calvo, Elías; Palacios-Chaves, Leyre; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Martirosyan, Anna; von Bargen, Kristine; Grilló, María-Jesús; Jerala, Roman; Brandenburg, Klaus; Llobet, Enrique; Bengoechea, José A.; Moreno, Edgardo

    2012-01-01

    Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines. PMID:22589715

  8. Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

    PubMed

    de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

    2015-04-01

    Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

  9. Investigating genetic diversity of Brucella abortus and Brucella melitensis in Italy with MLVA-16.

    PubMed

    Garofolo, Giuliano; Di Giannatale, Elisabetta; De Massis, Fabrizio; Zilli, Katiuscia; Ancora, Massimo; Cammà, Cesare; Calistri, Paolo; Foster, Jeffrey T

    2013-10-01

    Despite the eradication of brucellosis from most of Europe, the disease remains relatively common in a variety of livestock in southern European countries. It is therefore surprising that with such high prevalence rates, there have been few genetic characterizations of brucellosis outbreaks in this region. We conducted a genetic assessment of 206 isolates of Brucella abortus and B. melitensis from Italy using Variable Number Tandem Repeats (VNTRs). We determined genetic diversity and geographic distribution of these Brucella VNTR genotypes from 160 farms in eight regions of Southern Italy in a fine-scale analysis using 16 VNTR loci in a MLVA-16 methodology. In a broad scale analysis, we then used a reduced dataset of 11 VNTR loci (MLVA-11) to compare genotypes from Italy to a global database. In the 84 isolates of B. melitensis, there were 56 genotypes using MLVA-16; 43 of these genotypes were found only once. At a broad scale, 81 of these isolates were part of an Italian sub-group within the West Mediterranean group. One of the two B. melitensis isolates from a human patient shared the same genotype as a livestock isolate, suggesting a possible epidemiological connection. In 122 B. abortus isolates, there were 34 genotypes by MLVA-16; 16 of these genotypes were found only once. At a broad scale with MLVA-11, one genotype was predominant, comprising 77.8% of the isolates and was distributed throughout Southern Italy. These data on the current lineages of Brucella present in Italy should form the basis for epidemiological studies of Brucella throughout the country, while placing these strains in a global context.

  10. The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.

    PubMed

    Conde-Álvarez, Raquel; Arce-Gorvel, Vilma; Iriarte, Maite; Manček-Keber, Mateja; Barquero-Calvo, Elías; Palacios-Chaves, Leyre; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Martirosyan, Anna; von Bargen, Kristine; Grilló, María-Jesús; Jerala, Roman; Brandenburg, Klaus; Llobet, Enrique; Bengoechea, José A; Moreno, Edgardo; Moriyón, Ignacio; Gorvel, Jean-Pierre

    2012-01-01

    Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

  11. Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7

    PubMed Central

    2013-01-01

    Background Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Results Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. Conclusions In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further. PMID:23802650

  12. CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection

    PubMed Central

    Fernández, Andrea G.; Bonetto, Josefina; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.

    2015-01-01

    Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site. PMID:26448160

  13. CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection.

    PubMed

    Hielpos, M Soledad; Ferrero, Mariana C; Fernández, Andrea G; Bonetto, Josefina; Giambartolomei, Guillermo H; Fossati, Carlos A; Baldi, Pablo C

    2015-01-01

    Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.

  14. Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

    2016-01-04

    We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis.

  15. Infection of cattle with Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park.

    PubMed Central

    Forbes, L B; Tessaro, S V

    1996-01-01

    An experiment was conducted to determine if cattle could be infected with a strain of Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park. Three pregnant cows inoculated conjunctivally with 5.7 x 10(8) cfu of the bacterium, and their subsequent calves, showed seroconversion on standard serological tests for bovine brucellosis, and large numbers of the bacterium were isolated from numerous tissues at necropsy. A 4th cow that was moved into the pen that previously contained the inoculated cows subsequently showed seroconversion, and the same strain of B. abortus biovar 1 was isolated from numerous tissues. Although this strain from bison in Wood Buffalo National Park has existed in isolation from cattle for over 60 years, it remains infectious and contagious for cattle. PMID:8809394

  16. [Intensity of the epizootic process in sheep infected with S. abortus ovis and Chlamydia psittaci var. ovis].

    PubMed

    Vodas, K; Elitsina, P

    1986-01-01

    An attempt was made to analyze comparatively the intensity of the epizootic process in young female sheep, ewes (second pregnancy, with no records of abortions in their first pregnancy) and their lambs either with an infection of Salmonella abortus ovis only or with a mixed infection of S. abortus ovis and Chlamydia psittaci var. ovis. This was reached through following up and studying the parameters morbidity, mortality, lethality, index of infectedness, index of deadlines, and fertility. It was found that the intensity of the apparent epizootic process was highest with young females affected with a mixed infection, and it was lowest with ewes affected with a pure infection (Salmonella abortion). The intensity of the inapparent epizootic process was best manifested in the young females affected with a pure Salmonella abortion. With these animals both the index of infectedness and the index of deadlines had highest values.

  17. Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains.

    PubMed

    Szymańska-Czerwińska, Monika; Mitura, Agata; Niemczuk, Krzysztof; Zaręba, Kinga; Jodełko, Agnieszka; Pluta, Aneta; Scharf, Sabine; Vitek, Bailey; Aaziz, Rachid; Vorimore, Fabien; Laroucau, Karine; Schnee, Christiane

    2017-01-01

    Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

  18. Chlamydiaceae Genomics Reveals Interspecies Admixture and the Recent Evolution of Chlamydia abortus Infecting Lower Mammalian Species and Humans.

    PubMed

    Joseph, Sandeep J; Marti, Hanna; Didelot, Xavier; Castillo-Ramirez, Santiago; Read, Timothy D; Dean, Deborah

    2015-10-27

    Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae. Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis. In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk (Buteo jamaicensis), is an outlying strain with admixture of C. abortus, C. psittaci, and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus. Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages.

  19. Deletion of wboA Enhances Activation of the Lectin Pathway of Complement in Brucella abortus and Brucella melitensis

    PubMed Central

    Fernandez-Prada, Carmen M.; Nikolich, Mikeljon; Vemulapalli, Ramesh; Sriranganathan, Nammalwar; Boyle, Stephen M.; Schurig, Gerhardt G.; Hadfield, Ted L.; Hoover, David L.

    2001-01-01

    Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae. PMID:11401980

  20. Lack of endogenous IL-10 enhances production of proinflammatory cytokines and leads to Brucella abortus clearance in mice.

    PubMed

    Corsetti, Patrícia P; de Almeida, Leonardo A; Carvalho, Natália B; Azevedo, Vasco; Silva, Teane M A; Teixeira, Henrique C; Faria, Ana C; Oliveira, Sergio C

    2013-01-01

    IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.

  1. Chlamydiaceae Genomics Reveals Interspecies Admixture and the Recent Evolution of Chlamydia abortus Infecting Lower Mammalian Species and Humans

    PubMed Central

    Joseph, Sandeep J.; Marti, Hanna; Didelot, Xavier; Castillo-Ramirez, Santiago; Read, Timothy D.; Dean, Deborah

    2015-01-01

    Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae. Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis. In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk (Buteo jamaicensis), is an outlying strain with admixture of C. abortus, C. psittaci, and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus. Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages. PMID:26507799

  2. Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains

    PubMed Central

    Szymańska-Czerwińska, Monika; Mitura, Agata; Niemczuk, Krzysztof; Zaręba, Kinga; Jodełko, Agnieszka; Pluta, Aneta; Scharf, Sabine; Vitek, Bailey; Aaziz, Rachid; Vorimore, Fabien; Laroucau, Karine; Schnee, Christiane

    2017-01-01

    Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates. PMID:28350846

  3. Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus1

    PubMed Central

    Hatten, Betty A.; Sulkin, S. Edward

    1966-01-01

    Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285–296. 1966.—Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO2 was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus. Images PMID:16562102

  4. Seroprevalence of brucellosis in sheep and isolation of Brucella abortus biovar 6 in Kassala State, Eastern Sudan.

    PubMed

    Gumaa, M M; Osman, H M; Omer, M M; El Sanousi, E M; Godfroid, J; Ahmed, A M

    2014-12-01

    Brucellosis is one of the important zoonotic diseases among livestock. This study was carried out to estimate the prevalence of brucellosis and isolate Brucella spp. in sheep in Kassala State in the east of Sudan. Two thousand and five serum samples were randomly collected from nine different localities. All serum samples were examined by the Rose Bengal plate test (RBPT) and the modified RBPT (mRBPT). Forty-three (2.15%, 95% confidence interval [CI]: 1.6,3.0) and 68 (3.4%, 95% CI: 2.6, 4.2) samples were positive with the RBPT and the mRBPT, respectively. According to a known diagnostic sensitivity of 86.6% and a known diagnostic specificity of 97.6% for the mRBPT, the true prevalence was estimated to be 1.2% (95% CI: 0.3, 2.2). Different tissue samples were collected from 41 mRBPT seropositive animals. Brucella abortus biovar 6 was isolated from a pyometra of a seropositive ewe. It is important to note that B. abortus biovar 6 cannot be differentiated from Brucella melitensis biovar 2 by routine bacteriology. Only phage typing performed in reference laboratories will allow accurate identification of the strain. The fact that B. abortus biovar 6 does not require CO2 for growth, combined with the fact that it has been isolated from a small ruminant in this study, could easily have led to misidentification (as B. melitensis biovar 2), to wrong epidemiological inferences and to the implementation of inappropriate control measures. The results presented here suggest that sheep are spillover hosts, as previously described for camels, and that the actual reservoir of B. abortus biovar 6 is cattle in Kassala State, Eastern Sudan. This study highlights the importance of isolating and identifying Brucella spp. in different livestock species in order to accurately decipher brucellosis epidemiology in sub-Saharan Africa.

  5. Draft Genome Sequence of Brucella abortus S99: Designated Antigenic Smooth Reference Strain Used in Diagnostic Tests in India

    PubMed Central

    Shome, Rajeswari; Krithiga, Natesan; Padmashree, B. S.; Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S.; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rahman, Habibur

    2014-01-01

    Brucella abortus strain S99 is widely used for the preparation of colored, plain, recombinant and smooth lipopolysaccharide antigens for the preparation of Brucella diagnostic kits. The genome of this strain was sequenced and the length of the genome was 3,253,175 bp, with 57.2% G+C content. A total of 3,365 protein coding genes and 53 RNA genes were predicted. PMID:25146137

  6. Draft Genome Sequence of Brucella abortus S99: Designated Antigenic Smooth Reference Strain Used in Diagnostic Tests in India.

    PubMed

    Shome, Rajeswari; Krithiga, Natesan; Padmashree, B S; Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash; Rahman, Habibur

    2014-08-21

    Brucella abortus strain S99 is widely used for the preparation of colored, plain, recombinant and smooth lipopolysaccharide antigens for the preparation of Brucella diagnostic kits. The genome of this strain was sequenced and the length of the genome was 3,253,175 bp, with 57.2% G+C content. A total of 3,365 protein coding genes and 53 RNA genes were predicted.

  7. Abortion caused by Brucella abortus biovar 1 in a free-ranging bison (Bison bison) from Yellowstone National Park.

    PubMed

    Rhyan, J C; Quinn, W J; Stackhouse, L S; Henderson, J J; Ewalt, D R; Payeur, J B; Johnson, M; Meagher, M

    1994-07-01

    A near-term aborted bison (Bison bison) fetus was collected near Old Faithful geyser in Yellowstone National Park, Wyoming (USA). On necropsy, the fetus liver had a small capsular tear, and there was a small quantity of blood in the peritoneal cavity. Microscopic lesions included mild, purulent bronchopneumonia and mild, multifocal, interstitial pneumonia. Brucella abortus biovar 1 was isolated from fetal abomasal contents, lung, and heart blood.

  8. Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response.

    PubMed

    Pollak, Cora N; Delpino, M Victoria; Fossati, Carlos A; Baldi, Pablo C

    2012-01-01

    Outer membrane vesicles (OMVs) released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa) and monocytes (THP-1), and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8) to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively). Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

  9. Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

    PubMed Central

    Zaitseva, M B; Golding, H; Betts, M; Yamauchi, A; Bloom, E T; Butler, L E; Stevan, L; Golding, B

    1995-01-01

    Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells. PMID:7790090

  10. Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

    PubMed Central

    Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

    2001-01-01

    Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

  11. The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice.

    PubMed

    Gee, Jason M; Valderas, Michelle Wright; Kovach, Michael E; Grippe, Vanessa K; Robertson, Gregory T; Ng, Wai-Leung; Richardson, John M; Winkler, Malcolm E; Roop, R Martin

    2005-05-01

    Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.

  12. A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

    PubMed

    Wang, Jing-Yu; Wu, Ning; Liu, Wan-Hua; Ren, Juan-Juan; Tang, Pan; Qiu, Yuan-Hao; Wang, Chi-Young; Chang, Ching-Dong; Liu, Hung-Jen

    2014-01-01

    The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.

  13. Circulating Strains of Brucella abortus in Cattle in Santo Domingo De Los Tsáchilas Province – Ecuador

    PubMed Central

    Rodríguez-Hidalgo, Richar Ivan; Contreras-Zamora, Javier; Benitez Ortiz, Washington; Guerrero-Viracocha, Karina; Salcan-Guaman, Holger; Minda, Elizabeth; Ron Garrido, Lenin

    2015-01-01

    The Province of Santo Domingo de los Tsáchilas in Ecuador represents the largest informal cattle market. Because of its strategic position, cattle movement is very high and therefore we selected this region, to determine the strain variation of Brucella sp. Part of the study aimed at the isolation, biotyping, and genotyping of Brucella species from milk and supra-mammary lymph nodes of sero-positive bovines, using selective Farrell medium, biochemical assays, and IS711-PCR, AMOS-PCR, and HOOF-Prints techniques. In total, 656 animals from 12 sero-positive dairy herds and from the provincial slaughterhouse were diagnosed by Rose Bengal and Wright’s Slow Agglutination test with EDTA. Amongst these animals, 50 animals were sero-positive for brucellosis. Twenty-five lymph nodes and 25 milk samples from each group of positive reactors were transferred to culture medium. Isolation was possible from 4 (16%) lymph nodes and 9 (36%) milk samples; out of these, 10 isolates were diagnosed as Brucella sp. All four isolates of lymphatic tissue corresponded to Brucella abortus biotype 1, confirmed as field strains by molecular analysis. Milk isolations, showed biochemically a more dispersed pattern in which B. abortus biotypes 1 and 4 were found; yet four samples gave a pattern similar to B. abortus biotype 2; however, only biotypes 1 and 4 were confirmed by molecular analysis. The concordance between biochemical and molecular diagnostic tests reached 76.9%. PMID:25806363

  14. Detection of antibodies against Brucella abortus, Leptospira spp., and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina.

    PubMed

    Konrad, José L; Campero, Lucía M; Caspe, Gastón S; Brihuega, Bibiana; Draghi, Graciela; Moore, Dadin P; Crudeli, Gustavo A; Venturini, María C; Campero, Carlos M

    2013-11-01

    Water buffalo industry has become a profitable activity worldwide, including the Northeast of Argentina (NEA). However, research on diseases affecting this species is scarce. The aim of the present study was to detect antibodies against Brucella abortus, Leptospira spp., Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in 500 water buffalo cows from five ranches (100 animals each) in the NEA. Serum samples were tested for B. abortus by fluorescence polarization assay, Leptospira spp. by microagglutination test, and N. caninum, T. gondii, and Sarcocystis spp. by indirect fluorescent antibody tests. Overall, the proportion of seropositive animals was 6.4, 22.2, 42.2, 25.4, and 50.8 % for brucellosis, leptospirosis, neosporosis, toxoplasmosis, and sarcocystosis, respectively. The proportion of seropositive animals for all diseases was statistically different among herds (p < 0.05). Statistical differences were also detected among age groups for brucellosis and neosporosis (p < 0.05). The detection of specific antibodies to B. abortus, Leptospira spp., and several Apicomplexa protozoans in water buffaloes in the NEA is reported in this study.

  15. Brucella abortus Ornithine Lipids Are Dispensable Outer Membrane Components Devoid of a Marked Pathogen-Associated Molecular Pattern

    PubMed Central

    Palacios-Chaves, Leyre; Conde-Álvarez, Raquel; Gil-Ramírez, Yolanda; Zúñiga-Ripa, Amaia; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Arce-Gorvel, Vilma; Gorvel, Jean-Pierre; Moreno, Edgardo; de Miguel, María-Jesús; Grilló, María-Jesús

    2011-01-01

    The brucellae are α-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-α release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity. PMID:21249206

  16. Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil

    PubMed Central

    Barbosa Pauletti, Rebeca; Reinato Stynen, Ana Paula; Pinto da Silva Mol, Juliana; Seles Dorneles, Elaine Maria; Alves, Telma Maria; de Sousa Moura Souto, Monalisa; Minharro, Silvia; Heinemann, Marcos Bryan; Lage, Andrey Pereira

    2015-01-01

    This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains. PMID:26181775

  17. A potent Brucella abortus 2308 Δery live vaccine allows for the differentiation between natural and vaccinated infection.

    PubMed

    Zhang, Junbo; Yin, Shuanghong; Guo, Fei; Meng, Ren; Chen, Chuangfu; Zhang, Hui; Li, Zhiqiang; Fu, Qiang; Shi, Huijun; Hu, Shengwei; Ni, Wei; Li, Tiansen; Zhang, Ke

    2014-08-01

    Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. However, the current Brucella abortus vaccines (S19 and RB51) are deficient; they can cause abortion in pregnant animals. Moreover, when the vaccine S19 is used, tests cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent vaccine is needed. A Brucella abortus 2308 ery promoter mutant (Δery) was constructed to overcome these drawbacks. The growth of the Δery mutant was significantly attenuated in macrophages and mice and induced high protective immunity in mice. Moreover, Δery induced an anti-Brucella-specific IgG (immunoglobulin G) response and stimulated the expression of interferon-gamma (INF-γ) and interleukin-4 (IL-4). Furthermore, the expression of EryA antigen allowed for the serological differentiation between natural and vaccinated infection in mice. These results indicate that the Δery mutant is a potential attenuated live vaccine candidate against virulent Brucella abortus 2308 (S2308) infection.

  18. Circulating Strains of Brucella abortus in Cattle in Santo Domingo De Los Tsáchilas Province - Ecuador.

    PubMed

    Rodríguez-Hidalgo, Richar Ivan; Contreras-Zamora, Javier; Benitez Ortiz, Washington; Guerrero-Viracocha, Karina; Salcan-Guaman, Holger; Minda, Elizabeth; Ron Garrido, Lenin

    2015-01-01

    The Province of Santo Domingo de los Tsáchilas in Ecuador represents the largest informal cattle market. Because of its strategic position, cattle movement is very high and therefore we selected this region, to determine the strain variation of Brucella sp. Part of the study aimed at the isolation, biotyping, and genotyping of Brucella species from milk and supra-mammary lymph nodes of sero-positive bovines, using selective Farrell medium, biochemical assays, and IS711-PCR, AMOS-PCR, and HOOF-Prints techniques. In total, 656 animals from 12 sero-positive dairy herds and from the provincial slaughterhouse were diagnosed by Rose Bengal and Wright's Slow Agglutination test with EDTA. Amongst these animals, 50 animals were sero-positive for brucellosis. Twenty-five lymph nodes and 25 milk samples from each group of positive reactors were transferred to culture medium. Isolation was possible from 4 (16%) lymph nodes and 9 (36%) milk samples; out of these, 10 isolates were diagnosed as Brucella sp. All four isolates of lymphatic tissue corresponded to Brucella abortus biotype 1, confirmed as field strains by molecular analysis. Milk isolations, showed biochemically a more dispersed pattern in which B. abortus biotypes 1 and 4 were found; yet four samples gave a pattern similar to B. abortus biotype 2; however, only biotypes 1 and 4 were confirmed by molecular analysis. The concordance between biochemical and molecular diagnostic tests reached 76.9%.

  19. The ferrous iron transporter FtrABCD is required for the virulence of Brucella abortus 2308 in mice.

    PubMed

    Elhassanny, Ahmed E M; Anderson, Eric S; Menscher, Evan A; Roop, R Martin

    2013-06-01

    Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host.

  20. Mutation of purD and purF genes further attenuates Brucella abortus strain RB51.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Watarai, Masahisa; Hahn, Tae-Wook

    2015-02-01

    In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus.

  1. Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil.

    PubMed

    Barbosa Pauletti, Rebeca; Reinato Stynen, Ana Paula; Pinto da Silva Mol, Juliana; Seles Dorneles, Elaine Maria; Alves, Telma Maria; de Sousa Moura Souto, Monalisa; Minharro, Silvia; Heinemann, Marcos Bryan; Lage, Andrey Pereira

    2015-01-01

    This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains.

  2. An influenza viral vector Brucella abortus vaccine induces good cross-protection against Brucella melitensis infection in pregnant heifers.

    PubMed

    Tabynov, Kaissar; Ryskeldinova, Sholpan; Sansyzbay, Abylai

    2015-07-17

    Brucella melitensis can be transmitted and cause disease in cattle herds as a result of inadequate management of mixed livestock farms. Ideally, vaccines against Brucella abortus for cattle should also provide cross-protection against B. melitensis. Previously we created a novel influenza viral vector B. abortus (Flu-BA) vaccine expressing the Brucella ribosomal proteins L7/L12 or Omp16. This study demonstrated Flu-BA vaccine with adjuvant Montanide Gel01 provided 100% protection against abortion in vaccinated pregnant heifers and good cross-protection of the heifers and their calves or fetuses (90-100%) after challenge with B. melitensis 16M; the level of protection provided by Flu-BA was comparable to the commercial vaccine B. abortus S19. In terms of the index of infection and colonization of Brucella in tissues, both vaccines demonstrated significant (P=0.02 to P<0.0001) protection against B. melitensis 16M infection compared to the negative control group (PBS+Montanide Gel01). Thus, we conclude the Flu-BA vaccine provides cross-protection against B. melitensis infection in pregnant heifers.

  3. Profiling antibody responses to infections by Chlamydia abortus enables identification of potential virulence factors and candidates for serodiagnosis.

    PubMed

    Forsbach-Birk, Vera; Foddis, Corinna; Simnacher, Ulrike; Wilkat, Max; Longbottom, David; Walder, Gernot; Benesch, Christiane; Ganter, Martin; Sachse, Konrad; Essig, Andreas

    2013-01-01

    Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the "macrophage infectivity potentiator", MIP), CAB167 (homologue of the "translocated actin recruitment protein", TARP), CAB712 (homologue of the "chlamydial protease-like activity factor", CPAF), CAB776 (homologue of the "Polymorphic membrane protein D", PmpD), and the "hypothetical proteins" CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus.

  4. Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity.

    PubMed

    Sáez, Darwin; Fernández, Pablo; Rivera, Alejandra; Andrews, Edilia; Oñate, Angel

    2012-02-08

    Brucella infections mainly occur through mucosal surfaces. Thus, the development of mucosal administered vaccines could be instrumental for the control of brucellosis. Here, we evaluated the usefulness of recombinant Lactococcus lactis secreting Brucella abortus Cu-Zn superoxide dismutase (SOD) as oral antigen delivery system, when administered alone or in combination with L. lactis expressing IL-12. To this end, mice were vaccinated by oral route with L. lactis NZ9000 transformed with pSEC derivatives encoding for SOD (pSEC:SOD) and IL-12 (pSEC:scIL-12). In animals receiving L. lactis pSEC:SOD alone, anti-SOD-specific IgM antibodies were detected in sera at day 28 post-vaccination, together with an IgG2a dominated IgG response. SOD-specific sIgA was also detected in nasal and bronchoalveolar lavages. In addition, T-cell-proliferative responses upon re-stimulation with either recombinant SOD or crude Brucella protein extracts were observed up to 6 months after the last boost, suggesting the induction of long term memory. Vaccinated animals were also protected against challenge with the virulent B. abortus 2308 strain. Responses were mildly improved when L. lactis pSEC:SOD was co-administered with L. lactis pSEC:scIL-12. These results indicated that vaccines based on lactococci-derived live carriers are promising interventions against B. abortus infections.

  5. Immunogenicity and protective efficacy of Brucella abortus recombinant protein cocktail (rOmp19+rP39) against B. abortus 544 and B. melitensis 16M infection in murine model.

    PubMed

    Tadepalli, Ganesh; Singh, Amit Kumar; Balakrishna, Konduru; Murali, Harishchandra Sripathy; Batra, Harsh Vardhan

    2016-03-01

    In this study, the immunogenicity and protective efficacy of recombinant proteins Omp19 (rO) and P39 (rP) from Brucella abortus were evaluated individually and compared with the cocktail protein (rO+rP) against B. abortus 544 and Brucella melitensis 16M infection in BALB/c mouse model. Intra-peritoneal (I.P.) immunization with rO+rP cocktail developed substantially higher antibody titers predominant with Th1 mediated isotypes (IgG2a/2b). Western blot analysis using anti-rO+rP antibodies showed specific reactivity with native Omp19 (19 kDa) and P39 (39 kDa) among whole cell proteins of B. abortus and B. melitensis. Splenocytes extracted from rO+rP immunized mice induced significantly (P<0.001) higher proliferative responses at 30 μg/ml with considerable expression of pro-inflammatory cytokines (IFN-γ, IL-2 and IL-12) than rO and rP. Macrophage cell (RAW 264.7) monolayer supplemented with anti-rO+rP polysera exhibited enhanced viability against challenge with B. abortus 544 (72.27%) and B. melitensis 16M (68.57%). On the other hand, individual anti-rO and anti-rP polysera resulted in relatively lesser protection against the pathogens (64.79%, 54.45% and 47.13%, 45.11%, respectively). Immunized group of mice when I.P. challenged with 5 × 10(4) CFU of B. abortus 544 and B. melitensis 16M were found significantly (P<0.001) protected in the rO+rP group (log units of protection, spleen: 2.38, 2.12; liver: 1.04, 0.81, respectively) than in rO (spleen: 1.43, 1.21; liver: 0.7, 0.47) and rP (spleen: 1.24, 1.17; liver: 0.65, 0.34). Findings from this study depicted that rO+rP cocktail is highly immunogenic with the Th1 predominant serum antibody titers and T-cell mediated immune protection, would be a valuable intervention in the development of a safer and improved Brucella vaccine.

  6. Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection

    PubMed Central

    2014-01-01

    Background We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. Methods and Results Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was

  7. Immunotoxic effect of thiamethoxam in immunized mice with Brucella abortus cultural filtrate antigen

    PubMed Central

    Salema, L. H.; Alwan, M. J.; Yousif, Afaf Abdulrahman

    2016-01-01

    Aim: This study was planned for determination the toxic effect of thiamethoxam (TMX) in immunized mice with Brucella abortus culture filtrate antigen (CFBAgs) (as a vaccine) and its role of TMX on decrease activity of B. abortus antigen on eliciting of humoral and cellular immunity. Materials and Methods: To achieve these goals 60 female mice were used, 7-8 weeks age, they were divided equally into three groups (20 in each group) and treated as follows: 1st group: Mice were immunized with CFBAgs intraperitoneally in two doses, 2 weeks intervals with (protein concentration 2 mg\\ml), 2nd group: Mice immunized as in the 1st group and was administrated orally with 1/10 lethal dose 50% of TMX (83.7 mg/kg B.W.) for 4 weeks daily, 3rd group was administrated orally with 0.3 ml normal saline served as a control group. At day 28 post immunization (PI) delayed type hypersensitivity (skin test) was done, and serum samples were collected at day 30 (PI) for detection of passive hemagglutination test (PHA); interferon gamma (IFN-γ) which was done by enzyme-linked immunosorbent assay test in addition to phagocytes assay. Results: The results of skin test post injection with soluble antigen of B. abortus intradermally showed a high significantly mean values at p≤0.05 of footpad skin thickness in the 1st group of mice which recorded (0.51±0.002 mm) as compared with the 2nd group of mice which showed (0.08±0.002 mm) after 24 h; the mean values of skin thickness were declined in the 1st mice (0.46±0.002) and 2nd mice (0.070±0.001) at 48 h; control group showed a negative results. These results were agreed with results of serum levels of IFN-γ (pg/ml) that showed that a significant increase the vaccinated 1st group (406.36±1.52), than those values in the 2nd group (151.61±0.89) and negative result in 3rd group (46.47±0.60), in addition to results of PHA test which showed a significant increase in antibody titer in the 1st group (139±12.16) with low level of serum antibody

  8. Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: evaluation of protection in pregnant heifers.

    PubMed

    Tabynov, Kaissar; Yespembetov, Bolat; Sansyzbay, Abylai

    2014-10-14

    The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS+Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51.

  9. Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

    PubMed

    Hagemann, Jürgen Benjamin; Simnacher, Ulrike; Longbottom, David; Livingstone, Morag; Maile, Julia; Soutschek, Erwin; Walder, Gernot; Boden, Katharina; Sachse, Konrad; Essig, Andreas

    2016-07-01

    The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

  10. Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay

    PubMed Central

    Simnacher, Ulrike; Longbottom, David; Livingstone, Morag; Maile, Julia; Soutschek, Erwin; Walder, Gernot; Boden, Katharina; Sachse, Konrad; Essig, Andreas

    2016-01-01

    The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C. abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C. abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C. abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C. abortus infections. PMID:27194684

  11. Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

    PubMed

    Santiago-Rodríguez, María Del Rosario; Díaz-Aparicio, Efrén; Arellano-Reynoso, Beatriz; García-Lobo, Juan M; Gimeno, Miquel; Palomares-Reséndiz, Erika G; Hernández-Castro, Rigoberto

    2015-02-01

    The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×10⁸ colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×10⁸ CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

  12. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus.

    PubMed Central

    Ficht, T A; Bearden, S W; Sowa, B A; Adams, L G

    1989-01-01

    The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When

  13. Immune responses of bison and efficacy after booster vaccination with Brucella abortus strain RB51.

    PubMed

    Olsen, S C; McGill, J L; Sacco, R E; Hennager, S G

    2015-04-01

    Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 10(10) CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1β (IL-1β) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 10(7) CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against

  14. DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

    PubMed Central

    ONUMA, Selma Samiko Miyazaki; KANTEK, Daniel Luis Zanella; CRAWSHAW, Peter Gransden; MORATO, Ronaldo Gonçalves; MAY-JÚNIOR, Joares Adenilson; de MORAIS, Zenaide Maria; FERREIRA, José Soares; de AGUIAR, Daniel Moura

    2015-01-01

     This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection. PMID:25923900

  15. Detection of Leptospira spp. and Brucella abortus antibodies in free-living jaguars (Panthera onca) in two protected areas of northern Pantanal, Brazil.

    PubMed

    Onuma, Selma Samiko Miyazaki; Kantek, Daniel Luis Zanella; Crawshaw Júnior, Peter Gransden; Morato, Ronaldo Gonçalves; May-Júnior, Joares Adenilson; Morais, Zenaide Maria de; Ferreira Neto, José Soares; Aguiar, Daniel Moura de

    2015-01-01

    This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

  16. Effectiveness of Brucella abortus Strain 19 single calfhood vaccination in elk (Cervus elaphus)

    USGS Publications Warehouse

    Roffe, Thomas J.; Jones, Lee C.; Coffin, Kenneth; Sweeney, Steven J.; Williams, Beth; Quist, Charlotte

    2002-01-01

    Brucellosis in Greater Yellowstone Area (GYA) bison and elk has been a source of controversy and focus of the Greater Yellowstone Interagency Brucellosis Committee (GYIBC) for years. Brucellosis has been eradicated from cattle in the 3 states of Wyoming, Montana, and Idaho and all three states currently are classified as “brucellosis free” with regard to livestock. Yet free-ranging elk that attend feedgrounds in the GYA, and bison in Yellowstone and Grand Teton National Parks, still have high seroprevalence to the disease and are viewed as a threat to the state-federal cooperative national brucellosis eradication program. Recently, cattle in eastern Idaho were found infected with brucellosis and transmission was apparently from fed elk. The GYIBC, formed of state and federal agencies involved in wildlife and livestock management in the 3 states, has committed to eventual elimination of the disease from wildlife. Management tools to control or eliminate the disease are limited; however, wildlife vaccination is one of the methods currently employed. Effective wildlife vaccination depends on dose efficacy, deliverability, and safety to non-targeted species. We commenced a single-dose efficacy study of vaccine Brucella abortus strain 19 (S19) in elk in 1999.

  17. Immune responses of elk to vaccination with Brucella abortus strain RB51.

    PubMed

    Olsen, Steven C; Kreeger, Terry J; Palmer, Mitchell V

    2002-10-01

    In a study conducted from January to August 2000, elk (Cervus elaphus) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6) or injected with 0.15 M NaCl solution (n = 3) at approximately 6 mo of age. Beginning at 2 wk and continuing to 25 wk after vaccination, SRB51-vaccinated elk had greater antibody responses (P < 0.05) to SRB51 when compared to nonvaccinated elk. Peripheral blood mononuclear cells (PBMC) from SRB51-vaccinated elk had greater (P < 0.05) proliferative responses to SRB51 at 18 wk after vaccination when compared to responses of nonvaccinated elk. Strain RB51 was recovered from blood samples of all vaccinates at 2 wk, and three of six vaccinates at 4 wk after vaccination. The SRB51 vaccine strain was recovered from the superficial cervical lymph node of all vaccinates sampled at 6 wk after vaccination. but not from lymph node samples obtained from vaccinates at 12 or 18 wk after vaccination. At 34 wk after vaccination, SRB51 was recovered from the bronchial lymph node of one of five vaccinates but not from other tissues. Strain RB51 was not recovered at any time from samples obtained from nonvaccinated elk. This study suggests that following vaccination with SRB51, elk remain bacteremic for a prolonged period of time, rapidly develop high antibody titers, and are slower to develop detectable proliferative responses in PBMC when compared to responses of cattle or bison (Bison bison).

  18. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    PubMed Central

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

  19. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    PubMed

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  20. Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle.

    PubMed Central

    Denoel, P A; Vo, T K; Tibor, A; Weynants, V E; Trunde, J M; Dubray, G; Limet, J N; Letesson, J J

    1997-01-01

    Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39. PMID:9009303

  1. A proficiency testing method for detecting antibodies against Brucella abortus in quantitative and qualitative serological tests.

    PubMed

    Gall, D; Nielsen, K; Nicola, A; Renteria, T

    2008-12-01

    A proficiency testing panel for detecting antibodies against Brucella abortus was developed and evaluated by both primary binding and conventional serological tests, using the guidelines of the World Organisation for Animal Health and the International Organization for Standardization Guide 43-1. All serological tests were judged satisfactory. Among the primary binding tests, the competitive enzyme-linked immunosorbent assay (ELISA 2) and the indirect enzyme-linked immunosorbent assay (ELISA 1), with standard deviation indices (z-scores) of -0.06 and 0.10, respectively, performed best. Similarly, E(n) numbers (i.e. a way of comparing different measurements of performance) of 0 for both the competitive ELISA 2 and the indirect ELISA 1 indicated that these tests performed best in the initial round of proficiency testing. The conventional serological tests all passed the panel. Comparing data from both the quantitative and qualitative tests demonstrated that this proficiency testing scheme was fit for the purpose for which it was designed.

  2. Neutrophils Exert a Suppressive Effect on Th1 Responses to Intracellular Pathogen Brucella abortus

    PubMed Central

    Ordoñez-Rueda, Diana; Arce-Gorvel, Vilma; Alfaro-Alarcón, Alejandro; Lepidi, Hubert; Malissen, Bernard; Malissen, Marie; Gorvel, Jean-Pierre; Moreno, Edgardo

    2013-01-01

    Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity. PMID:23458832

  3. Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats.

    PubMed

    Leal-Klevezas, D S; Martínez-Vázquez, I O; García-Cantú, J; López-Merino, A; Martínez-Soriano, J P

    2000-07-03

    The polymerase chain reaction (PCR) was used to diagnose goat brucellosis and compare its sensitivity against some of the most commonly used serological and bacteriological techniques. Twenty two female and one male out of 300 clinically healthy, mixed-breed goats were randomly chosen from a ranch located at Marín, Nuevo León, Mexico. Milk and blood samples were taken from each animal and used to obtain both microbiological cultures and DNA of the pathogen, and sera was tested against Rose Bengal antigen (RBT). Results showed that 86% of the blood samples were positive on the PCR test, while 60% were positive on the serological test. The pathogen was isolated from only one blood culture. Sixty four percent of the milk samples were positive on PCR tests, but failed to yield bacteria in culture. Biochemical and PCR specific assay demonstrated that Brucella abortus biovar 1 was associated with the infection. This study demonstrates the higher sensitivity of PCR over RBT and blood culture and its potential towards a rapid identification of Brucella strains.

  4. Mouse running activity is lowered by Brucella abortus treatment: a potential model to study chronic fatigue.

    PubMed

    Ottenweller, J E; Natelson, B H; Gause, W C; Carroll, K K; Beldowicz, D; Zhou, X D; LaManca, J J

    1998-03-01

    Chronic fatigue syndrome, which can occur after acute infection and last for years, is characterized by severe and persistent fatigue. Others have reported decreases in mouse running activity following infection and have suggested this may provide an animal model for studying chronic fatigue. Voluntary running is a highly motivated activity in mice, which will often run 5-7 mi/day in our laboratory. Following 2 weeks of acclimation to running wheels with food and water available ad lib, female BALB/c mice received 0.2-mL tail vein injections of killed Brucella abortus (BA) or saline vehicle. Subsequently the effects on voluntary running and grooming behavior were determined. Injection of BA caused an immediate large decrease in running and a lack of grooming. Vehicle injections produced no changes in behavior. After the first several days of reduced running behavior, levels of running and grooming slowly returned back to normal over the next 2-4 weeks, with substantial individual differences in the rate of recovery. The pattern of running during recovery was intriguing in that BA mice first ran at normal levels just after the lights went out, but they stopped after only 1-2 h. As recovery proceeded, they gradually increased the duration of the running bout during the night. Because this model uses voluntary exertion and the ability to run for longer periods of time characterizes recovery, the model may be a good one for studying the biologic underpinnings of chronic fatigue.

  5. Neutrophils exert a suppressive effect on Th1 responses to intracellular pathogen Brucella abortus.

    PubMed

    Barquero-Calvo, Elías; Martirosyan, Anna; Ordoñez-Rueda, Diana; Arce-Gorvel, Vilma; Alfaro-Alarcón, Alejandro; Lepidi, Hubert; Malissen, Bernard; Malissen, Marie; Gorvel, Jean-Pierre; Moreno, Edgardo

    2013-02-01

    Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.

  6. Improvement of the Brucella abortus B19 vaccine by its preparation in a glycerol based medium.

    PubMed

    Sangari, F J; Agüero, J; García-Lobo, J M

    1996-03-01

    The Brucella abortus B19 vaccine strain differs from other Brucella strains in its sensitivity to erythritol. However, erythritol tolerant (Eri(t)) mutants arise from sensitive cultures of B19 at high rate, and may cause persistence and/or abortion when the vaccine is inoculated on adult cattle. Twelve different batches of B19 have been examined for the presence of Eri(t) mutants. All contained Eri(t) variants at a proportion ranging from 10(-4) to 10(-6). In order to eliminate these mutants from the vaccine cultures, we have developed a minimal medium with glycerol as the sole carbon source, named MMG30. Growth of the parental strain B19 (erythritol sensitive) in this medium was fairly good compared with the growth of its Eri(t) derivatives. Culture of the 12 different batches of B19 in liquid MMG30 produced up to a thousandfold decrease in the proportion of Eri(t) mutants present in the vaccine cultures. Use of this medium to grow B19 could represent an easy and considerable improvement of the vaccine, by the reduction of the presence of potentially dangerous Eri(t) mutants.

  7. Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

    PubMed

    Opota, Onya; Jaton, Katia; Branley, James; Vanrompay, Daisy; Erard, Veronique; Borel, Nicole; Longbottom, David; Greub, Gilbert

    2015-10-01

    Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml(- 1)). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

  8. Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model.

    PubMed

    Pan, Qing; Pais, Roshan; Ohandjo, Adaugo; He, Cheng; He, Qing; Omosun, Yusuf; Igietseme, J U; Eko, F O

    2015-04-08

    Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826+FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG+FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1β and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ, IgA and IgG2c antibody responses compared to CpG+FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG+FL adjuvants.

  9. Comparison of Buffered, Acidified Plate Antigen to Standard Serologic Tests for the Detection of Serum Antibodies to Brucella abortus in Elk (Cervus canadensis).

    PubMed

    Clarke, P Ryan; Edwards, William H; Hennager, Steven G; Block, Jean F; Yates, Angela M; Ebel, Eric; Knopp, Douglas J; Fuentes-Sanchez, Antonio; Jennings-Gaines, Jessica; Kientz, Rebecca L; Simunich, Marilyn

    2015-07-01

    Brucellosis (caused by the bacterium Brucella abortus) is a zoonotic disease endemic in wild elk (Cervus canadensis) of the Greater Yellowstone Ecosystem, US. Because livestock and humans working with elk or livestock are at risk, validated tests to detect the B. abortus antibody in elk are needed. Using the κ-statistic, we evaluated the buffered, acidified plate antigen (BAPA) assay for agreement with the results of the four serologic tests (card test [card], complement fixation test [CF], rivanol precipitation plate agglutination test [RIV], standard plate agglutination test [SPT]) that are approved by the US Department of Agriculture for the detection of the B. abortus antibody in elk. From 2006 to 2010, serum samples collected from elk within B. abortus-endemic areas (n = 604) and nonendemic areas (n = 707) and from elk culture-positive for B. abortus (n = 36) were split and blind tested by four elk serum diagnostic laboratories. κ-Values showed a high degree of agreement for the card (0.876), RIV (0.84), and CF (0.774) test pairings and moderate agreement for the SPT (0.578). Sensitivities for the BAPA, card, RIV, CF, and SPT were 0.859, 0.839, 0.899, 1.00, and 0.813, whereas specificities were 0.986, 0.993, 0.986, 0.98, and 0.968, respectively. The positive predictive values and the negative predictive values were calculated for 2.6%, 8.8%, and 16.2% prevalence levels. These findings suggest the BAPA test is a suitable screening test for the B. abortus antibodies in elk.

  10. Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model

    PubMed Central

    Pan, Qing; Pais, Roshan; Ohandjo, Adaugo; He, Cheng; He, Qing; Omosun, Yusuf; Igietseme, J. U.; Eko, F. O.

    2015-01-01

    Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826+FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG+FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1β and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ IgA and IgG2c antibody responses compared to CpG+FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG+FL adjuvants. PMID:25698486

  11. Effectiveness of natural and synthetic complexes of porin and O polysaccharide as vaccines against Brucella abortus in mice.

    PubMed Central

    Winter, A J; Rowe, G E; Duncan, J R; Eis, M J; Widom, J; Ganem, B; Morein, B

    1988-01-01

    A single vaccination of mice with a complex of porin and smooth lipopolysaccharide (porin-S-LPS) extracted from virulent Brucella abortus 2308 provided significant protection (P less than 0.01 to P less than 0.001) against challenge with the same strain, equivalent to that achieved by vaccination with living attenuated B. abortus 19. The porin-S-LPS vaccine given without adjuvant or in several adjuvants (trehalose dimycolate and muramyl dipeptide; the pluronic polymer L-121 and muramyl dipeptide; or complexed with Quil A in immunostimulating complexes) provided equivalent protection. In contrast, one vaccination with porin complexed with rough LPS (porin-R-LPS) from a rough mutant of strain 2308 provided no protection with any adjuvant tested. In one experiment, two inoculations with the porin-R-LPS resulted in a low level of protection, probably owing to priming of the animals for production of O-polysaccharide-specific antibodies. However, one vaccination with rough-strain porin covalently bound to purified O polysaccharide conferred protection equal to that obtained with natural complexes of porin-S-LPS or with living strain 19. A synthetic vaccine containing long chains of O polysaccharide was more effective than one prepared with short chains. Protective vaccines caused the formation of increased concentrations of circulating O-polysaccharide-specific antibodies, although there were individual exceptions to the quantitative association between O-polysaccharide-specific antibodies and protection. Antibodies specific for porin or R-LPS were found in negligible quantities in vaccinated mice. These results provide additional evidence that the O polysaccharide will constitute an essential component of an effective subcellular vaccine against B. abortus and that O-polysaccharide-specific antibodies play an important role in protective immunity in brucellosis. PMID:2844673

  12. Variables Associated with Infections of Cattle by Brucella abortus., Leptospira spp. and Neospora spp. in Amazon Region in Brazil.

    PubMed

    Chiebao, D P; Valadas, S Y O B; Minervino, A H H; Castro, V; Romaldini, A H C N; Calhau, A S; De Souza, R A B; Gennari, S M; Keid, L B; Soares, R M

    2015-10-01

    The frequency of Neospora spp., Leptospira spp. and Brucella abortus infections in adult cattle was determined in herds of the State of Pará, Brazil, which is an important region for cattle production located in the Amazon region. A total of 3466 adult female cattle from 176 herds were tested, leading to a frequency of seropositive animals of 14.7%, 3.7% and 65.5% and a herd positivity of 87.4%, 41.3% and 98.8% for infections caused by Neospora spp., B. abortus and Leptospira spp., respectively. The five most frequently diagnosed serologic responses to Leptospira spp. were those against serovars hardjo, wolfii, grippotyphosa, hebdomadis and shermani. The following associations were found: practice of artificial insemination, large farm size, large herd size, large number of dogs and high number of total abortions per year with the presence of antibodies against serovar hardjo; positive results to serovar grippotyphosa with the presence of dogs; inappropriate disposal of aborted foetuses with positivity to serovar hebdomadis. Serovar grippotyphosa was also associated with number of episodes of abortions. Neospora spp. positive herds were associated with episodes of abortion and B. abortus infection with the disposal of dead animals and aborted foetuses on pastures and with the use of artificial insemination. In conclusion, the high frequency of brucellosis, leptospirosis and neosporosis in the region may be a consequence of social, natural and raising conditions as: (i) climate conditions that favour the survival and spread of pathogens in the environment; (ii) farms located in regions bordering forest areas; (iii) farms in areas of difficult access to the veterinary service; (iv) extensive beef herds raised at pastures with different age and productive groups inter-mingled; and (v) minimal concerns regarding hygiene practices and disease prevention measures.

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

    PubMed

    Kianmehr, Zahra; Soleimanjahi, Hoorieh; Ardestani, Susan Kaboudanian; Fotouhi, Fatemeh; Abdoli, Asghar

    2015-04-01

    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.

  14. Profiling Antibody Responses to Infections by Chlamydia abortus Enables Identification of Potential Virulence Factors and Candidates for Serodiagnosis

    PubMed Central

    Forsbach-Birk, Vera; Foddis, Corinna; Simnacher, Ulrike; Wilkat, Max; Longbottom, David; Walder, Gernot; Benesch, Christiane; Ganter, Martin; Sachse, Konrad; Essig, Andreas

    2013-01-01

    Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the “macrophage infectivity potentiator”, MIP), CAB167 (homologue of the “translocated actin recruitment protein”, TARP), CAB712 (homologue of the “chlamydial protease-like activity factor”, CPAF), CAB776 (homologue of the “Polymorphic membrane protein D”, PmpD), and the “hypothetical proteins” CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus. PMID:24260366

  15. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide.

    PubMed

    Dabral, Neha; Jain-Gupta, Neeta; Seleem, Mohamed N; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2015-01-01

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

  16. Survey of Omp19 immunogenicity against Brucella abortus and Brucella melitensis: influence of nanoparticulation versus traditional immunization.

    PubMed

    Abkar, Morteza; Lotfi, Abbas Sahebghadam; Amani, Jafar; Eskandari, Khadijeh; Ramandi, Mehdi Fasihi; Salimian, Jafar; Brujeni, Gholamreza Nikbakht; Alamian, Saeed; Kamali, Mehdi; Koushki, Hamid

    2015-12-01

    Brucellosis is the most common zoonotic bacterial disease. Prevention of human brucellosis is achieved through pasteurization of dairy products, appropriate sanitation and vaccination of domestic animals against the Brucella species. B. abortus unlipidated 19 kDa outer membrane protein (U-Omp19) is a promising candidate for a subunit vaccine against brucellosis. This study investigates immunogenicity of Omp19 alone and with Freund's adjuvant (Omp19-IFA) and N-trimethyl chitosan (TMC/Omp19) nanoparticles, as well as the effect of Omp19 administration route on immunological responses and protection. The omp19 gene was expressed in E. coli BL21 (DE3). After purification, the recombinant Omp19 was loaded onto TMC nanoparticles by ionic gelation with tripolyphosphate. Particle size and loading efficiency of the nanoparticles were determined. Omp19-IFA was administered intraperitoneally while TMC/Omp19 nanoparticles were administered orally and intraperitoneally. The results indicated that intraperitoneal (i.p.) immunization by Omp19-IFA and TMC/Omp19 nanoparticles induced Th1 and Th2 immune responses, respectively, whereas oral immunization of TMC/Omp19 nanoparticles induced a mixed Th1/Th17 immune response. Moreover, oral immunization increased IgA levels in feces. Immunized mice were challenged with virulent B. melitensis 16 M and B. abortus 544. Oral immunization with TMC/Omp19 nanoparticles induced a remarkably high protection level against B. melitensis and B. abortus. The results showed that immunization route has a pivotal role in immune response polarization and protective efficiency of Omp19 antigen. Also, it was deduced that the higher protection level achieved through oral administration of TMC/Omp19 nanoparticles may be due to the elicited Th17 response.

  17. Brucella abortus ΔrpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis.

    PubMed

    Willett, Jonathan W; Herrou, Julien; Czyż, Daniel M; Cheng, Jason X; Crosson, Sean

    2016-09-30

    The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection.

  18. Biosafety of parenteral Brucella abortus RB51 vaccine in bison calves

    USGS Publications Warehouse

    Roffe, T.J.; Olsen, S.C.; Gidlewski, T.; Jensen, A.E.; Palmer, M.V.; Huber, R.

    1999-01-01

    Vaccination is considered among the primary management tools for reducing brucellosis prevalence in Greater Yellowstone Area (GYA) ungulates. Before their use, however, vaccine safety and efficacy must be demonstrated. Twenty-seven female bison (Bison bison) calves (approx 5 months old) were vaccinated with Brucella abortus Strain RB51 (1.5 x 1010 colony forming units [CFU], subcutaneously) as part of routine management. We assessed the persistence, pathology, shedding, and transmission associated with RB51 by serial necropsy, bacteriology, histopathology, and serology of 20 of these 27 vaccinated calves, and RB51 serology of 10 nonvaccinated, commingling adult females. With the exception of 1 calf, RB51 dot-blot titers at necropsy were <1:80. Strain RB51 was cultured from lymph nodes in 4 of 4 calves at 14 weeks postvaccination (PV), 4 of 4 calves at 18 weeks PV, 1 of 4 calves at 22 weeks PV, 3 of 4 at 26 weeks PV, and 0 of 4 calves at 30 weeks PV. No gross lesions were observed. Mild histologic changes occurred only in a few draining lymph nodes early in sampling. Adverse clinical effects were not observed in vaccinates. Swabs from nasopharynx, conjunctiva, rectum, and vagina were uniformly culture negative for RB51. Strain RB51 dot-blot assays of bison cows were negative at a 1:20 dilution at 26 weeks PV. Our results suggest that RB51 persists longer in bison calves than in domestic cattle and is systemically distributed within lymphatic tissues. However, bison apparently clear the RB51 vaccine strain without shedding, transmission, or significant adverse reactions.

  19. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    SciTech Connect

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  20. Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strains RB51 or RB51 overexpressing superoxide dismutase and Glycosyltransferase genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus stra...

  1. Comparative analysis of the early transcriptome of Brucella abortus - infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis

    PubMed Central

    Rossetti, C.A.; Galindo, C.L.; Everts, R.E.; Lewin, H.A.; Garner, H.R.; Adams, L.G.

    2010-01-01

    Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that 1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, 2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and 3) the host cell activity was not altered after 12h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12h p.i. These results contribute to understand of how host responses may be manipulated to prevent infection by brucellae. PMID:20932540

  2. Phenotypic and genotypic characterization of Brucella strains isolated from autochthonous livestock reveals the dominance of B. abortus biovar 3a in Nigeria.

    PubMed

    Bertu, Wilson J; Ducrotoy, Marie J; Muñoz, Pilar M; Mick, Virginie; Zúñiga-Ripa, Amaia; Bryssinckx, Ward; Kwaga, Jacob K P; Kabir, Junaid; Welburn, Susan C; Moriyón, Ignacio; Ocholi, Reuben A

    2015-10-22

    Brucellosis is a worldwide widespread zoonosis caused by bacteria of the genus Brucella. Control of this disease in a given area requires an understanding of the Brucella species circulating in livestock and humans. However, because of the difficulties intrinsic to Brucella isolation and typing, such data are scarce for resource-poor areas. The paucity of bacteriological data and the consequent imperfect epidemiological picture are particularly critical for Sahelian and Sub-Sahara African countries. Here, we report on the characterization of 34 isolates collected between 1976 and 2012 from cattle, sheep and horses in Nigeria. All isolates were identified as Brucella abortus by Bruce-ladder PCR and assigned to biovar 3 by conventional typing. Further analysis by enhanced AMOS-ERY PCR showed that all of them belonged to the 3a sub-biovar, and MLVA analysis grouped them in a cluster clearly distinct from that formed by European B. abortus biovar 3b strains. Nevertheless, MLVA detected heterogeneity within the Nigerian biovar 3a strains. The close genetic profiles of the isolates from cattle, sheep and horses, suggest that, at least in some parts of Nigeria, biovar 3a circulates among animal species that are not the preferential hosts of B. abortus. Consistent with previous genetic analyses of 7 strains from Ivory Cost, Gambia and Togo, the analysis of these 34 Nigerian strains supports the hypothesis that the B. abortus biovar 3a lineage is dominant in West African countries.

  3. Induction of immune responses by two recombinant proteins of brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model.

    PubMed

    Sung, Kyung Yong; Jung, Myunghwan; Shin, Min-Kyoung; Park, Hyun-Eui; Lee, Jin Ju; Kim, Suk; Yoo, Han Sang

    2014-06-28

    The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

  4. Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice.

    PubMed

    Cabrera, Alex; Sáez, Darwin; Céspedes, Sandra; Andrews, Edilia; Oñate, Angel

    2009-01-01

    Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.

  5. Serological response to administration of Brucella abortus strain RB51 vaccine in beef and dairy heifers, using needle-free and standard needle-based injection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to compare immunologic responses of heifers vaccinated with 10**10 colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51) by standard needle-and-syringe system or a needle-free injection system. Heifers were randomly assigned to control and vaccination gro...

  6. Comparison of abortion and infection after experimental challenge of pregnant bison and cattle with Brucella abortus strain 2308.

    PubMed

    Olsen, S C; Johnson, C

    2011-12-01

    A comparative study was conducted using data from naive bison (n = 45) and cattle (n = 46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered during midgestation. The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (P < 0.05) in bison than in cattle. In animals that did abort, the time between experimental challenge and abortion was shorter (P < 0.05) for bison than for cattle. Brucella colonization of four target tissues and serologic responses on the standard tube agglutination test at the time of abortion did not differ (P > 0.05) between cattle and bison. The results of our study suggest that naive bison and cattle have similarities and differences after experimental exposure to a virulent B. abortus strain. Although our data suggest that bison may be more susceptible to infection with Brucella, some pathogenic characteristics of brucellosis were similar between bison and cattle.

  7. Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

    PubMed

    Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

    2009-07-01

    The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

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

    PubMed

    Zhang, Min; Han, Xiangan; Liu, Haiwen; Tian, Mingxing; Ding, Chan; Song, Jun; Sun, Xiaoqing; Liu, Zongping; Yu, Shengqing

    2013-06-28

    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.

  9. Molecular characterization, occurrence, and immunogenicity in infected sheep and cattle of two minor outer membrane proteins of Brucella abortus.

    PubMed Central

    Tibor, A; Saman, E; de Wergifosse, P; Cloeckaert, A; Limet, J N; Letesson, J J

    1996-01-01

    Screening of a Brucella abortus genomic library with two sets of monoclonal antibodies allowed the isolation of the genes corresponding to two minor outer membrane proteins (OMP10 and OMP19) found in this bacterial species. Sequence analysis of the omp10 gene revealed an open reading frame capable of encoding a protein of 126 amino acids. The nucleotide sequence of the insert producing the OMP19 protein contains two overlapping open reading frames, the largest of which (177 codons) was shown to encode the protein of interest. Analysis of the N-terminal sequences of both putative proteins revealed features of a bacterial signal peptide, and homology to the bacterial lipoprotein processing sequence was also observed. Immunoblotting with monoclonal antibodies specific for OMP10 or OMP19 showed that both proteins are present in the 34 Brucella strains tested, representing all six Brucella species and all their biovars. The OMP19 detected in the five Brucella ovis strains examined migrated at an apparent molecular weight that is slightly higher than those of the other Brucella species, confirming the divergence of B. ovis from these species. OMP10 and OMP19 were produced in recombinant Escherichia coli and purified to homogeneity for serological analysis. A large fraction of sera from sheep naturally infected with Brucella melitensis were reactive with these proteins in an enzyme-linked immunosorbent assay, whereas sera from B. abortus-infected cattle were almost completely unreactive in this assay. PMID:8557326

  10. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

    PubMed

    Kim, Hye-Sook; Willett, Jonathan W; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-11-01

    In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.

  11. Generation and characterization of murine monoclonal antibodies to genus-specific 31-kilodalton recombinant cell surface protein of Brucella abortus.

    PubMed

    Kumar, Sanjay; Tuteja, Urmil; Batra, Harsh Vardhan

    2007-08-01

    In the present study hybridomas were produced from fusion with splenocytes of BALB/c mice immunized with the recombinant 31-kDa cell surface protein (r31CSP) specific for Brucella species. A set of eight stabilized hybridoma cell lines was generated against r31CSP. Monoclonal antibodies (MAbs) produced by all these clones exhibited reactivity for r31CSP as well as with the protein of 31-kDa, derived from whole-cell lysate of 31-kDa Brucella abortus 544. Four of eight MAbs were IgG1, two IgG2b, and two IgM in nature. These MAbs did not show any cross-reaction with whole-cell lysate of Yersinia enterocolitica O: 9, Vibrio cholerae, Salmonella typhimurium and Escherichia coli 0157 by Western blotting. Reactivity of these MAbs was further assessed with other organisms of Brucella species namely, B. abortus S99, B. canis, B. melitensis 16M, B. suis, and a clinical isolate of B. melitensis. Collectively, these data suggest that these MAbs may have the potential for use in the detection of Brucella species with high specificity.

  12. Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

    PubMed

    Polkinghorne, Adam; Vaughan, Lloyd

    2011-01-01

    The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.

  13. Expression of human serum albumin--L7/L12 (Brucella abortus ribosomal protein) fusion protein in Saccharomyces cerevisiae.

    PubMed

    Pakzad, Iraj; Rezaee, Abbas; Emaneini, Mohammad; Hosseini, Ahmad Zavaran; Tabbaraee, Bahman; Taherikalani, Morovat

    2009-01-01

    Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. It has already been expressed in several bacteria and has been used as DNA vaccine. In order to construct yeast expressing vector for the tHSA-L7/L12 fusion protein, the l7/l12 ribosomal gene was amplified by PCR. The expression plasmid pYtHSA-L7/L12 was constructed by inserting the L7/L12 gene into the pYHSA5 shuttle vector (containing inulinase signal sequence, HSA gene and Gal10 promoter). The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The secreted recombinant fusion protein was detected in supernatant by SDS-PAGE and confirmed by western blot analysis using anti-HSA and anti-L7/L12 antibodies. Fusion protein was purified by affinity chromatography and its amount was approximately 500 microg/liter.

  14. Pseudorabies Virus and Brucella abortus from an Expanding Wild Pig ( Sus scrofa ) Population in Southern Oklahoma, USA.

    PubMed

    Gaskamp, Joshua A; Gee, Kenneth L; Campbell, Tyler A; Silvy, Nova J; Webb, Stephen L

    2016-04-28

    Wild pigs ( Sus scrofa ) are causing increasing ecologic and economic damage at a global scale. Because wild pigs can carry ≥65 diseases that affect livestock, their widespread expansion threatens native wildlife and livestock. We screened wild pigs from south-central Oklahoma, US for antibodies against Brucella abortus , pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRS). These pathogens were chosen because they are part of eradication programs in the US and could have large economic impacts on domestic livestock if transmitted from wild animals. We tested 282 serum samples during spring 2010 (n=149) and 2011 (n=133) and found an overall exposure rate to PRV of 24.1% (n=68); PRV was detected at two of three study sites. Two wild pigs had detectable antibody to B. abortus , and one had detectable antibody to PRRS. On average, 27% of wild pigs within a sounder were positive for PRV antibody, with 44% of the sounders (16/36) having at least one positive individual. These data highlight that wild pigs could carry pathogens that affect domestic livestock. Because the US is free of these pathogens in commercial livestock operations, continued surveillance and vaccination of domestic livestock are needed. Commercial livestock producers at the wildlife-livestock interface may benefit from spatial prioritization of risk zones to facilitate strategic control efforts.

  15. Serological Diagnosis of Ovine Enzootic Abortion by Enzyme-Linked Immunosorbent Assay with a Recombinant Protein Fragment of the Polymorphic Outer Membrane Protein POMP90 of Chlamydophila abortus

    PubMed Central

    Longbottom, David; Fairley, Susan; Chapman, Stephanie; Psarrou, Evgenia; Vretou, Evangelia; Livingstone, Morag

    2002-01-01

    Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with Chlamydophila abortus is of major economic importance worldwide. Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep, Chlamydophila pecorum. In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 of C. abortus was assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected with C. abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes of C. pecorum. The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT. Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C. pecorum subtypes had been isolated. ELISAs with both POMP90 fragments outperformed CFT with serum samples from C. pecorum-infected animals, producing no false-positive results. However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples. The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy

  16. Evaluation of immunogenicity and protective efficacy of a liposome containing Brucella abortus S19 outer membrane protein in BALB/c mice

    PubMed Central

    Mukherjee, F.; Prasad, A.; Bahekar, V. S.; Rana, S. K.; Rajendra, L.; Sharma, G. K.; Srinivasan, V. A.

    2016-01-01

    The use of liposome as an adjuvant and a vaccine carrier has been cited previously in the literature. It has also been shown to be effective in enhancing the immunogenicity of vaccine candidates. BALB/c mice immunized subcutaneously with outer membrane protein (OMP) of Brucella abortus S19 vaccine strain entrapped in a commercial cationic liposome (S19-OMP-liposome) for vaccine delivery, showed enhanced protection (P<0.05) compared to groups of mice inoculated with S19 OMP alone, S19 live B. abortus vaccine and liposome alone, when challenged intra-peritoneally with virulent B. abortus strain 544 at 30 days post-immunization (DPI). The S19-OMP-liposome preparation was found to be safer compared to the live B. abortus S19 vaccine at 15 days post challenge (DPC), as evidenced by the significant difference in spleen weight between S19-OMP-liposome, S19 OMP and S19 live as well as the liposome control groups (P<0.01). Antibody isotype response profiles of the experimental groups indicated that the immune response was Th1 cell mediated. The protective advantage conferred to mice immunized with S19-OMP entrapped in liposome over those immunized with the live B. abortus S19 version, could probably be related to the significantly different response of IgG2b at 30 DPI (P<0.01), IgG2a (P<0.01), IgG2b (P<0.01) and IgG3 (P<0.05) at the DPC stages, respectively. PMID:27656221

  17. Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2.

    PubMed

    Barrionuevo, Paula; Cassataro, Juliana; Delpino, M Victoria; Zwerdling, Astrid; Pasquevich, Karina A; García Samartino, Clara; Wallach, Jorge C; Fossati, Carlos A; Giambartolomei, Guillermo H

    2008-01-01

    The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-gamma)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.

  18. Key role of Toll-like receptor 2 in the inflammatory response and major histocompatibility complex class ii downregulation in Brucella abortus-infected alveolar macrophages.

    PubMed

    Ferrero, Mariana C; Hielpos, M Soledad; Carvalho, Natalia B; Barrionuevo, Paula; Corsetti, Patricia P; Giambartolomei, Guillermo H; Oliveira, Sergio C; Baldi, Pablo C

    2014-02-01

    Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1β (IL-1β), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival.

  19. Experimental infection of nontarget species of rodents and birds with Brucella abortus strain RB51 vaccine

    USGS Publications Warehouse

    Januszewski, M.C.; Olsen, S.C.; McLean, R.G.; Clark, L.; Rhyan, Jack C.

    2001-01-01

    The Brucella abortus vaccine strain RB51 (SRB51) is being considered for use in the management of bnucellosis in wild bison (Bison bison) and elk (Cervus elaphus) populations in the Greater Yellowstone Area (USA). Evaluation of the vaccines safety in non-target species was considered necessary prior to field use. Between June 1998 and December 1999, ground squirrels (Spermophilus richardsonii, n = 21), deer mice (Peromyscus maniculatus, n = 14), prairie voles (Microtus ochrogaster, n = 21), and ravens (Corvus corax, n = 13) were orally inoculated with SRB51 or physiologic saline. Oral and rectal swabs and blood samples were collected for bacteriologic evaluation. Rodents were necropsied at 8 to 10 wk and 12 to 21 wk post inoculation (PI), and ravens at 7 and 11 wk PI. Spleen, liver and reproductive tissues were collected for bacteriologic and histopathologic evaluation. No differences in clinical signs, appetite, weight loss or gain, or activity were observed between saline- and SRB51-inoculated animals in all four species. Oral and rectal swabs from all species were negative throughout the study. In tissues obtained from SRB51-inoculated animals, the organism was isolated from six of seven (86%) ground squirrels, one of six (17%) deer mice, none of seven voles, and one of five (20%) ravens necropsied at 8, 8, 10, and 7 wk PI, respectively. Tissues from four of seven (57%) SRB51-inoculated ground squirrels were culture positive for the organism 12 wk PI; SRB51 was not recovered from deer mice, voles. or ravens necropsied 12, 21, or 11 wk, respectively, PI. SRB51 was not recovered from saline-inoculated ground squirrels, deer mice, or voles at any time but was recovered from one saline-inoculated raven at necropsy, 7 wk PI, likely attributable to contact with SRB51-inoculated ravens in an adjacent aviary room. Spleen was time primary tissue site of colonization in ground squirrels, followed by the liver and reproductive organs. The results indicate oral exposure to

  20. Structural, functional and immunogenic insights on Cu,Zn superoxide dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

    DOE PAGES

    Pratt, Ashley J.; DiDonato, Michael; Shin, David S.; ...

    2015-10-12

    Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomicmore » details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against reactive oxygen

  1. Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

    PubMed Central

    Pratt, Ashley J.; DiDonato, Michael; Shin, David S.; Cabelli, Diane E.; Bruns, Cami K.; Belzer, Carol A.; Gorringe, Andrew R.; Langford, Paul R.; Tabatabai, Louisa B.; Kroll, J. Simon; Tainer, John A.

    2015-01-01

    ABSTRACT Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against

  2. Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models

    PubMed Central

    KWON, Ae Jeong; MOON, Ja Young; KIM, Won Kyong; KIM, Suk; HUR, Jin

    2016-01-01

    Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 108 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 108 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 109 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B–D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus. PMID:27349900

  3. Seminal vesiculitis and orchitis caused by Brucella abortus biovar 1 in young bison bulls from South Dakota.

    PubMed

    Rhyan, J C; Holland, S D; Gidlewski, T; Saari, D A; Jensen, A E; Ewalt, D R; Hennager, S G; Olsen, S C; Cheville, N F

    1997-10-01

    Specimens of blood, lymph nodes, spleens, and genitalia were collected at slaughter from seven 3- and 4-year-old male bison that had recently become seropositive for brucellosis. The animals were from a captive herd of approximately 3,500 bison located in central South Dakota. Brucella abortus biovar 1 was isolated from 2 or more specimens from each of 6 bison. Severe necrotizing and pyogranulomatous orchitis was present in 1 testicle from 1 bull, and 4 animals had mild to marked seminal vesiculitis. Immunohistochemical staining labeled organisms in seminal vesicles and the testicle with orchitis. Ultrastructurally, intact bacilli were present in cytoplasmic vacuoles of some macrophages; other macrophages contained intracytoplasmic aggregates of calcified coccobacilli.

  4. Microarray-based identification of differentially expressed genes in intracellular Brucella abortus within RAW264.7 cells.

    PubMed

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Zhang, Min; Ding, Chan; Ding, Jiabo; Chen, Guanghua; Yu, Shengqing

    2013-01-01

    Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella's virulence and potential for chronic infection include the ability to circumvent the host cell's internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292) had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.

  5. Efficacy of dart or booster vaccination with strain RB51 in protecting bison against experimental Brucella abortus challenge.

    PubMed

    Olsen, S C; Johnson, C S

    2012-06-01

    This study characterized the efficacy of the Brucella abortus strain RB51 vaccine in bison when delivered by single intramuscular vaccination (hand RB51), by single pneumatic dart delivery (dart RB51), or as two vaccinations approximately 13 months apart (booster RB51) in comparison to control bison. All bison were challenged intraconjunctivally in midgestation with 10(7) CFU of B. abortus strain 2308 (S2308). Bison were necropsied and sampled within 72 h of abortion or delivery of a live calf. Compared to nonvaccinated bison, bison in the booster RB51 treatment had a reduced (P < 0.05) incidence of abortion, uterine infection, or infection in maternal tissues other than the mammary gland at necropsy. Bison in single-vaccination treatment groups (hand RB51 and dart RB51) did not differ (P > 0.05) from the control group in the incidence of abortion or recovery of S2308 from uterine, mammary, fetal, or maternal tissues at necropsy. Compared to nonvaccinated animals, all RB51 vaccination groups had reduced (P < 0.05) mean colonization or incidence of infection in at least 2 of 4 target tissues, with the booster RB51 group having reduced (P < 0.05) colonization and incidence of infection in all target tissues. Our data suggest that booster vaccination of bison with RB51 enhances protective immunity against Brucella challenge compared to single vaccination with RB51 by hand or by pneumatic dart. Our study also suggests that an initial vaccination of calves followed by booster vaccination as yearlings should be an effective strategy for brucellosis control in bison.

  6. Immune responses and safety after dart or booster vaccination of bison with Brucella abortus strain RB51.

    PubMed

    Olsen, S C; Johnson, C

    2012-05-01

    One alternative for management of brucellosis in Yellowstone National Park bison (Bison bison) is vaccination of calves and yearlings. Although Brucella abortus strain RB51 vaccination protects bison against experimental challenge, the effect of booster vaccinations was unknown. This study characterized immunologic responses after dart or booster vaccination of bison with Brucella abortus strain RB51. In two studies, 8- to 10-month-old female bison were inoculated with saline (n = 14), hand vaccinated with 1.1 × 10(10) to 2.0 × 10(10) CFU of RB51 (n = 21), or dart vaccinated with 1.8 × 10(10) CFU of RB51 (n = 7). A subgroup of hand vaccinates in study 1 was randomly selected for booster vaccination 15 months later with 2.2 × 10(10) CFU of RB51. Compared to single vaccinates, booster-vaccinated bison had greater serologic responses to RB51. However, there was a trend for antigen-specific proliferative responses of peripheral blood mononuclear cells (PBMC) from booster vaccinates to be reduced compared to responses of PBMC from single vaccinates. PBMC from booster vaccinates tended to have greater gamma interferon (IFN-γ) production than those from single vaccinates. In general, dart vaccination with RB51 induced immunologic responses similar to those of hand vaccination. All vaccinates (single hand, dart, or booster) demonstrated greater (P < 0.05) immunologic responses at various times after vaccination than nonvaccinated bison. Booster vaccination with RB51 in early gestation did not induce abortion or fetal infection. Our data suggest that booster vaccination does not induce strong anamnestic responses. However, phenotypic data on resistance to experimental challenge are required to fully assess the effect of booster vaccination on protective immunity.

  7. Purified native haptens of Brucella abortus B19 and B. melitensis 16M reveal the lipopolysaccharide origin of the antigens.

    PubMed

    Zygmunt, M S; Dubray, G; Bundle, D R; Perry, M P

    1988-01-01

    Purification of the Brucella polysaccharide referred to as native hapten (NH) and extracted from cells by the autoclaving procedure, was accomplished by ultrafiltration, followed by repetitive gel filtration using high-performance liquid chromatography on a "TSK-G2000-SW" column. The purified NH was analysed by SDS-PAGE, gas-liquid chromatography mass spectroscopy, and 13C and 1H NMR spectroscopy. NH from B. abortus B19 (NH-A) was shown to have a structure identical to that of A polysaccharide from B. abortus 1119-3, a linear homopolymer of alpha-1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues. The structure of the NH from B. melitensis 16M (NH-M) was identified as a linear homopolysaccharide of the same sugar but composed of a pentasaccharide repeating unit in which four alpha 1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues are linked alpha-1,3 to the last monosaccharide of the sequence. This structure is similar to that determined for the Brucella M polysaccharide from B. melitensis 16M. The discovery in highly purified NH preparations of covalently bound monosaccharides characteristic of lipopolysaccharide inner core regions e.g., quinovosamine, mannose and 3-deoxy-D-manno-octulosonate (KDO), indicates that this polysaccharide is derived from lipopolysaccharides (LPS) by hydrolytic conditions fortuitously generated during the extraction protocol. The antigenically important polysaccharides of Brucella are now established to be either A or M antigens. Polysaccharide B is a cyclic glucan with no structural or serological relationship to A or M polysaccharides, its apparent activity in diagnostic tests of infected cattle results from O polysaccharide contamination. This artefact, previously referred to as NH, results from LPS hydrolysis under the extraction conditions used to prepare polysaccharide B.

  8. Intracellular Production of Brucella L Forms II. Induction and Survival of Brucella abortus L Forms in Tissue Culture1

    PubMed Central

    Hatten, Betty A.; Sulkin, S. Edward

    1966-01-01

    Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. II. Induction and survival of Brucella abortus L forms in tissue culture. J. Bacteriol. 91:14–20. 1966.—Intracellular survival of altered brucellae, possibly L forms, was not greatly affected by penicillin or streptomycin in concentrations ranging from 5.0 to 40 μg/ml, but a combination of these two antibiotics (2.5 to 20 μg/ml each) reduced the number of positive L-form cultures. Tetracycline (2.0 μg/ml) decreased the number of positive L-form cultures at about the same rate as combinations of the higher concentrations of penicillin and streptomycin. Various concentrations of tetracycline (0.1 to 2.0 μg/ml) with 5.0 μg/ml of penicillin or streptomycin significantly reduced the number of positive L-form cultures. L forms were recovered for several days after elimination of bacteria from the cultures by all of the antibiotics tested. L-form production was not dependent upon the presence of antibiotics in the culture medium, but they were recovered in greater numbers when bacteria were still present in the hamster kidney cells. Addition of thallium acetate to infected cells (at varying intervals of time after infection) to control bacterial growth and conversion to the L phase during cellular disintegration decreased the number of positive L-form cultures obtained over a 10-day period. Comparison of the antibiotic sensitivity of bacteria recovered from infected tissue culture cells with the stock strain of Brucella abortus indicated that some resistance to penicillin and tetracycline had developed. A marked resistance to streptomycin was observed in those bacteria recovered from cells maintained in the presence of this antibiotic. PMID:4955246

  9. Performance of skin tests with allergens from B. melitensis B115 and rough B. abortus mutants for diagnosing swine brucellosis.

    PubMed

    Dieste-Pérez, L; Blasco, J M; De Miguel, M J; Marín, C M; Barberán, M; Conde-Álvarez, R; Moriyón, I; Muñoz, P M

    2014-01-10

    Swine brucellosis by Brucella suis biovar 2 is an emerging disease whose control is based on serological testing and culling. However, current serological tests detect antibodies to the O-polysaccharide (O/PS) moiety of Brucella smooth lipopolysaccharide (S-LPS), and thus lack specificity when infections by Yersinia enterocolitica O:9 and other gram-negative bacteria carrying cross-reacting O/PS occur. The skin test with the protein-rich brucellin extract obtained from rough B. melitensis B115 is assumed to be specific for discriminating these false positive serological reactions (FPSR). However, B115 strain, although unable to synthesize S-LPS, accumulates O/PS internally, which could cause diagnostic problems. Since the brucellin skin test has been seldom used in pigs and FPSR are common in these animals, we assessed its performance using cytosoluble protein extracts obtained from B. abortus rough mutants in manBcore or per genes (critical for O/PS biosynthesis) and B. melitensis B115. The diagnostic sensitivity and specificity were determined in B. suis biovar 2 culture positive and brucellosis free sows, and apparent prevalence in sows of unknown individual bacteriological and serological status belonging to B. suis biovar 2 naturally infected herds. Moreover, the specificity in discriminating brucellosis from FPSR was assessed in brucellosis free boars showing FPSR. The skin test with B. abortus ΔmanBcore and B. melitensis B115 allergens performed similarly, and the former one resulted in 100% specificity when testing animals showing FPSR in indirect ELISA, Rose Bengal and complement fixation serological tests. We conclude that O/PS-free genetically defined mutants represent an appropriate alternative to obtain Brucella protein extracts for diagnosing swine brucellosis.

  10. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    SciTech Connect

    Serer, María I.; Bonomi, Hernán R.; Guimarães, Beatriz G.; Rossi, Rolando C.; Goldbaum, Fernando A.; Klinke, Sebastián

    2014-05-01

    This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

  11. Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

    SciTech Connect

    Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

    2003-08-01

    Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

  12. Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation.

    PubMed

    Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

    2015-01-01

    The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M.

  13. FixL-like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells.

    PubMed

    Roset, Mara S; Almirón, Marta A

    2013-09-17

    Replication of Brucella inside eukaryotic cells is essential for pathogenesis, and successful infection requires rapid adaptation to the intracellular milieu. Close relatives of Brucella use the two-component system FixLJ to survive inside the host. We aimed to identify a homologous sensor in Brucella abortus. A predicted protein with transmembrane and conserved histidine kinase domains was identified as the Fix-like Brucella sensor, FlbS. Although it lacks the PAS domain, recombinant FlbS binds haem in vitro. An internal in-frame deletion in flbS severely decreased B. abortus survival inside professional and non-professional phagocytes. This phenotype was reverted by genetic complementation. These results indicate the critical role of this haemoprotein in the intracellular lifestyle of Brucella.

  14. Evaluation of strain-specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus (Chalmydia psittaci) for the detection of EAE by PCR.

    PubMed

    Creelan, J L; McCullough, S J

    2000-09-01

    Strain-specific primer sequences derived from the helicase gene of an ovine abortifacient strain (S26/3) of Chlamydophila abortus (Chlamydia psittaci) were evaluated for the diagnosis of enzootic abortion in ewes (EAE) by polymerase chain reaction (PCR). C abortus DNA was amplified from tissues submitted from ovine abortion cases using genus-specific and strain-specific primers in a standard thermal cycler. Amplification was followed by Southern blotting and hybridisation with a strain-specific probe. Real-time PCR was also evaluated using strain-specific primers in a microvolume fluorimeter-based thermal cycler (LightCycler). Detection using both PCR methods was compared with other diagnostic methods against the standard of McCoy cell culture isolation. In this paper we report the application of strain-specific PCR as a fast, sensitive, specific method for the detection of EAE.

  15. Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice

    PubMed Central

    Escalona, Emilia; Sáez, Darwin; Oñate, Angel

    2017-01-01

    Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus. PMID:28232837

  16. Molecular Characterization of a Brucella Species Large DNA Fragment Deleted in Brucella abortus Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide

    PubMed Central

    Vizcaíno, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernández-Lago, Luis

    1999-01-01

    A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the

  17. Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

    PubMed Central

    Matrone, M.; Keid, L.B.; Rocha, V.C.M.; Vejarano, M.P.; Ikuta, C.Y.; Rodriguez, C.A.R.; Ferreira, F.; Dias, R.A.; Ferreira Neto, J.S

    2009-01-01

    The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p< 0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and vice-versa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol. PMID:24031391

  18. Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu-Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice.

    PubMed

    Escalona, Emilia; Sáez, Darwin; Oñate, Angel

    2017-01-01

    Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu-Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4(+) T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus.

  19. Oral immunization of mice with gamma-irradiated Brucella neotomae induces protection against intraperitoneal and intranasal challenge with virulent B. abortus 2308.

    PubMed

    Dabral, Neha; Martha-Moreno-Lafont; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2014-01-01

    Brucella spp. are Gram-negative, facultative intracellular coccobacilli that cause one of the most frequently encountered zoonosis worldwide. Humans naturally acquire infection through consumption of contaminated dairy and meat products and through direct exposure to aborted animal tissues and fluids. No vaccine against brucellosis is available for use in humans. In this study, we tested the ability of orally inoculated gamma-irradiated B. neotomae and B. abortus RB51 in a prime-boost immunization approach to induce antigen-specific humoral and cell mediated immunity and protection against challenge with virulent B. abortus 2308. Heterologous prime-boost vaccination with B. abortus RB51 and B. neotomae and homologous prime-boost vaccination of mice with B. neotomae led to the production of serum and mucosal antibodies specific to the smooth LPS. The elicited serum antibodies included the isotypes of IgM, IgG1, IgG2a, IgG2b and IgG3. All oral vaccination regimens induced antigen-specific CD4(+) and CD8(+) T cells capable of secreting IFN-γ and TNF-α. Upon intra-peritoneal challenge, mice vaccinated with B. neotomae showed the highest level of resistance against virulent B. abortus 2308 colonization in spleen and liver. Experiments with different doses of B. neotomae showed that all tested doses of 10(9), 10(10) and 10(11) CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, a dose of 10(11) CFU-equivalent of B. neotomae was required for affording protection against intranasal challenge as shown by the reduced bacterial colonization in spleens and lungs. Taken together, these results demonstrate the feasibility of using gamma-irradiated B. neotomae as an effective and safe oral vaccine to induce protection against respiratory and systemic infections with virulent Brucella.

  20. BrucELISA: an enzyme-antibody immunoassay for detection of Brucella abortus antibodies in milk: correlation with the Brucella ring test and with shedding of viable organisms.

    PubMed Central

    Boraker, D K; Stinebring, W R; Kunkel, J R

    1981-01-01

    An indirect enzyme-antibody immunosorbent assay (BrucELISA) is described for the detection of antibody to Brucella abortus in cow's milk. Three series of milk samples were obtained from an adult-vaccinated dairy herd infected with B. abortus. The BrucELISA system was used as a screening test for individual milks diluted 1:200 (BE 200 test), for undiluted bulk milks, and to determine antibody titer (BrucELISA titration assay). The BrucELISA results correlated highly with positive Brucella ring test reactions and culture positivity, eliminated false-positive Brucella ring test reactions, detected antibody in some samples which were Brucella ring test negative, and distinguished between vaccinated and infected animals. BrucELISA titration assay titers of greater than 1:800 were correlated with shedding, or were prognostic for animals which eventually became shedders. Binding of the enzyme-antibody conjugate to bovine immunoglobulin in the absence of rabbit anti-bovine immunoglobulin occurred with culture-positive or -negative milks showing titers of greater than 1:1,600 (the beta effect); the effect was also of predictive value in identifying eventual shedders. The BrucELISA system is a sensitive, specific, and inexpensive method for screening large numbers of individual or bulk milk samples for the presence of antibody to B. abortus. PMID:6793622

  1. Transcription of non-classic major histocompatibility complex (MHC) class I in the bovine placenta throughout gestation and after Brucella abortus infection.

    PubMed

    Dos Santos, Larissa Sarmento; da Silva Mol, Juliana Pinto; de Macedo, Auricélio Alves; Silva, Ana Patrícia Carvalho; Dos Santos Ribeiro, Diego Luiz; Santos, Renato Lima; da Paixão, Tatiane Alves; de Carvalho Neta, Alcina Vieira

    2015-10-15

    Transcription of non-classical major histocompatibility complex class I (MHC-I) was assessed in the bovine placenta throughout gestation. Additionally, the effect of Brucella abortus infection on expression of non-classical MHC-I was also evaluated using a chorioallantoic membrane explant model of infection. The non-classical MHC-I genes MICB and NC3 had higher levels of transcription in the intercotyledonary region when compared to the placentome, which had higher levels of transcription at the second trimester of gestation. NC1 and classical MHC-I had very low levels of transcription throughout gestation. Trophoblastic cells of B. abortus-infected chorioallantoic membrane explants had an increase in transcription of non-classical MHC-I at 4h post infection. Therefore, this study provides an analysis of non-classical MHC-I transcription at different stages of gestation and different placental tissues, and during B. abortus infection. These findings provide additional knowledge on immune regulation in placental tissues, a known immune-privileged site.

  2. Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

    PubMed

    Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

    2014-04-01

    The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel.

  3. MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain S2 from cattle in China.

    PubMed

    Jiang, Hai; Wang, Heng; Xu, Liqing; Hu, Guiying; Ma, Junying; Xiao, Pei; Fan, Weixing; Di, Dongdong; Tian, Guozhong; Fan, Mengguang; Mi, Jingchuan; Yu, Ruiping; Song, Litao; Zhao, Hongyan; Piao, Dongri; Cui, Buyun

    2013-01-01

    In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

  4. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  5. The Outer Membrane of Brucella ovis Shows Increased Permeability to Hydrophobic Probes and Is More Susceptible to Cationic Peptides than Are the Outer Membranes of Mutant Rough Brucella abortus Strains

    PubMed Central

    Freer, Enrique; Pizarro-Cerdá, Javier; Weintraub, Andrej; Bengoechea, José-Antonio; Moriyón, Ignacio; Hultenby, Kjell; Gorvel, Jean-Pierre; Moreno, Edgardo

    1999-01-01

    The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-l-lysine, and poly-l-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells. PMID:10531286

  6. Immune Response of Calves Vaccinated with Brucella abortus S19 or RB51 and Revaccinated with RB51.

    PubMed

    Dorneles, Elaine M S; Lima, Graciela K; Teixeira-Carvalho, Andréa; Araújo, Márcio S S; Martins-Filho, Olindo A; Sriranganathan, Nammalwar; Al Qublan, Hamzeh; Heinemann, Marcos B; Lage, Andrey P

    2015-01-01

    Brucella abortus S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with B. abortus S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6-1.2 x 1011 CFU) or RB51 (1.3 x 1010 CFU) on day 0, and revaccinated with RB51 (1.3 x 1010 CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4+ and CD8+ T-cells; IFN-ɣ and IL-17A production by CD4+ T-cells; cytotoxic CD8+ T-cells; IL-6 secretion; CD4+ and CD8+ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4+ memory cells, induction of CD21+ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4+ and CD8+ T-cells, cytotoxic CD8+ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4+- or CD8+-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.

  7. Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization.

    PubMed

    Jain, Lata; Rawat, Mayank; Ramakrishnan, Saravanan; Kumar, Bablu

    2017-01-01

    Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage 'ϕLd'. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 μg protein and 120 μg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

  8. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR.

    PubMed Central

    Bricker, B J; Halling, S M

    1994-01-01

    Several PCR assays which identify the genus Brucella but do not discriminate among species have been reported. We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella. Individual biovars within a species are not differentiated. The assay can identify three biovars (1, 2, and 4) of B. abortus, all three biovars of B. melitensis, biovar 1 of B. suis, and all B. ovis biovars. These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research. The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome. Identity is determined by the size(s) of the product(s) amplified from primers hybridizing at various distances from the element. The performance of the assay with U.S. field isolates was highly effective. When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods. Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhodospirillum rubrum) and two control bacteria (Bordetella bronchiseptica and Escherichia coli) tested negative by the assay. Images PMID:7852552

  9. The unexpected discovery of Brucella abortus Buck 19 vaccine in goats from Ecuador underlines the importance of biosecurity measures.

    PubMed

    Ron-Román, Jorge; Berkvens, Dirk; Barzallo-Rivadeneira, Daniela; Angulo-Cruz, Alexandra; González-Andrade, Pablo; Minda-Aluisa, Elizabeth; Benítez-Ortíz, Washington; Brandt, Jef; Rodríguez-Hidalgo, Richar; Saegerman, Claude

    2017-03-01

    Very few, mostly old, and only preliminary serological studies of brucellosis in goats exist in Ecuador. In order to assess the current epidemiological situation, we performed a cross-sectional serological study in the goat populations of Carchi (n = 160 animals), Pichincha (n = 224 animals), and Loja provinces (n = 2024 animals). Only two positive serological results (RB negative and SAT-EDTA ≥400 IU/ml) were obtained in lactating goats from the same farm in Quito (Pichincha province). Additionally, milk was sampled from 220 animals in Pichincha province. The present study indicates a low apparent prevalence in Pichincha province and absence in Carchi and Loja provinces. A total of 25 positive milk ring tests (MRT) were obtained in Pichincha province yielding a prevalence of MRT of 11.16%. Subsequent culture was performed on the positive MRT samples. All results were negative, apart from a single sample, obtained from a serologically positive goat in Quito, that was positive for Brucella abortus strain 19 (B19). Several hypotheses are forwarded concerning this unexpected result. The most likely hypothesis is the possible accidental use of a needle, previously used for vaccination of cattle with the said vaccine, for the administration of drug treatment to the goat. This hypothesis underlines the necessity of biosecurity measures to prevent this type of accidents.

  10. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    PubMed Central

    Serer, María I.; Bonomi, Hernán R.; Guimarães, Beatriz G.; Rossi, Rolando C.; Goldbaum, Fernando A.; Klinke, Sebastián

    2014-01-01

    Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C 3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity. PMID:24816110

  11. In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

    PubMed Central

    2016-01-01

    Purpose At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. Materials and Methods In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. Results The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 µg/mL of the culture. Conclusion Our results indicate that Omp2b protein has a potential to induce both B-cell– and T-cell–mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis. PMID:26866027

  12. Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections.

    PubMed

    Abkar, Morteza; Amani, Jafar; Sahebghadam Lotfi, Abbas; Nikbakht Brujeni, Gholamreza; Alamian, Saeed; Kamali, Mehdi

    2015-08-01

    Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1-Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC-vaccinated mice displayed a strong recall proliferative response with high amounts of IL-4, IL-12 and IFN-γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.

  13. Comparison of fetal and maternal inflammatory responses in the ovine placenta after experimental infection with Chlamydophila abortus.

    PubMed

    Sammin, D J; Markey, B K; Quinn, P J; McElroy, M C; Bassett, H F

    2006-01-01

    Placentae from 13 pregnant ewes infected intravenously with Chlamydophila abortus, together with placentae from nine uninfected control ewes, were examined at 14, 21 or 28 days post-inoculation (p.i.). Chlamydial inclusions were present in the trophoblast at 14 days p.i. and were widespread by 21 days p.i. Chorioallantoic lesions (oedema, arteritis and thrombosis) were severe at 28 days p.i., the changes being particularly marked in the membrane surrounding placentomes. Lymphocytes constituted only a small proportion of the cellular infiltrate in the chorioallantois; neutrophil infiltration of the chorionic surface was evident where the trophoblast layer had sloughed, whereas macrophages represented the predominant cell type in the deeper stroma. In contrast, on the maternal side of the placenta, chlamydial inclusions were sparse at all timepoints, and even at 28 days p.i., lesions were restricted to focal endometritis at the placentomal limbus and occasional foci of septal necrosis. T lymphocytes were numerous within endometrial and septal lesions, the infiltrate consistently containing more CD8(+) than CD4(+) cells. The fetal response to chlamydial invasion of the placenta was innate in character, whereas the maternal response appeared to represent an acquired, chlamydia-specific immune response.

  14. Transcriptional regulator GntR of Brucella abortus regulates cytotoxicity, induces the secretion of inflammatory cytokines and affects expression of the type IV secretion system and quorum sensing system in macrophages.

    PubMed

    Li, Zhiqiang; Wang, Shuli; Zhang, Hui; Zhang, Jinliang; Xi, Li; Zhang, Junbo; Chen, Chuangfu

    2017-03-01

    The pathogenic mechanisms of Brucella are still poorly understood. GntR is a transcriptional regulator and plays an important role in the intracellular survival of Brucella. To investigate whether GntR is involved in the cytotoxicity of Brucella abortus (B. abortus), we created a 2308ΔgntR mutant of B. abortus 2308 (S2308). Lactate dehydrogenase (LDH) cytotoxicity assays using a murine macrophage cell line (RAW 264.7) show that high-dose infection with the parental strain produces a high level of cytotoxicity to macrophages, but the 2308ΔgntR mutant exhibits a very low level of cytotoxicity, indicating that mutation of GntR impairs the cytotoxicity of B. abortus to macrophages. After the macrophages are infected with 2308ΔgntR, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) increase and are slightly higher than that for the S2308 infected group, indicating that the 2308ΔgntR mutant could induce the secretion of inflammatory cytokines. The virulence factor detection experiments indicate that genes involved in the type IV secretion system (T4SS) and quorum sensing system (QSS) are down-regulated in 2308ΔgntR. The lower levels of survival of 2308ΔgntR under various stress conditions and the increased sensitivity of 2308ΔgntR to polymyxin B suggest that GntR is a virulence factor and that deletion of gntR reduces of B. abortus to stress conditions. Taken together, our results demonstrate that GntR is involved in the cytotoxicity, virulence and intracellular survival of B. abortus during its infection.

  15. SodA is a major metabolic antioxidant in Brucella abortus 2308 that plays a significant, but limited, role in the virulence of this strain in the mouse model.

    PubMed

    Martin, Daniel W; Baumgartner, John E; Gee, Jason M; Anderson, Eric S; Roop, R Martin

    2012-07-01

    The gene designated BAB1_0591 in the Brucella abortus 2308 genome sequence encodes the manganese-cofactored superoxide dismutase SodA. An isogenic sodA mutant derived from B. abortus 2308, designated JB12, displays a small colony phenotype, increased sensitivity in vitro to endogenous superoxide generators, hydrogen peroxide and exposure to acidic pH, and a lag in growth when cultured in rich and minimal media that can be rescued by the addition of all 20 amino acids to the growth medium. B. abortus JB12 exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, but this attenuation is limited to the early stages of infection. Addition of the NADPH oxidase inhibitor apocynin to infected macrophages does not alleviate the attenuation exhibited by JB12, suggesting that the basis for the attenuation of the B. abortus sodA mutant is not an increased sensitivity to exogenous superoxide generated through the oxidative burst of host phagocytes. It is possible, however, that the increased sensitivity of the B. abortus sodA mutant to acid makes it less resistant than the parental strain to killing by the low pH encountered during the early stages of the development of the brucella-containing vacuoles in macrophages. These experimental findings support the proposed role for SodA as a major cytoplasmic antioxidant in brucella. Although this enzyme provides a clear benefit to B. abortus 2308 during the early stages of infection in macrophages and mice, SodA appears to be dispensable once the brucellae have established an infection.

  16. Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004.

    PubMed

    Runge, Martin; Binder, Alfred; Schotte, Ulrich; Ganter, Martin

    2012-01-01

    The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.

  17. Roles of genomic island 3 (GI-3) BAB1_0267 and BAB1_0270 open reading frames (ORFs) in the virulence of Brucella abortus 2308.

    PubMed

    Ortiz-Román, Luisa; Riquelme-Neira, Roberto; RobertoVidal; Oñate, Angel

    2014-08-06

    One of the properties of bacteria is their capacity to acquire large fragments of genomic DNA from other bacteria or to loose important parts of their own genome. Such fragments include genomic islands (GIs); nine GIs are present in Brucella, including genomic island 3 (GI-3), present in B. abortus, B. melitensis and B. ovis. The GI-3 have 29 open reading frames (ORFs) most of them with unknown function. Within the GI-3, the ORFs BAB1_0267 encodes a hypothetical protein sharing a SH3 domain and BAB1_270 a zinc-dependent metallopeptidase. We have obtained deletion mutants for BAB1_0267 and BAB1_0270 ORFs present within GI-3, which have been named the Δ0267 and Δ0270, respectively; in both cases the mutation did not affect the growth of bacteria. Both mutants were evaluated with respect to their growth rates, their ability to invade and replicate in the non-professional and professional phagocytes, HeLa and J774.A1 cells, respectively. Their persistence in the spleens of mice was also evaluated. The mutants efficiently invaded HeLa and J774.A1 cells but both mutants showed a decreased intracellular survival in macrophages and HeLa cells 72 and 96 h post-infection, respectively, and were non-detected in J774.A1 cells 120 h post infection. With respect to in vivo persistence Δ0267 was detected through the fourth week while Δ0270 decreased at 7 days disappearing the second week. Our results indicated that deletion of BAB1_0267 and BAB1_270 are necessary to establish an optimal infectious process in B. abortus 2308, having more effect the deletion of ORF BAB1_0270. Therefore these ORFs, principally BAB1_0270 are important virulent of B. abortus.

  18. Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases ▿

    PubMed Central

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; De Simone, Emilio A.; Vanzulli, Silvia I.; Fossati, Carlos A.; Baldi, Pablo C.; Delpino, M. Victoria

    2011-01-01

    Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators. PMID:21730088

  19. Evaluation of the Recombinant 10-Kilodalton Immunodominant Region of the BP26 Protein of Brucella abortus for Specific Diagnosis of Bovine Brucellosis ▿

    PubMed Central

    Tiwari, Arvind Kumar; Kumar, Subodh; Pal, Vijai; Bhardwaj, Bhupendra; Rai, Ganga Prasad

    2011-01-01

    Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohen's kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals. PMID:21852548

  20. Molecular prevalence of putative virulence-associated genes in Brucella melitensis and Brucella abortus isolates from human and livestock specimens in Iran.

    PubMed

    Hashemifar, Iman; Yadegar, Abbas; Jazi, Faramarz Masjedian; Amirmozafari, Nour

    2017-04-01

    Molecular prevalence of nine putative virulence factors in two more prevalent Brucella species in Iranian patients and livestock was investigated. During five years (2010-2015), 120 human and animal specimens were collected from three geographical areas of Iran. All samples were cultured in blood culture media and subcultured into Brucella agar medium. Nine primer pairs were designed for detection of VirB2, VirB5, VceC, BtpA, BtpB, PrpA, BetB, BPE275 and BSPB virulence factors using PCR and sequence analysis. Totally, 68 Brucella isolates including 60 B. melitensis and 8 B. abortus were isolated from the human and animal specimens examined. Approximately, all B. melitensis and B. abortus strains were positive (100%) regarding btpA, btpB, virB5, vceC, bpe275, bspB, and virB2 genes except for prpA and betB that were detected in 86% and 97% of the strains, respectively. Significant relationships were found between the presence of prpA and human B. melitensis isolates (P = 0.04), and also between the presence of betB and human isolates of B. abortus (P = 0.03). In conclusion, our results revealed that Iranian Brucella strains, regardless of human or animal sources, are extremely virulent due to high prevalence of virulence attributes in almost all strains studied.

  1. Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 in Chlamydophila abortus-Infected Pregnant Sheep

    PubMed Central

    Livingstone, Morag; Entrican, Gary; Wattegedera, Sean; Buxton, David; McKendrick, Iain J.; Longbottom, David

    2005-01-01

    Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans. PMID:15939753

  2. Critical role of ASC inflammasomes and bacterial type IV secretion system in caspase-1 activation and host innate resistance to Brucella abortus infection.

    PubMed

    Gomes, Marco Tulio R; Campos, Priscila C; Oliveira, Fernanda S; Corsetti, Patricia P; Bortoluci, Karina R; Cunha, Larissa D; Zamboni, Dario S; Oliveira, Sergio C

    2013-04-01

    Pathogens are detected by innate immune receptors that, upon activation, orchestrate an appropriate immune response. Recent studies revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella abortus infection. However, no report has elucidated the role of inflammasome receptors in Brucella recognition. Therefore, we decided to investigate the function of NLRC4, NLRP3, and AIM2 in sensing Brucella. In this study, we showed that NLRC4 is not required to induce caspase-1 activation and further secretion of IL-1β by B. abortus in macrophages. In contrast, we determined that AIM2, which senses Brucella DNA, and NLRP3 are partially required for caspase-1 activation and IL-1β secretion. Additionally, mitochondrial reactive oxygen species induced by Brucella were implicated in IL-1β production. Furthermore, AIM2, NLRP3, ASC, and caspase-1 knockout mice were more susceptible to B. abortus infection than were wild-type animals, suggesting that multiple ASC-dependent inflammasomes contribute to host protection against infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than pyroptosis, because we observed augmented bacterial burden in IL-1R and IL-18 knockout mice. Finally, we determined that bacterial type IV secretion system VirB and live, but not heat-killed, Brucella are required for full inflammasome activation in macrophages during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.

  3. T cell regulation of the thymus-independent antibody response to trinitrophenylated-Brucella abortus (TNP-BA)

    SciTech Connect

    Tanay, A.; Strober, S.

    1985-06-01

    The authors have previously observed a reduction of the T cell-dependent primary antibody response to dinitrophenylated keyhole limpet hemocyanin, and an enhancement of the T cell-independent response to trinitrophenylated Brucella abortus (TNP-BA) in BALB/c mice after treatment with total lymphoid irradiation (TLI). To elucidate the relative contribution of T and B cells to the enhanced T cell-independent antibody responses after TLI, a syngeneic primary adoptive transfer system was utilized whereby irradiated hosts were reconstituted with unfractionated spleen cells or a combination of purified T and B cells from TLI-treated and untreated control mice. Antibody responses of purified splenic B cells from TLI-treated BALB/c mice (TLI/B) to TNP-BA were enhanced 10-fold as compared with those of unfractionated (UF) spleen cells or B cells from normal (NL) BALB/c mice (NL/UF and NL/B, respectively). Splenic T cells from normal animals (NL/T) suppressed the anti-TNP-BA response of TLI/B by more than 100-fold. NL/T neither suppressed nor enhanced the response of NL/B. On the other hand, T cells from TLI-treated mice (TLI/T) enhanced by 100-fold the anti-TNP-BA response of NL/B, but neither suppressed nor enhanced the response of TLI/B. Thus, T cells can regulate the T cell-independent antibody response to TNP-BA. However, experimental manipulation of the T and B cell populations is needed to demonstrate the regulatory functions.

  4. Comparison between Immunization Routes of Live Attenuated Salmonella Typhimurium Strains Expressing BCSP31, Omp3b, and SOD of Brucella abortus in Murine Model

    PubMed Central

    Kim, Won K.; Moon, Ja Y.; Kim, Suk; Hur, Jin

    2016-01-01

    Live, attenuated Salmonella Typhimurium vaccine candidate expressing BCSP31, Omp3b, and SOD proteins of Brucella abortus was constructed. Thirty BALB/c mice were divided equally into three groups, Group A, were intraperitoneally (IP) inoculated with 100 μl of approximately 1.2 × 106 colony-forming units (CFUs)/ml of the Salmonella containing vector only in 100 μl as a control. And groups B and C mice were orally and IP immunized with approximately 1.2 × 109 CFU/ml of the mixture of three delivery strains in 10 μl and IP immunized with approximately 1.2 × 106 CFU/ml of the mixture in 100 μl, respectively. The serum IgG, TNF-α and IFN-γ concentrations in groups B (except Omp3b) and C were significantly higher than those in group A. Following challenge with B. abortus strain 544; challenge strain was detected <103 CFU from the spleen of all mice of group C. These results suggest that IP immunization with the mixture of the vaccine candidate can induce immune responses, and can effectively protect mice against brucellosis. PMID:27148232

  5. Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice.

    PubMed

    Lalsiamthara, Jonathan; Gogia, Neha; Goswami, Tapas K; Singh, R K; Chaudhuri, Pallab

    2015-05-21

    Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis.

  6. Vaccination of adult animals with a reduced dose of Brucella abortus S19 vaccine to control brucellosis on dairy farms in endemic areas of India.

    PubMed

    Chand, Puran; Chhabra, Rajesh; Nagra, Juhi

    2015-01-01

    Bovine brucellosis is an economically important disease which seriously affects dairy farming by causing colossal losses. It can be controlled by practicing vaccination of animals with Brucella abortus S19 vaccine (S19 vaccine). In the present study, adult bovines were vaccinated on seven dairy farms with a reduced dose of S19 vaccine to control brucellosis. Serological screening of adult animals (N = 1,082) by Rose Bengal test (RBT) and ELISA prior to vaccination revealed the presence and absence of brucellosis on five and two farms, respectively. The positive animals (N = 171) were segregated and those which tested negative (N = 911) were vaccinated by conjunctival route with a booster after 4 months. The conjunctival vaccination induced weak antibody response in animals, which vanished within a period of 9 to 12 weeks. Abortion in 12 animals at various stages of pregnancy and post-vaccination was recorded, but none was attributed to S19 vaccine. However, virulent B. abortus was incriminated in six heifers, and the cause of abortion could not be established in six animals. The six aborted heifers perhaps acquired infection through in utero transmission or from the environment which remained undetected until abortion. These findings suggested that vaccination of adult animals with a reduced dose of S19 vaccine by conjunctival route did not produce adverse effects like abortion in pregnant animals and persistent vaccinal antibody titers, which are the major disadvantages of subcutaneous vaccination of adult animals.

  7. Exploring the Molecular Basis for Binding of Inhibitors by Threonyl-tRNA Synthetase from Brucella abortus: A Virtual Screening Study

    PubMed Central

    Li, Ming; Wen, Fang; Zhao, Shengguo; Wang, Pengpeng; Li, Songli; Zhang, Yangdong; Zheng, Nan; Wang, Jiaqi

    2016-01-01

    Targeting threonyl-tRNA synthetase (ThrRS) of Brucella abortus is a promising approach to developing small-molecule drugs against bovine brucellosis. Using the BLASTp algorithm, we identified ThrRS from Escherichia coli (EThrRS, PDB ID 1QF6), which is 51% identical to ThrRS from Brucella abortus (BaThrRS) at the amino acid sequence level. EThrRS was used as the template to construct a BaThrRS homology model which was optimized using molecular dynamics simulations. To determine the residues important for substrate ATP binding, we identified the ATP-binding regions of BaThrRS, docked ATP to the protein, and identified the residues whose side chains surrounded bound ATP. We then used the binding site of ATP to virtually screen for BaThrRS inhibitors and got seven leads. We further characterized the BaThrRS-binding site of the compound with the highest predicted inhibitory activity. Our results should facilitate future experimental effects to find novel drugs for use against bovine brucellosis. PMID:27447614

  8. The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model.

    PubMed

    González-González, Edith; García-Hernández, Ana Lilia; Flores-Mejía, Raúl; López-Santiago, Rubén; Moreno-Fierros, Leticia

    2015-02-25

    Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.

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

    PubMed

    Gil-Ramírez, Yolanda; Conde-Álvarez, Raquel; Palacios-Chaves, Leyre; Zúñiga-Ripa, Amaia; Grilló, María-Jesús; Arce-Gorvel, Vilma; Hanniffy, Sean; Moriyón, Ignacio; Iriarte, Maite

    2014-08-01

    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.

  10. A diagnostic protocol to identify water buffaloes (Bubalus bubalis) vaccinated with Brucella abortus strain RB51 vaccine.

    PubMed

    Tittarelli, Manuela; Atzeni, Marcello; Calistri, Paolo; Di Giannatale, Elisabetta; Ferri, Nicola; Marchi, Enrico; Martucciello, Alessandra; De Massis, Fabrizio

    2015-01-01

    The use of live vaccine strain RB51 for vaccination of domestic water buffaloes (Bubalus bubalis) at risk of infection with Brucella abortus is permitted notwithstanding the plans for the eradication and only under strict veterinary control. The antibodies induced by RB51 vaccination are not detectable using conventional diagnostic techniques; therefore, it is necessary to have a specific diagnostic tool able to discriminate vaccinated from unvaccinated animals. The combination of a complement fixation test (CFT) with specific RB51 antigen (RB51-CFT) and a brucellin skin test has been demonstrated to be a reliable diagnostic system to identify single cattle (Bos taurus) vaccinated with RB51. So far, no data are available in the international scientific literature regarding the use of this test association in water buffalo. For this reason the suitability of this test combination has been evaluated in a water buffalo herd. One hundred twenty-seven animals farmed in a herd of Salerno province (Campania, Southern Italy), in the context of a presumptive unauthorized use of RB51 vaccine were chosen for this study. All tested animals resulted negative to Rose Bengal test (RBT) and complement fixation test (CFT) used for the detection of specific antibodies against Brucella field strains. Seventy-one animals (56%) developed RB51 antigen-specific CFT (RB51-CFT) antibodies against RB51 vaccine in a first sampling, while 104 animals (82%) gave positive result to a second serum sampling conducted 11 days after the intradermal inoculation of the RB51 brucellin. One hundred and seven animals (84%) showed a positive reaction to the RB51-CFT in at least 1 sampling, while 111 animals (87%) resulted positive to the RB51 brucellin skin test. Thus, analysing the results of the 3 testing in parallel, 119 animals (94%) were positive to at least 1 of the performed tests. The results suggest that the use in parallel of the RB51 brucellin skin test with RB51-CFT may represent a reliable

  11. Structural, functional and immunogenic insights on Cu,Zn superoxide dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

    SciTech Connect

    Pratt, Ashley J.; DiDonato, Michael; Shin, David S.; Cabelli, Diane E.; Bruns, Cami K.; Belzer, Carol A.; Gorringe, Andrew R.; Langford, Paul R.; Tabatabai, Louisa B.; Kroll, J. Simon; Tainer, John A.; Getzoff, Elizabeth D.

    2015-10-12

    Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens.

    IMPORTANCE By

  12. Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests.

    PubMed

    Nardi, G Júnior; Ribeiro, M G; Jorge, A M; Megid, J; Silva, L M P

    2012-03-01

    The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers.

  13. Polymorphonuclear Neutrophils Are Necessary for the Recruitment of CD8+ T Cells in the Liver in a Pregnant Mouse Model of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection

    PubMed Central

    de Oca, Roberto Montes; Buendía, Antonio J.; Del Río, Laura; Sánchez, Joaquín; Salinas, Jesús; Navarro, Jose A.

    2000-01-01

    The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+ cell populations decreased, particularly the CD8+ T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response. PMID:10679002

  14. Antigenic, Immunologic and Genetic Characterization of Rough Strains B.abortus RB51, B.melitensis B115 and B.melitensis B18

    PubMed Central

    Adone, Rosanna; Muscillo, Michele; La Rosa, Giuseppina; Francia, Massimiliano; Tarantino, Michela

    2011-01-01

    The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B.abortus RB51, B.melitensis B115 and B.melitensis B18. RB51 derived from B.abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B.melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis. PMID:22065984

  15. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice.

    PubMed

    Luo, Deyan; Ni, Bing; Li, Peng; Shi, Wei; Zhang, Songle; Han, Yue; Mao, Liwei; He, Yangdong; Wu, Yuzhang; Wang, Xiliang

    2006-05-01

    This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

  16. Oral vaccination with microencapsuled strain 19 vaccine confers enhanced protection against Brucella abortus strain 2308 challenge in red deer (Cervus elaphus elaphus).

    PubMed

    Arenas-Gamboa, Angela M; Ficht, Thomas A; Davis, Donald S; Elzer, Philip H; Kahl-McDonagh, Melissa; Wong-Gonzalez, Alfredo; Rice-Ficht, Allison C

    2009-10-01

    Bison (Bison bison) and elk (Cervus elaphus nelsoni) in the Greater Yellowstone Area (GYA), USA, are infected with Brucella abortus, the causative agent of bovine brucellosis, and they serve as a wildlife reservoir for the disease. Bovine brucellosis recently has been transmitted from infected elk to cattle in Montana, Wyoming, and Idaho and has resulted in their loss of brucellosis-free status. An efficacious Brucella vaccine with a delivery system suitable for wildlife would be a valuable tool in a disease prevention and control program. We evaluated Strain 19 (S19) in a sustained release vehicle consisting of alginate microspheres containing live vaccine. In a challenge study using red deer (Cervus elaphus elaphus) as a model for elk, alginate, a naturally occurring polymer combined with a protein of Fasciola hepatica vitelline protein B was used to microencapsulate S19. Red deer were orally or subcutaneously immunized with 1.5 x 10(10) colony-forming units (CFUs) using microencapsulated S19. Humoral and cellular profiles were analyzed bimonthly throughout the study. The vaccinated red deer and nonvaccinated controls were challenged 1 yr postimmunization conjunctivally with 1 x 10(9) CFUs of B. abortus strain 2308. Red deer vaccinated with oral microencapsulated S19 had a statistically significant lower bacterial tissue load compared with controls. These data indicate for the first time that protection against Brucella-challenge can be achieved by combining a commonly used vaccine with a novel oral delivery system such as alginate-vitelline protein B microencapsulation. This system is a potential improvement for efficacious Brucella-vaccine delivery to wildlife in the GYA.

  17. Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice.

    PubMed

    Li, Xianbo; Xu, Jie; Xie, Yongfei; Qiu, Yefeng; Fu, Simei; Yuan, Xitong; Ke, Yuehua; Yu, Shuang; Du, Xinying; Cui, Mingquan; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Huang, Kehe; Huang, Liuyu; Peng, Guangneng; Chen, Zeliang; Wang, Yufei

    2012-12-28

    Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

  18. Identification of an IS711 Element Interrupting the wboA Gene of Brucella abortus Vaccine Strain RB51 and a PCR Assay To Distinguish Strain RB51 from Other Brucella Species and Strains

    PubMed Central

    Vemulapalli, Ramesh; McQuiston, John R.; Schurig, Gerhardt G.; Sriranganathan, Nammalwar; Halling, Shirley M.; Boyle, Stephen M.

    1999-01-01

    Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested. PMID:10473532

  19. The bhuQ gene encodes a heme oxygenase that contributes to the ability of Brucella abortus 2308 to use heme as an iron source and is regulated by Irr.

    PubMed

    Ojeda, Jenifer F; Martinson, David A; Menscher, Evan A; Roop, R Martin

    2012-08-01

    The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl(3), but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source.

  20. Herd-level prevalence and associated risk factors for Toxoplasma gondii, Neospora caninum, Chlamydia abortus and bovine viral diarrhoea virus in commercial dairy and beef cattle in eastern, northern and northeastern China.

    PubMed

    Sun, Wu-Wen; Meng, Qing-Feng; Cong, Wei; Shan, Xiao-Feng; Wang, Chun-Feng; Qian, Ai-Dong

    2015-11-01

    Although the seroprevalence of Toxoplasma gondii, Neospora caninum, Chlamydia abortus and bovine viral diarrhea virus infection in cattle have been reported in some areas in China, most of them were conducted with small number of cattle samples and very limited districts and neglected the assessment of herd management factors associated with herd-level prevalence of these pathogen infections. Thus, from September 2013 to December 2014, a large-scale seroprevalence study was conducted to determine the animal-level and herd-level seroprevalence and identify herd-level risk factors associated with these pathogen infections in 4487 cattle from 134 herds in five provinces (Heilongjiang, Jilin, Liaoning, Shandong, Hebei) and Inner Mongolia Autonomous Region of China. At animal level, the true prevalence of antibodies against T. gondii, N. caninum, C. abortus and bovine viral diarrhoea virus (BVDV) was 10.48, 17.14, 11.92 and 50.10%, respectively. At herd level, the true prevalence of antibodies against T. gondii, N. caninum, C. abortus and BVDV was 27.16, 29.10, 37.31 and 40.30%, respectively. Multivariate analysis of these characteristics showed that source of water and presence of felids were significantly associated with T. gondii infection in the studied cattle herds. Source of water was significantly associated with N. caninum infection in the studied cattle herds. While herd size and management system were significantly associated with BVDV infection in the studied cattle herds, this is the first report of herd-level prevalence and associated risk factors of T. gondii, N. caninum, C. abortus and BVDV infection in cattle in China.

  1. Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection.

    PubMed

    Tabynov, Kaissar; Kydyrbayev, Zhailaubay; Ryskeldinova, Sholpan; Yespembetov, Bolat; Zinina, Nadezhda; Assanzhanova, Nurika; Kozhamkulov, Yerken; Inkarbekov, Dulat; Gotskina, Tatyana; Sansyzbay, Abylai

    2014-04-11

    This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus - a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1-1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4(+) and CD8(+) cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

  2. Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice

    PubMed Central

    Gómez, Leonardo A.; Alvarez, Francisco I.; Fernández, Pablo A.; Flores, Manuel R.; Molina, Raúl E.; Coloma, Roberto F.; Oñate, Angel A.

    2016-01-01

    Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis. PMID:27747197

  3. Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice.

    PubMed

    Riquelme-Neira, Roberto; Retamal-Díaz, Angello; Acuña, Francisca; Riquelme, Pablo; Rivera, Alejandra; Sáez, Darwin; Oñate, Angel

    2013-08-12

    The immunogenicity of a DNA vaccine containing an open reading frame (ORF) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the open reading frame with homology to an ABC-type transporter (pV278a) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pV278a had a dominant immunoglobulin G2a (IgG2a) response. This DNA vaccine elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 278a protein. Upon stimulation with an appropriate recombinant protein or crude Brucella protein, the vaccine did not induce IL-4, suggesting a typical T-helper (TH1) response. Furthermore, the vaccine induced protection in BALB/c mice when challenged with the virulent strain Brucella abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery of the homologous of an ABC-type transporter antigen, and provides the first evidence of a protective effect of this antigen in the construction of vaccines against B. abortus.

  4. Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

    PubMed Central

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Ding, Chan; Wang, Shaohui; Peng, Daxin

    2014-01-01

    We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants. PMID:24752516

  5. Chlamydophila abortus (Chlamydia psittaci serotype 1) clearance is associated with the early recruitment of neutrophils and CD8(+)T cells in a mouse model.

    PubMed

    Del Río, L; Buendía, A J; Sánchez, J; Garcés, B; Caro, M R; Gallego, M C; Bernabé, A; Cuello, F; Salinas, J

    2000-01-01

    The immune mechanisms in response to Chlamydophila abortus (Chlamydia psittaci serotype 1) infection were studied in C57BL/6 and CBA mice. The infection was monitored and the following aspects of the immune response were evaluated: the nature of the leucocyte infiltrate in the liver, the percentages of polymorphonuclear neutrophils (PMNs), macrophages and lymphocytes in the spleen, and the concentrations of cytokines in serum. In addition, the serum concentrations of IgG1 and IgG2a were determined. Both mouse strains showed a Th1-like immune response, with high concentrations of IFN-gamma and minimal levels of IL-4; however, C57 mice differed from CBA mice in showing milder clinical signs and earlier resolution of infection. The greater ability of C57 mice than CBA mice to eliminate chlamydophilae was related to the establishment of an earlier innate immunity, based on a more pronounced PMN response, and on a greater presence of CD8(+)T cells.

  6. Update: potential exposures to attenuated vaccine strain Brucella abortus RB51 during a laboratory proficiency test--United States and Canada, 2007.

    PubMed

    2008-01-18

    In November 2007, New York State Department of Health (NYSDOH) officials notified CDC of potential exposures to attenuated vaccine strain Brucella abortus RB51 (RB51) in multiple clinical laboratories that participated in a Laboratory Preparedness Survey (LPS) proficiency test. NYSDOH conducted a survey of participating laboratories and identified 17 laboratories that reported handling the RB51 sample in a manner placing lab workers at potential risk for exposure. Subsequently, CDC recommended that public health officials conduct a review of biosafety practices at all LPS-participating laboratories to identify any additional RB51 exposures. This report summarizes the results of investigations in 36 states, two cities, one county, and the District of Columbia. As of January 14, 2008, follow-up by public health officials with LPS-participating laboratories throughout the United States identified a total of 916 laboratory workers in 254 laboratories with potential RB51 exposure. The results highlight the need for routine adherence to recommended biosafety practices when working with infectious organisms, particularly during widespread infectious-disease events, including bioterrorism attacks.

  7. A serological study on Brucella abortus, caprine arthritis-encephalitis virus and Leptospira in dairy goats in Rio de Janeiro, Brazil.

    PubMed

    Lilenbaum, Walter; de Souza, Guilherme Nunes; Ristow, Paula; Moreira, Madelayne Cortez; Fráguas, Suzana; Cardoso, Verônica da Silva; Oelemann, Walter Martin Roland

    2007-03-01

    In spite of the large number of goats found in several developing tropical countries, milk production remains unsatisfactory. The occurrence of infectious diseases, such as leptospirosis, brucellosis and caprine arthritis-encephalitis (CAE) may in part be responsible for sub-optimal production. In this study, 1000 serum samples were tested for leptospirosis, 953 for brucellosis and 562 for CAE. All tested flocks presented at least one seroreactive animal for leptospirosis and for CAE. Reactivity to leptospirosis was 11.1%, and serovar hardjo was the most frequently found. Anti-B. abortus agglutinins were found in 0.5% of the samples presented and 14.1% were seroreactive to CAE. Leptospirosis was considered to represent the major infectious problem in the studied goat flocks. The occurrence of infectious diseases in the tested flocks may represent an important factor contributing to the decreased productivity of the animals. These findings may be similar to those observed in other developing countries and require further study to define the relationship between seropositivity and reduced production.

  8. The two-component systems PrrBA and NtrYX co-ordinately regulate the adaptation of Brucella abortus to an oxygen-limited environment.

    PubMed

    Carrica, Mariela Del Carmen; Fernandez, Ignacio; Sieira, Rodrigo; Paris, Gastón; Goldbaum, Fernando Alberto

    2013-04-01

    Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortus PrrBA is a functional two-component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two-component systems PrrBA and NtrYX co-ordinately regulate the expression of denitrification and high-affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild-type B. abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.

  9. A new cis-encoded sRNA, BsrH, regulating the expression of hemH gene in Brucella abortus 2308.

    PubMed

    Peng, Xiaowei; Dong, Hao; Wu, Qingmin

    2015-01-01

    A total of 129 sRNA candidates were identified in Brucella abortus 2308 in our previous work, and one candidate with potential to regulate expression of hemH gene was further analyzed in this study. We found that the novel sRNA can inhibit the expression of hemH and called it BsrH (Brucella sRNA regulating HemH). The expression level of BsrH was tested in four different stress conditions. A significant upregulation was detected during the growth in acidic and Brucella minimal media, as well as in the presence of hydroxyl peroxide, while iron deficiency caused the opposite effect. As expected, BsrH strongly affected the survival ratio of the Brucella cells under iron-limitation conditions, though overexpression of BsrH did not affect Brucella virulence. Thus, we conclude that BsrH plays a regulatory role in bacterial heme biosynthesis and can be considered as the first Brucella sRNA involved in stress responses.

  10. Evaluation of immunogenicity and protective efficacy of a plasmid DNA vaccine encoding ribosomal protein L9 of Brucella abortus in BALB/c mice.

    PubMed

    Jain, Shikha; Afley, Prachiti; Dohre, Sudhir K; Saxena, Nandita; Kumar, Subodh

    2014-07-31

    Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans and existing animal vaccines have limitations. We have previously described the ribosomal protein L9 to have the vaccine potential. In this study, L9 based DNA vaccine (pVaxL9) was generated and evaluated in mouse model. Intramuscular immunisation of pVaxL9 was able to elicit the anti-L9 IgG antibody response of both IgG1 and IgG2a isotypes when compared with PBS and pVax immunised control animals. Heightened antibody response was observed in mice groups immunised with pVaxL9 priming and recombinant L9 boosting (PB) and where pDNA immunisation was carried out by in vivo electroporation (EP). The vaccine groups proliferated splenocytes and released Th1 type cytokines e.g. IFN-γ, TNF-α, IL-2. Further, flow cytometric analysis revealed that IFN-γ was released by both by CD4+ and CD8+ T cells particularly in PB and EP groups when compared with mice immunised with empty control vector. The L9 based pDNA vaccine was able to confer significant protection in mice against challenge with virulent B. abortus with PB and EP groups offering better protection. Taken together, it can be concluded that L9 based DNA vaccine is immunogenic and confer protection in mouse model.

  11. Immunization of BALB/c mice with Brucella abortus 2308ΔwbkA confers protection against wild-type infection

    PubMed Central

    Li, Zhi-qiang; Gui, Dan; Sun, Zhi-hua; Zhang, Jun-bo; Zhang, Wen-zhi; Guo, Fei

    2015-01-01

    Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308ΔwbkA) and evaluated its virulence. The survival of 2308ΔwbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308ΔwbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔwbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep. PMID:26040616

  12. A novel Omp25-binding peptide screened by phage display can inhibit Brucella abortus 2308 infection in vitro and in vivo

    PubMed Central

    Zhang, Junbo; Guo, Fei; Huang, Xiaoqiang; Zhang, Hui; Wang, Yuanzhi; Yin, Shuanghong; Li, Zhiqiang

    2014-01-01

    Brucellosis is a globally distributed zoonotic disease affecting animals and humans, and current antibiotic and vaccine strategies are not optimal. The surface-exposed protein Omp25 is involved in Brucella virulence and plays an important role in Brucella pathogenesis during infection, suggesting that Omp25 could be a useful target for selecting potential therapeutic molecules to inhibit Brucella pathogenesis. In this study, we identified, we believe for the first time, peptides that bind specifically to the Omp25 protein of pathogens, using a phage panning technique, After four rounds of panning, 42 plaques of eluted phages were subjected to pyrosequencing. Four phage clones that bound better than the other clones were selected following confirmation by ELISA and affinity constant determination. The peptides selected could significantly inhibit Brucella abortus 2308 (S2308) internalization and intracellular growth in RAW264.7 macrophages, and significantly induce secretion of TNF-α and IL-12 in peptide- and S2308-treated cells. Any observed peptide (OP11, OP27, OP35 or OP40) could significantly inhibit S2308 infection in BALB/c mice. Moreover, the peptide OP11 was the best candidate peptide for inhibiting S2308 infection in vitro and in vivo. These results suggest that peptide OP11 has potential for exploitation as a peptide drug in resisting S2308 infection. PMID:24722798

  13. Immunization of BALB/c mice with Brucella abortus 2308ΔwbkA confers protection against wild-type infection.

    PubMed

    Li, Zhi-qiang; Gui, Dan; Sun, Zhi-hua; Zhang, Jun-bo; Zhang, Wen-zhi; Zhang, Hui; Guo, Fei; Chen, Chuang-fu

    2015-01-01

    Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308ΔwbkA) and evaluated its virulence. The survival of 2308ΔwbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308ΔwbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔwbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep.

  14. The Immunization of Guinea-pigs and Mice with a Whole-Culture Extract of a Smooth and a Rough Strain of Brucella abortus

    PubMed Central

    Keppie, J.; Witt, K.; Smith, H.

    1972-01-01

    A purified killed Br. abortus vaccine developed for use in man from the smooth strain 544 and previously tested in mice (Keppie, Witt and Smith, 1971) has now been shown to immunize guinea-pigs without the addition of adjuvant. A similar vaccine prepared from the rough strain 45/20 also immunized guinea-pigs but to a slightly lesser degree. The activity of both vaccines in the guinea-pig was markedly enhanced by the use of water-in-oil emulsions. A commercially available killed vaccine, “Duphavac”, prepared from strain 45/20 for use in cattle, was slightly more active in guinea-pigs than the emulsified whole-culture extract of “544” possibly because its oily adjuvant was different. In mice, all the “45/20” vaccines were only feebly protective even when given with oily adjuvant, in sharp contrast to the “544” vaccines which were highly active in this animal, with or without adjuvant. As expected, the “45/20” vaccines were much less agglutinogenic than the “544” vaccines but both produced a similar degree of delayed hypersensitivity in guinea-pigs. It is suggested that the whole-extract vaccine from strain 544 is suitable for a trial in man. PMID:4628447

  15. Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation.

    PubMed

    Paweska, J T; Potts, A D; Harris, H J; Smith, S J; Viljoen, G J; Dungu, B; Brett, O L; Bubb, M; Prozesky, L

    2002-03-01

    An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than

  16. The Lipopolysaccharide of Brucella abortus BvrS/BvrR Mutants Contains Lipid A Modifications and Has Higher Affinity for Bactericidal Cationic Peptides

    PubMed Central

    Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S.; Koch, Michel H. J.; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

    2005-01-01

    The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic β(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants. PMID:16077108

  17. Brucella abortus surveillance of cattle in Fiji, Papua New Guinea, Vanuatu, the Solomon Islands and a case for active disease surveillance as a training tool.

    PubMed

    Tukana, Andrew; Hedlefs, Robert; Gummow, Bruce

    2016-10-01

    There have been no surveys of the cattle population for brucellosis in the Pacific Island Countries and Territories (PICTs) for more than 15 years. This study used disease surveillance as a capacity building training tool and to examine some of the constraints that impede surveillance in PICTs. The study also developed and implemented a series of surveys for detecting antibodies to B. abortus in cattle in Fiji, Papua New Guinea, Vanuatu and the Solomon Islands contributing to OIE requirements. The findings indicated lack of funds, lack of technical capacity, shortage of veterinarians, high turnover of in-country officials and lack of awareness on the impacts of animal diseases on public health that were constraining active disease surveillance. During the development and implementation of the surveys, constraints highlighted were outdated census data on farm numbers and cattle population, lack of funds for mobilisation of officials to carry out the surveys, lack of equipment for collecting and processing samples, lack of staff knowledge on blood sampling, geographical difficulties and security in accessing farms. Some of the reasons why these were constraints are discussed with likely solutions presented. The detection surveys had the objectives of building capacity for the country officials and demonstrating freedom from brucellosis in cattle for PNG, Vanuatu and the Solomon Islands. PNG, Vanuatu and the Solomon Islands all demonstrated freedom from bovine brucellosis in the areas surveyed using the indirect ELISA test. Fiji had an outbreak of brucellosis, and the objective was to determine its distribution and prevalence on untested farms. The Muaniweni district surveyed during the training had a 95 % confidence interval for true prevalence between 1.66 and 5.45 %. The study showed that active disease surveillance could be used as a tool for training officials thus, improves surveillance capacity in resource poor countries.

  18. The lipopolysaccharide of Brucella abortus BvrS/BvrR mutants contains lipid A modifications and has higher affinity for bactericidal cationic peptides.

    PubMed

    Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S; Koch, Michel H J; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

    2005-08-01

    The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

  19. Investigating the Use of Protein Saver Cards for Storage and Subsequent Detection of Bovine Anti-Brucella abortus Smooth Lipopolysaccharide Antibodies and Gamma Interferon

    PubMed Central

    Commander, Nicola J.; Erdenlig, Sevil; McGiven, John A.; Stack, Judy

    2013-01-01

    Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature. PMID:23986318

  20. Immunological assay after vaccination of goats with Brucella melitensis Rev. I and Brucella abortus 45/20 vaccines at Saudi Arabia.

    PubMed

    Taher, S A; Ewais, M A

    1997-01-01

    The living smooth Brucella melitensis Rev. I vaccine given in the normal dose (7x10(8) organism) to goats 3 to 5 months of age stimulated a marked increase in agglutinin titer. Fractionation of pools of serum by gel filtration by anion exchange chromatography, using diethylaminoethyl (DEAE -) cellulose showed that both IgM and IgG agglutinins were present from 12 to 47 days after goats were vaccinated. Only mercaptoethanol (ME)-sensitive agglutinins were detected in most goats 4 months after vaccination, but 2 of 30 goats retained ME-resistant agglutinins for the 13 1/2 - month observation period. Fractionation of serums from goats representative of these 2 types of serologic response indicated that most goats had only IgM agglutinins, whereas the 2 given goats had activity in the IgG fraction. Adult goats given Brucella abortus 42/20 adjuvant vaccine and revaccinated 5 months later developed low agglutination titers to smooth antigen, and all became test positive to the antiglobulin test. Fractionation of serum of pools taken 1 week and 8 months after revaccination indicated that antibody activity was restricted to the IgG fraction. Sera from 5 vaccinated and nonvaccinated goats which were positive by bacteriological culture examination at necropsy 31 to 47 days after goats were given conjunctival inoculation of virulent B. melitensis had antibody activity in both IgG and IgM fractions. Test positive reaction to the ME, complement-fixation (CF), and antiglobulin (AG) test were restricted to the IgG-containing fractions of serum, whereas reactions to the agglutination (STT) and the card tests appeared with either or both fractions. The effect of these findings on the choice of tests for the differentiation of vaccination response is discussed.

  1. Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay.

    PubMed

    Ramírez-Pfeiffer, C; Díaz-Aparicio, E; Rodríguez-Padilla, C; Morales-Loredo, A; Alvarez-Ojeda, G; Gomez-Flores, R

    2008-06-15

    The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p<0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24-72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples.

  2. First evaluation of an influenza viral vector based Brucella abortus vaccine in sheep and goats: Assessment of safety, immunogenicity and protective efficacy against Brucella melitensis infection.

    PubMed

    Tabynov, Kaissar; Yespembetov, Bolat; Matikhan, Nurali; Ryskeldinova, Sholpan; Zinina, Nadezhda; Kydyrbayev, Zhailaubay; Assanzhanova, Nurika; Tabynov, Kairat; Renukaradhya, Gourapura J; Mukhitdinova, Gulnara; Sansyzbay, Abylai

    2016-12-25

    Previously we developed and evaluated a candidate influenza viral vector based Brucella abortus vaccine (Flu-BA) administered with a potent adjuvant Montanide Gel01 in cattle, which was found safe and highly effective. This study was aimed to establish a proof-of-concept of the efficacy of Flu-BA vaccine formulation in sheep and goats. We vaccinated sheep and goats with Flu-BA vaccine and as a positive control vaccinated a group of animals with a commercial B. melitensis Rev.1 vaccine. Clinically, both Flu-BA and Rev.1 vaccines were found safe. Serological analysis showed the animals received Flu-BA vaccine did not induce antibody response against Brucella Omp16 and L7/L12 proteins during the period of our study (56days post-initial vaccination, PIV). But observed significant antigen-specific T cell response indicated by increased lymphocyte stimulation index and enhanced secretion of IFN-γ at day 56 PIV in Flu-BA group. The Flu-BA vaccinated animals completely protected 57.1% of sheep and 42.9% of goats against B. melitensis 16M challenge. The severity of brucellosis in terms of infection index and colonization of Brucella in tissues was significantly lower in the Flu-BA group compared to negative control animals group. Nevertheless, positive control commercial Rev.1 vaccine provided strong antigen-specific T cell immunity and protection against B. melitensis 16M infection. We conclude that the Flu-BA vaccine induces a significant antigen-specific T-cell response and provides complete protection in approximately 50% of sheep and goats against B. melitensis 16M infection. Further investigations are needed to improve the efficacy of Flu-BA and explore its practical application in small ruminants.

  3. Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice.

    PubMed

    Retamal-Díaz, Angello; Riquelme-Neira, Roberto; Sáez, Darwin; Rivera, Alejandra; Fernández, Pablo; Cabrera, Alex; Guzmán, Carlos A; Oñate, Angel

    2014-11-01

    This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.

  4. Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

    PubMed Central

    Guidolin, Leticia S.; Morrone Seijo, Susana M.; Guaimas, Francisco F.

    2015-01-01

    ABSTRACT Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification. IMPORTANCE In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella

  5. TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro.

    PubMed

    Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

    2014-09-01

    Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4(+) T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2(-/-) and TLR4(-/-) mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2(-/-) and TLR4(-/-) mice. In addition, CD4(+) T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4(+) T cells from TLR2(-/-) and TLR4(-/-) mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2(-/-) and TLR4(-/-) mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2(-/-) and TLR4(-/-) mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.

  6. Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters ▿ †

    PubMed Central

    Sieira, Rodrigo; Arocena, Gastón M.; Bukata, Lucas; Comerci, Diego J.; Ugalde, Rodolfo A.

    2010-01-01

    Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (PvirB). Using different procedures, we isolated a 27-kDa protein that binds specifically to PvirB. This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses. PMID:19854911

  7. Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models

    PubMed Central

    Zúñiga-Ripa, Amaia; Barbier, Thibault; Conde-Álvarez, Raquel; Martínez-Gómez, Estrella; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Grilló, María Jesús; Letesson, Jean-Jacques; Iriarte, Maite

    2014-01-01

    The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt. PMID:24936050

  8. Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models.

    PubMed

    Zúñiga-Ripa, Amaia; Barbier, Thibault; Conde-Álvarez, Raquel; Martínez-Gómez, Estrella; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Grilló, María Jesús; Letesson, Jean-Jacques; Iriarte, Maite; Moriyón, Ignacio

    2014-08-15

    The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt.

  9. Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system

    PubMed Central

    Zhang, Hongtao; Palma, Angelina S; Zhang, Yibing; Childs, Robert A; Liu, Yan; Mitchell, Daniel A; Guidolin, Leticia S; Weigel, Wilfried; Mulloy, Barbara; Ciocchini, Andrés E; Feizi, Ten; Chai, Wengang

    2016-01-01

    The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2–13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems. PMID:27053576

  10. S-SAD phasing of monoclinic histidine kinase from Brucella abortus combining data from multiple crystals and orientations: an example of data-collection strategy and a posteriori analysis of different data combinations.

    PubMed

    Klinke, Sebastián; Foos, Nicolas; Rinaldi, Jimena J; Paris, Gastón; Goldbaum, Fernando A; Legrand, Pierre; Guimarães, Beatriz G; Thompson, Andrew

    2015-07-01

    The histidine kinase (HK) domain belonging to the light-oxygen-voltage histidine kinase (LOV-HK) from Brucella abortus is a member of the HWE family, for which no structural information is available, and has low sequence identity (20%) to the closest HK present in the PDB. The `off-edge' S-SAD method in macromolecular X-ray crystallography was used to solve the structure of the HK domain from LOV-HK at low resolution from crystals in a low-symmetry space group (P21) and with four copies in the asymmetric unit (∼108 kDa). Data were collected both from multiple crystals (diffraction limit varying from 2.90 to 3.25 Å) and from multiple orientations of the same crystal, using the κ-geometry goniostat on SOLEIL beamline PROXIMA 1, to obtain `true redundancy'. Data from three different crystals were combined for structure determination. An optimized HK construct bearing a shorter cloning artifact yielded crystals that diffracted X-rays to 2.51 Å resolution and that were used for final refinement of the model. Moreover, a thorough a posteriori analysis using several different combinations of data sets allowed us to investigate the impact of the data-collection strategy on the success of the structure determination.

  11. PLGA (85:15) nanoparticle based delivery of rL7/L12 ribosomal protein in mice protects against Brucella abortus 544 infection: A promising alternate to traditional adjuvants.

    PubMed

    Singh, Damini; Somani, Vikas Kumar; Aggarwal, Somya; Bhatnagar, Rakesh

    2015-12-01

    There is a compelling need for the development of suitable adjuvants for human use to enhance the efficacy of the upcoming vaccines for the prevention of life threatening infections. In the current study, we have tried to explore the immunogenic potential of nanoparticles (NPs) made of PLGA (poly lactic-co-glycolic acid), a biodegradable and biocompatible polymer approved by FDA for human use after entrapping rL7/L12 protein, an immunodominant antigen of Brucella. Adjuvant properties were exhibited by the formulation as it elicited high IgG antibody titers just after first immunization which increased significantly after the booster administration. A good elicitation of the Th1 cytokines especially IFN-γ was recorded. Amongst the IgG antibody subclasses, IgG1 remained the predominant subclass to be elicited in mice serum after immunization; however IgG1/2a ratio showed a mixed profile of Th1/Th2 response. Lymphocyte proliferation assay as a marker of amplification in cellular immunity demonstrated that the splenocytes of the immunized mice had a high proliferation index with reference to the control, revealing that L7/L12 entrapping PLGA nanoparticles are potent inducer of inflammatory cell response indispensable to combat Brucella infection. Enumeration of splenic CFU after 14 days of infection with Brucella abortus 544 showed a significant reduction in log CFU of splenic bacteria in the vaccinated mice as compared to the control group. Therefore it is evident that PLGA nano formulations delivering the entrapped vaccine candidate in mice elicit specific humoral as well as cellular responses specific to the entrapped Brucella antigen. So there is much promise in this approach and this work by highlighting the adjuvant properties of the PLGA nanospheres will accelerate the development of improved vaccines safe for human as well as veterinary use.

  12. "Necesita una vacuna": what Spanish-speakers want in text-message immunization reminders.

    PubMed

    Ahlers-Schmidt, Carolyn R; Chesser, Amy; Brannon, Jennifer; Lopez, Venessa; Shah-Haque, Sapna; Williams, Katherine; Hart, Traci

    2013-08-01

    Appointment reminders help parents deal with complex immunization schedules. Preferred content of text-message reminders has been identified for English-speakers. Spanish-speaking parents of children under three years old were recruited to develop Spanish text-message immunization reminders. Structured interviews included questions about demographic characteristics, use of technology, and willingness to receive text reminders. Each participant was assigned to one user-centered design (UCD) test: card sort, needs analysis or comprehension testing. Respondents (N=54) were female (70%) and averaged 27 years of age (SD=7). A card sort of 20 immunization-related statements resulted in identification of seven pieces of critical information, which were compiled into eight example texts. These texts were ranked in the needs assessment and the top two were assessed for comprehension. All participants were able to understand the content and describe intention to act. Utilizing UCD testing, Spanish-speakers identified short, specific text content that differed from preferred content of English-speaking parents.

  13. Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

    PubMed Central

    Pacheco, W. A.; Genovez, M. E.; Pozzi, C. R.; Silva, L. M. P.; Azevedo, S. S.; Did, C. C.; Piatti, R. M.; Pinheiro, E. S.; Castro, V.; Miyashiro, S.; Gambarini, M. L.

    2012-01-01

    This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years. PMID:24031869

  14. Prevalence of Brucella abortus antibodies in equines of a tropical region of Mexico.

    PubMed

    Acosta-González, Rosa I; González-Reyes, Ismael; Flores-Gutiérrez, Gerardo H

    2006-10-01

    A cross-sectional study was conducted to determinate the seroprevalence rate of equine brucellosis in the state of Tamaulipas, Mexico. Serum samples from 420 equines were analyzed with the Rose Bengal test at cell concentrations of 3% (RBT-3%) and 8% (RBT-8%), and positive results were confirmed with the Rivanol test (RT). Risk factors were determined with the prevalence ratio (PR) and the use of variables generated from a questionnaire administered to the animals' owners. Serum from 1 stallion had positive results with both the RBT-8% and the RT, for a seroprevalence rate of 0.238%. Drinking of water from a pond that was also used by cattle and dogs was the only associated risk factor for this animal (PR = 0.25). However, the results were considered false-positive, because the results for other horses in the same environmental conditions were negative. Although brucellosis is considered endemic in ruminants in the study area, the results obtained suggest that equines are not a reservoir of brucellosis and do not play an important role in the epidemiologic patterns of this disease in northeastern Mexico.

  15. Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

    PubMed

    De Santis, Riccardo; Ancora, Massimo; De Massis, Fabrizio; Ciammaruconi, Andrea; Zilli, Katiuscia; Di Giannatale, Elisabetta; Pittiglio, Valentina; Fillo, Silvia; Lista, Florigio

    2013-10-01

    Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

  16. LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus.

    PubMed

    Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A; Briggs, Winslow R; Goldbaum, Fernando A; Paris, Gastón

    2015-01-01

    Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.

  17. U-Omp19 from Brucella abortus Is a Useful Adjuvant for Vaccine Formulations against Salmonella Infection in Mice

    PubMed Central

    Risso, Gabriela S.; Carabajal, Marianela V.; Bruno, Laura A.; Ibañez, Andrés E.; Coria, Lorena M.; Pasquevich, Karina A.; Lee, Seung-Joo; McSorley, Stephen J.; Briones, Gabriel; Cassataro, Juliana

    2017-01-01

    Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1–Th17 protective immune responses. PMID:28261222

  18. U-Omp19 from Brucella abortus Is a Useful Adjuvant for Vaccine Formulations against Salmonella Infection in Mice.

    PubMed

    Risso, Gabriela S; Carabajal, Marianela V; Bruno, Laura A; Ibañez, Andrés E; Coria, Lorena M; Pasquevich, Karina A; Lee, Seung-Joo; McSorley, Stephen J; Briones, Gabriel; Cassataro, Juliana

    2017-01-01

    Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1-Th17 protective immune responses.

  19. Immune responses and safety after dart or booster vaccination of bison with Brucella abortus strain RB51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One alternative in the Bison remote vaccination environmental impact statement (EIS) for Yellowstone National Park includes inoculation of both calves and yearlings. Although RB51 vaccination of bison does protect against experimental challenge, it was unknown whether booster vaccination might enhan...

  20. Identification of the Quorum-Sensing Target DNA Sequence and N-Acyl Homoserine Lactone Responsiveness of the Brucella abortus virB promoter▿

    PubMed Central

    Arocena, Gastón M.; Sieira, Rodrigo; Comerci, Diego J.; Ugalde, Rodolfo A.

    2010-01-01

    VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (PvirB) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in PvirB by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in PvirB increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of PvirB. Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella. PMID:20400542

  1. Expression of tfx and sensitivity to the rhizobial peptide antibiotic trifolitoxin in a taxonomically distinct group of alpha-proteobacteria including the animal pathogen Brucella abortus.

    PubMed Central

    Triplett, E W; Breil, B T; Splitter, G A

    1994-01-01

    Three phylogenetically distinct groups within the alpha-proteobacteria which differ in trifolitoxin sensitivity are described. Trifolitoxin sensitivity was found in strains of Agrobacterium, Brucella, Mycoplana, Ochrobactrum, Phyllobacterium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, and Rhizobium. Strains of Agrobacterium, Brucella, Phyllobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of tfxABCDEFG. PMID:7527627

  2. Topology prediction of Brucella abortus Omp2b and Omp2a porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods.

    PubMed

    Paquet, J Y; Vinals, C; Wouters, J; Letesson, J J; Depiereux, E

    2000-02-01

    In order to propose a reliable model for Brucella porin topology, several structure prediction methods were evaluated in their ability to predict porin topology. Four porins of known structure were selected as test-cases and their secondary structure delineated. The specificity and sensitivity of 11 methods were separately evaluated. Our critical assessment shows that some secondary structure prediction methods (PHD, Dsc, Sopma) originally designed to predict globular protein structure are useful on porin topology prediction. The overall best prediction is obtained by combining these three "generalist" methods with a transmembrane beta strand prediction technique. This "consensus" method was applied to Brucella porins Omp2b and Omp2a, sharing no sequence homology with any other porin. The predicted topology is a 16-stranded antiparallel beta barrel with Omp2a showing a higher number of negatively charged residue in the exposed loops than Omp2b. Experiments are in progress to validate the proposed topology and the functional hypotheses. The ability of the proposed consensus method to predict topology of complex outer membrane protein is briefly discussed.

  3. Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model

    PubMed Central

    2004-01-01

    Abstract Q fever and chlamydiosis often affect ovine and caprine flocks simultaneously or successively. Combination vaccines effective against these 2 diseases would be of great value in veterinary medicine. Unfortunately, the current effective vaccines are a live vaccine for chlamydiosis and killed vaccine for Q fever. Vaccination of mice with live chlamydiosis vaccine 1B and killed phase I vaccine against Q fever at 2 points on the back at the same time produced good protection against chlamydial abortion. This suggests that it may be possible to vaccinate ewes and goats against chlamydiosis and Q fever simultaneously. PMID:15352550

  4. Identification of Protective Epitopes by Sequencing of the Major Outer Membrane Protein Gene of a Variant Strain of Chlamydia psittaci Serotype 1 (Chlamydophila abortus)

    PubMed Central

    Vretou, Evangelia; Psarrou, Evgenia; Kaisar, Maria; Vlisidou, Isabella; Salti-Montesanto, Viviane; Longbottom, David

    2001-01-01

    Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the family Chlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs. PMID:11119563

  5. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  6. Demonstration of a Robot-Based Raman Spectroscopic Detector for the Identification of CBE Threat Agents

    DTIC Science & Technology

    2006-11-01

    Brucella abortus Bacillus anthracis (LD Viable) Raman...Yersinia pestis Burkholderia mallei Brucella abortus Bacillus anthracis (LD Viable) Raman Shift (cm-1 ) O f f s e t I n t e n s i t y © ChemImage...Hierarchical Cluster Analysis 0 0.5 1 1.5 2 2.5 3 Burkholderia mallei Ricin Brucella abortus Francisella tularensis Yersina pestis Bacillus anthracis

  7. Identification of Protective Brucella Antigens and their Expressions in Vaccinia Virus to Prevent Disease in Animals and Humans.

    DTIC Science & Technology

    1996-05-01

    cattle, sheep, goats , dogs, and camels. Important species of Brucella are: suis, abortus, ovis, melitensis , canis, and neotomae, each with certain...B. abortus is strain 19 and for protecting goats against B. melitensis is strain Rev 1. Vaccination with these strains leads to seroconversion...encoding the C-terminus portion of the Brucella protein. Western blot analysis of B. abortus and B. melitensis was performed using antibodies raised against

  8. The Brucellosis Eradication Program in Texas

    DTIC Science & Technology

    1983-09-01

    no reported exposures consistent with brucellosis. ....... Brucella organism was cultured from all the human infections. Brucella melitensis is the...abortus; (b) Brucella suis; and (c) Brucella melitensis . The most common organism that has caused the greatest threat in Texas is Brucella abortus...veterinarians. Initially the program consisted of calfhood vaccination with Strain 19 Brucella abortus vaccine and test and slaughter of infected herds and

  9. The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae

    PubMed Central

    Guzmán-Verri, C.; Manterola, L.; Sola-Landa, A.; Parra, A.; Cloeckaert, A.; Garin, J.; Gorvel, J.-P.; Moriyón, I.; Moreno, E.; López-Goñi, I.

    2002-01-01

    The Brucella BvrR/BvrS two-component regulatory system is homologous to the ChvI/ChvG systems of Sinorhizobium meliloti and Agrobacterium tumefaciens necessary for endosymbiosis and pathogenicity in plants. BvrR/BvrS controls cell invasion and intracellular survival. Probing the surface of bvrR and bvrS transposon mutants with monoclonal antibodies showed all described major outer membrane proteins (Omps) but Omp25, a protein known to be involved in Brucella virulence. Absence of Omp25 expression was confirmed by two-dimensional electrophoresis of envelope fractions and by gene reporter studies. The electrophoretic analysis also revealed reduction or absence in the mutants of a second set of protein spots that by matrix-assisted laser desorption ionization MS and peptide mass mapping were identified as a non-previously described Omp (Omp3b). Because bvrR and bvrS mutants are also altered in cell-surface hydrophobicity, permeability, and sensitivity to surface-targeted bactericidal peptides, it is proposed that BvrR/BvrS controls cell envelope changes necessary to transit between extracellular and intracellular environments. A genomic search revealed that Omp25 (Omp3a) and Omp3b belong to a family of Omps of plant and animal cell-associated α-Proteobacteria, which includes Rhizobium leguminosarum RopB and A. tumefaciens AopB. Previous work has shown that RopB is not expressed in bacteroids, that AopB is involved in tumorigenesis, and that dysfunction of A. tumefaciens ChvI/ChvG alters surface properties. It is thus proposed that the BvrR/BvrS and Omp3 homologues of the cell-associated α-Proteobacteria play a role in bacterial surface control and host cell interactions. PMID:12218183

  10. Colorectal Cancer: What You Should Know

    MedlinePlus

    ... Médicos Dispositivos que Emiten Radiación Fraude en la Salud Medicamentos Nutrición Productos Veterinarios Productos de Tabaco Salud Infantil Salud de la Mujer Suplementos Dietéticos Vacunas, ...

  11. Immunizations

    MedlinePlus

    ... vacunas Why Are Vaccinations Important? Measles, mumps, and whooping cough may seem like quaint old illnesses confined to ... pregnancy to help protect the baby against pertussis (whooping cough). The good news is you can still get ...

  12. In vivo differences in the virulence, pathogenicity, and induced protective immunity of wboA mutants from genetically different parent Brucella spp.

    PubMed

    Wang, Zhen; Niu, Jianrui; Wang, Shuangshan; Lv, Yanli; Wu, Qingmin

    2013-02-01

    To explore the effects of the genetic background on the characteristics of wboA gene deletion rough mutants generated from different parent Brucella sp. strains, we constructed the rough-mutant strains Brucella melitensis 16 M-MB6, B. abortus 2308-SB6, B. abortus S19-RB6, and B. melitensis NI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks for B. abortus S19-RB6 to 11 weeks for B. abortus 2308-SB6 and B. melitensis NI-NB6. However, B. abortus S19-RB6 and B. melitensis 16 M-MB6, with a shorter survival time in mice, offered better protection against challenges with B. abortus 2308 in protection tests than B. abortus 2308-SB6 and B. melitensis NI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival time in vivo. In the cross-protection assay, both B. melitensis 16 M-MB6 and B. abortus S19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated with B. abortus S19-RB6 and B. melitensis 16 M-MB6, B. abortus S19-RB6 did not induce abortion, whereas B. melitensis 16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunity in vivo in the wboA deletion mutants from genetically different parent Brucella spp. and also indicated that future rough vaccine strain development could be promising if suitable parent Brucella strains and/or genes were selected.

  13. Genotyping of Ochrobactrum spp. by AFLP analysis.

    PubMed

    Leal-Klevezas, Diana Sara; Martínez-de-la-Vega, Octavio; Ramírez-Barba, Ector Jaime; Osterman, Björn; Martínez-Soriano, Juan Pablo; Simpson, June

    2005-04-01

    AFLP was used to analyze the genetic diversity among Ochrobactrum strains. AFLP patterns showed a great genomic variability that separated the samples into three distinct clusters. Ochrobactrum intermedium was found to be closely related to Brucella abortus S99.

  14. INL Researchers Advance Detection of Brucellosis

    SciTech Connect

    Roberto, Frank; Newby, Deborah

    2008-08-06

    What do cattle ranchers in the greater Yellowstone region have in common with British soldiers garrisoned on the island of Malta in the late 1800s? Hint: it's a pathogen that starts with the letter B. It's Brucella Abortus.

  15. INL Researchers Advance Detection of Brucellosis

    ScienceCinema

    Roberto, Frank; Newby, Deborah

    2016-07-12

    What do cattle ranchers in the greater Yellowstone region have in common with British soldiers garrisoned on the island of Malta in the late 1800s? Hint: it's a pathogen that starts with the letter B. It's Brucella Abortus.

  16. Immunofluorescent studies of the local immune response in the mammary glands of rats.

    PubMed Central

    Lee, C G; Ladds, P W; Watson, D L; Goddard, M E

    1979-01-01

    Two irritants, phosphate-buffered saline and alcohol, and antigens including killed Brucella abortus, live B. abortus, Staphylococcus aureus, and rat parvovirus were separately infused into rat mammary glands during pregnancy, and by using immunofluorescent techniques, the numbers of immunoglobulin-containing cells in glands during lactation and involution were determined. The study provided basic information on the local immune response of the mammary gland to antigens of various types. In all experiments, the number of immunoglobulin M (IgM) cells present was small and no trends were apparent. IgA cells were always more prevalent than IgG cells. Fewer IgA cells were in the glands of rats infused with phosphate-buffered saline and alcohol than in normal rats. IgA cell prevalence was greatest in response to infusion of live B. abortus. Responses to live S. aureus and parvovirus were less pronounced, and infusion of killed B. abortus did not induce an elevation in IgA cell prevalence. IgG cell prevalence was greatest in response to infusion of live B. abortus or S. aureus and was decreasingly less pronounced in response to killed B. abortus and rat parvovirus. With the exception of parvovirus infusion, in regard to IgA cells, all glands locally infused with antigen had elevated IgA and IgG cell numbers when compared with noninfused glands in the same animal. Images PMID:106012

  17. Brucella cultures typed by the Who Brucellosis Centre at the Commonwealth Serum Laboratories, Melbourne.

    PubMed

    Ekers, B M

    1978-09-01

    Results of the typing of Brucella cultures received at the WHO Brucellosis Centre, CSL, Melbourne, from 1968-1976 are presented. The distribution of the biotypes of cultures recovered throughout Australia is shown on a host and State basis and atypical cultures are discussed. Cultures identified from Australia were Br. abortus, biotypes 1, 2 and 4 and Strain 19; Br. suis, biotype 1 and Br. ovis. Br. melitensis biotypes 1, 2 and 3 were recorded only as exotic human infections from the Mediterranean area and from laboratory infections. Br. abortus, biotype 1 was the most common bovine and human isolate and was found in horses, a goat and a sheep. There was a low incidence of Br. abortus, biotype 2 in cattle and it was found in 1 horse. Br. abortus, biotype 3 was not found in Australia but was submitted from Tanzania. Br. abortus, biotype 4 was rare in cattle and was found once in a horse. Br. abortus, Strain 19 was found occasionally in cattle and once from a test guinea pig. Br. suis, biotype 1 was found in both man and pigs in Queensland and New South Wales whereas biotype 3 was isolated only in New Guinea from pigs and cattle. Br. ovis was submitted from 3 States. Br. canis has not been found in Australia.

  18. Detection and genotyping of Chlamydia species responsible for reproductive disorders in Algerian small ruminants.

    PubMed

    Merdja, Salah-Eddine; Khaled, Hamza; Aaziz, Rachid; Vorimore, Fabien; Bertin, Claire; Dahmani, Ali; Bouyoucef, Abdallah; Laroucau, Karine

    2015-02-01

    Chlamydiosis in small ruminants is a zoonotic disease mainly related to Chlamydia abortus. This bacterium is responsible for abortions and reproductive disorders in sheep and goats. Stillbirth and infertility, leading to important economic losses, are also associated with this pathology. In Algeria, abortion cases are frequently reported by veterinarians but, except for brucellosis which is a notifiable disease in this country, abortive diseases are in general poorly studied. In order to detect and genotype Chlamydia species in small ruminants in different areas of Algeria, a study was conducted on samples collected from females (164 blood samples and 199 vaginal swabs) between October 2011 and March 2013. Serum samples were tested with a C. abortus-specific indirect ELISA test. Fourteen samples (8.5 %), from six farms (6/20, 30 %) were tested positive. Vaginal swabs were analysed with a real-time PCR targeting all Chlamydiaceae spp. Thirty samples (15 %) were diagnosed positive in 16 farms (16/25, 64 %). Positive samples were all re-tested with a C. abortus- and a C. pecorum-specific real-time PCR. Finally, 13/30 (43.3 %) and 6/30 (20 %) were identified as C. abortus and C. pecorum, respectively. Enough concentrated C. abortus samples were genotyped by multi-loci variable number of tandem repeat (VNTR) analysis (MLVA), and all were related to the genotype [2] group which mainly includes French C. abortus isolates. C. pecorum-positive samples were genotyped by multi-locus sequence typing (MLST). Interestingly, two of them were successfully genotyped and showed identical MLST sequences to VB2, AB10, E58 and SBE, a group which includes C. pecorum isolates considered as highly pathogenic. These findings suggest a possible role of C. abortus and C. pecorum strains in the aetiology of abortion in Algerian small ruminants.

  19. Pathology of brucellosis in bison from Yellowstone National Park

    USGS Publications Warehouse

    Rhyan, Jack C.; Gidlewski, T.; Roffe, T.J.; Aune, K.; Philo, L.M.; Ewalt, D.R.

    2001-01-01

    Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six

  20. Brucellosis Transmission between Wildlife and Livestock in the Greater Yellowstone Ecosystem: Inferences from DNA Genotyping.

    PubMed

    O'Brien, Michael P; Beja-Pereira, Albano; Anderson, Neil; Ceballos, Ruben M; Edwards, William H; Harris, Beth; Wallen, Rick L; Costa, Vânia

    2017-04-01

    The wildlife of the Greater Yellowstone Ecosystem carries brucellosis, which was first introduced to the area by cattle in the 19th century. Brucellosis transmission between wildlife and livestock has been difficult to study due to challenges in culturing the causative agent, Brucella abortus . We examined B. abortus transmission between American bison ( Bison bison ), Rocky Mountain elk ( Cervus elaphus nelsoni), and cattle ( Bos taurus ) using variable number tandem repeat (VNTR) markers on DNA from 98 B. abortus isolates recovered from populations in Idaho, Montana, and Wyoming, US. Our analyses reveal interspecies transmission. Two outbreaks (2007, 2008) in Montana cattle had B. abortus genotypes similar to isolates from both bison and elk. Nevertheless, similarity in elk and cattle isolates from the 2008 outbreak suggest that elk are the likely source of brucellosis transmission to cattle in Montana and Wyoming. Brucella abortus isolates from sampling in Montana appear to be divided in two clusters: one found in local Montana elk, cattle, and bison; and another found mainly in elk and a bison from Wyoming, which is consistent with brucellosis having entered Montana via migration of infected elk from Wyoming. Our findings illustrate complex patterns of brucellosis transmission among elk, bison, and cattle as well as the utility of VNTRs to infer the wildlife species of origin for disease outbreaks in livestock.

  1. Molecular identification of chlamydial cause of abortion in small ruminants in Jordan.

    PubMed

    Ababneh, Huthaifa Salah; Ababneh, Mustafa Mohammed Kheir; Hananeh, Wael Mahmoud; Alsheyab, Fawzi Mohammad; Jawasreh, Khaleel Ibraheem; Al-Gharaibeh, Moath Ahmad; Ababneh, Mohammed Mahmoud

    2014-12-01

    Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.

  2. Serologic Survey of Snowshoe Hares (Lepus americanus) in the Greater Yellowstone Area for Brucellosis, Tularemia, and Snowshoe Hare Virus.

    PubMed

    Tyers, Dan; Zimmer, Jeremy; Lewandowski, Kristen; Hennager, Steve; Young, John; Pappert, Ryan; Panella, Amanda; Kosoy, Olga

    2015-07-01

    We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population.

  3. Blue-light-activated histidine kinases: two-component sensors in bacteria.

    PubMed

    Swartz, Trevor E; Tseng, Tong-Seung; Frederickson, Marcus A; Paris, Gastón; Comerci, Diego J; Rajashekara, Gireesh; Kim, Jung-Gun; Mudgett, Mary Beth; Splitter, Gary A; Ugalde, Rodolfo A; Goldbaum, Fernando A; Briggs, Winslow R; Bogomolni, Roberto A

    2007-08-24

    Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

  4. Comparative evaluation of the enzyme-linked immunosorbent assay in the laboratory diagnosis of brucellosis.

    PubMed Central

    De Klerk, E; Anderson, R

    1985-01-01

    An enzyme-linked immunosorbent assay was adapted to measure total and Brucella abortus-specific immunoglobulin M antibodies. The results were compared with those of conventional serological tests for B. abortus antibody on the sera of a number of normal controls, apparently healthy occupationally exposed workers, and patients with suspected acute brucellosis. Relative to other tests, the B. abortus enzyme-linked immunosorbent assay was found to be both highly sensitive and highly specific. The serological results obtained in occupationally exposed workers indicate a higher "normal range" for this group and therefore a possibility of false-positive results and overdiagnosis. It is therefore important to establish a separate "normal range" for occupationally exposed workers. Investigation of patients with acute brucellosis showed that the enzyme-linked immunosorbent assay for immunoglobulin M was the most sensitive serodiagnostic test and was likely to be of value in the serodiagnosis of acute brucellosis in occupationally exposed workers. PMID:3920241

  5. Evaluating the virulence of a Brucella melitensis hemagglutinin gene in the caprine model.

    PubMed

    Perry, Quinesha L; Hagius, Sue D; Walker, Joel V; Elzer, Philip H

    2010-10-01

    With the completion of the genomic sequence of Brucella melitensis 16M, a putative hemagglutinin gene was identified which is present in 16M and absent in Brucella abortus. The possibility of this hemagglutinin being a potential virulence factor was evaluated via gene replacement in B. melitensis yielding 16MΔE and expression in trans in B. abortus 2308-QAE. Utilizing the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate these strains. B. melitensis 16M hemagglutinin gene expression in trans in 2308-QAE revealed a significant (p≤0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking B. melitensis 16M virulence in pregnant goats. The B. melitensis disruption mutant's colonization and abortion rates demonstrated no attenuation in colonization but displayed a 28% reduction in abortions when compared to parental B. melitensis 16M.

  6. Maintenance of brucellosis in Yellowstone bison: linking seasonal food resources, host–pathogen interaction, and life-history trade-offs

    PubMed Central

    Treanor, John J; Geremia, Chris; Ballou, Michael A; Keisler, Duane H; White, Patrick J; Cox, John J; Crowley, Philip H

    2015-01-01

    The seasonal availability of food resources is an important factor shaping the life-history strategies of organisms. During times of nutritional restriction, physiological trade-offs can induce periods of immune suppression, thereby increasing susceptibility to infectious disease. Our goal was to provide a conceptual framework describing how the endemic level bovine brucellosis (Brucella abortus) may be maintained in Yellowstone bison based on the seasonality of food resources and the life-history strategies of the host and pathogen. Our analysis was based on active B. abortus infection (measured via bacterial culture), nutritional indicators (measured as metabolites and hormones in plasma), and carcass measurements of 402 slaughtered bison. Data from Yellowstone bison were used to investigate (1) whether seasonal changes in diet quality affect nutritional condition and coincide with the reproductive needs of female bison; (2) whether active B. abortus infection and infection intensities vary with host nutrition and nutritional condition; and (3) the evidence for seasonal changes in immune responses, which may offer protection against B. abortus, in relation to nutritional condition. Female bison experienced a decline in nutritional condition during winter as reproductive demands of late gestation increased while forage quality and availability declined. Active B. abortus infection was negatively associated with bison age and nutritional condition, with the intensity of infection negatively associated with indicators of nutrition (e.g., dietary protein and energy) and body weight. Data suggest that protective cell-mediated immune responses may be reduced during the B. abortus transmission period, which coincides with nutritional insufficiencies and elevated reproductive demands during spring. Our results illustrate how seasonal food restriction can drive physiological trade-offs that suppress immune function and create infection and transmission opportunities

  7. Maintenance of brucellosis in Yellowstone bison: linking seasonal food resources, host-pathogen interaction, and life-history trade-offs.

    PubMed

    Treanor, John J; Geremia, Chris; Ballou, Michael A; Keisler, Duane H; White, Patrick J; Cox, John J; Crowley, Philip H

    2015-09-01

    The seasonal availability of food resources is an important factor shaping the life-history strategies of organisms. During times of nutritional restriction, physiological trade-offs can induce periods of immune suppression, thereby increasing susceptibility to infectious disease. Our goal was to provide a conceptual framework describing how the endemic level bovine brucellosis (Brucella abortus) may be maintained in Yellowstone bison based on the seasonality of food resources and the life-history strategies of the host and pathogen. Our analysis was based on active B. abortus infection (measured via bacterial culture), nutritional indicators (measured as metabolites and hormones in plasma), and carcass measurements of 402 slaughtered bison. Data from Yellowstone bison were used to investigate (1) whether seasonal changes in diet quality affect nutritional condition and coincide with the reproductive needs of female bison; (2) whether active B. abortus infection and infection intensities vary with host nutrition and nutritional condition; and (3) the evidence for seasonal changes in immune responses, which may offer protection against B. abortus, in relation to nutritional condition. Female bison experienced a decline in nutritional condition during winter as reproductive demands of late gestation increased while forage quality and availability declined. Active B. abortus infection was negatively associated with bison age and nutritional condition, with the intensity of infection negatively associated with indicators of nutrition (e.g., dietary protein and energy) and body weight. Data suggest that protective cell-mediated immune responses may be reduced during the B. abortus transmission period, which coincides with nutritional insufficiencies and elevated reproductive demands during spring. Our results illustrate how seasonal food restriction can drive physiological trade-offs that suppress immune function and create infection and transmission opportunities

  8. Protective Properties of Rifampin-Resistant Rough Mutants of Brucella melitensis

    PubMed Central

    Adone, R.; Ciuchini, F.; Marianelli, C.; Tarantino, M.; Pistoia, C.; Marcon, G.; Petrucci, P.; Francia, M.; Riccardi, G.; Pasquali, P.

    2005-01-01

    Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species. PMID:15972510

  9. Protective properties of rifampin-resistant rough mutants of Brucella melitensis.

    PubMed

    Adone, R; Ciuchini, F; Marianelli, C; Tarantino, M; Pistoia, C; Marcon, G; Petrucci, P; Francia, M; Riccardi, G; Pasquali, P

    2005-07-01

    Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.

  10. Feasibility of quarantine procedures for bison (Bison bison) calves from Yellowstone National Park for conservation of brucellosis-free bison.

    PubMed

    Ryan Clarke, P; Frey, Rebecca K; Rhyan, Jack C; McCollum, Matt P; Nol, Pauline; Aune, Keith

    2014-03-01

    OBJECTIVE--To determine the feasibility of qualifying individuals or groups of Yellowstone National Park bison as free from brucellosis. DESIGN--Cohort study. SAMPLE--Serum, blood, and various samples from live bison and tissues taken at necropsy from 214 bison over 7 years. PROCEDURES--Blood was collected from bison every 30 to 45 days for serologic tests and microbiological culture of blood for Brucella abortus. Seropositive bison were euthanized until all remaining bison had 2 consecutive negative test results. Half the seronegative bison were randomly euthanized, and tissues were collected for bacteriologic culture. The remaining seronegative bison were bred, and blood was tested at least twice per year. Cow-calf pairs were sampled immediately after calving and 6 months after calving for evidence of B abortus. RESULTS--Post-enrollment serial testing for B abortus antibodies revealed no bison that seroconverted after 205 days (first cohort) and 180 days (second cohort). During initial serial testing, 85% of bison seroconverted within 120 days after removal from the infected population. Brucella abortus was not cultured from any euthanized seronegative bison (0/88). After parturition, no cows or calves had a positive test result for B abortus antibodies, nor was B abortus cultured from any samples. CONCLUSIONS AND CLINICAL RELEVANCE--Results suggested it is feasible to qualify brucellosis-free bison from an infected herd following quarantine procedures as published in the USDA APHIS brucellosis eradication uniform methods and rules. Latent infection was not detected in this sample of bison when applying the USDA APHIS quarantine protocol.

  11. Diagnosis of placental pathogens in small ruminants by immunohistochemistry and PCR on paraffin-embedded samples.

    PubMed

    Navarro, J A; Ortega, N; Buendia, A J; Gallego, M C; Martínez, C M; Caro, M R; Sánchez, J; Salinas, J

    2009-08-08

    A histological study was carried out on 58 formalin-fixed and paraffin-embedded samples of placenta from sheep and goats that had aborted, and the placental lesions were graded. Sequential histological sections of each cotyledon were then immunostained with specific antibodies and used for PCR detection of Chlamydophila abortus, Coxiella burnetii, Salmonella Abortusovis, Brucella melitensis, Listeria monocytogenes and Toxoplasma gondii. Most of the cotyledons showed different degrees of placentitis. The proportional agreement between the two techniques was 0.879 (kappa value 0.746). C abortus was the most prevalent pathogen. Mixed infections were common.

  12. Chlamydial infections in small ruminants.

    PubMed

    Nietfeld, J C

    2001-07-01

    Chlamydophila abortus (formerly Chlamydia psittaci) is one of the most important causes of reproductive failure in sheep and goats, especially in intensively managed flocks. The disease is usually manifested as abortion in the last 2 to 3 weeks of gestation, regardless of when the animal was infected. Ewes that abort are resistant to future reproductive failure due to C. abortus, but they become inapparent carriers and persistently shed the organism from their reproductive tracts during estrus. Chlamydophila pecorum is the other member of the genus that affects small ruminants, and it is recognized as a primary cause of keratoconjunctivitis in sheep and goats and of polyarthritis in sheep.

  13. Brucellosis in the United States: Role and Significance of Wildlife Reservoirs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Regulatory programs for brucellosis in domestic livestock have been active in the United States for almost 80 years. Wildlife reservoirs of brucellosis include bison (Bison bison) and elk (Cervus elaphus nelsonii) for B. abortus whereas B. suis is the predominant species infecting feral swine. The...

  14. Neonatal brucellosis: A case report.

    PubMed

    Alnemri, Abdul Rahman M; Hadid, Adnan; Hussain, Shaik Asfaq; Somily, Ali M; Sobaih, Badr H; Alrabiaah, Abdulkarim; Alanazi, Awad; Shakoor, Zahid; AlSubaie, Sarah; Meriki, Naema; Kambal, Abdelmageed M

    2017-02-28

    Although brucellosis is not uncommon in Saudi Arabia, neonatal brucellosis has been infrequently reported. In this case of neonatal brucellosis, Brucella abortus was isolated by blood culture from both the mother and the neonate. Serology was positive only in the mother.

  15. Bovine Brucellosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brucella abortus is an intracellular pathogen that causes reproductive losses in cattle and zoonotic infections in people. An eradication program based on serologic detection and vaccination has been in place for decades in the United States. Brucella use multiple molecular mechanisms to modify th...

  16. Pathogenesis and epidemiology of Brucellosis in Yellowstone bison: serologic and culture results from adult females and their offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this prospective study was to follow the natural course of Brucella abortus infection in cohorts of seropositive and seronegative female bison and their offspring in Yellowstone National Park over a 5 year period. Specimens were collected from 53 adult, female bison at least once a...

  17. DNA Genotyping Suggests Recent Brucellosis Outbreaks in the Greater Yellowstone Area Originated from Elk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brucellosis is a disease caused by bacteria of the genus Brucella. Brucella species infect a variety of livestock animals and humans world wide. In the United States, the disease with the greatest economic impact is caused by Brucella abortus in cattle. Although the disease has been mostly eradic...

  18. B Lymphocyte Subpopulation Defined by a Rat Monoclonal Antibody, 14G8

    DTIC Science & Technology

    1982-05-01

    Bethesda. MO). Proprtis an exresion f mmbrae atigns (, 2 In The goat anti-p antiserum from which the specifically purified antibodies and xpresio of embane...cell * Independent responses in B-cell defective CsA/N mice to Brucella abortus cycle by ProPiumn iodide staining. J. Cell. Bil. 66:188. and to TNP

  19. Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9.

    PubMed

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

    2003-07-01

    Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

  20. Recurrent spontaneous abortions due to a homologous Robertsonian translocation (14q14q)

    PubMed Central

    Gracias-Espinal, R; Roberts, S H; Duckett, D P; Laurence, K M

    1982-01-01

    A female with a history of recurrent spontaneous abortions was shown to carry a balanced Robertsonian translocation involving the No 14 homologues. One abortus had trisomy 14 with a 46,XX,-14,+t(14q14q)mat karyotype. Images PMID:7154046

  1. Serologic detection of antibodies to Brucella spp. using a commercial ELISA in cattle in Grenada, West Indies.

    PubMed

    Chikweto, A; Tiwari, K; Kumthekar, S; Stone, D; Louison, B; Thomas, D; Sharma, R; Hariharan, H

    2013-06-01

    Bovine brucellosis, caused mainly by Brucella abortus, a zoonotic bacterium, has been reported from many areas of the world, including Central and South America, and the Caribbean island state of Trinidad and Tobago. Although brucellosis has been eradicated from domestic cattle in Canada it still exists in one or two herds in the United States. Serological tests are important in estimating prevalence of Brucella exposure in order to target eradication programmes. In this study, serum samples from 150 cattle were tested using a commercial competitive enzyme linked immunosorbent assay (SVANOVIR®Brucella-Ab C-ELISA) which detects antibodies to both B. abortus and Brucella melitensis. All cattle tested were greater than 6 months old and were unvaccinated. Sampled cattle were from 35 herds representing animals from all 6 parishes of Grenada. Nine of the 150 animals (6%) were positive for antibodies to B. abortus and/or melitensis by the C-ELISA. Of the 35 herds, 7 (20%) had C-ELISA- positive animals. Three of the 6 parishes contained positive herds. Based on the high sensitivity (98%) and specificity (99.7%) of the C-ELISA, these results strongly indicate the presence of cattle exposed to B. abortus and/or melitensis in Grenada.

  2. Advancement of knowledge of Brucella over the past 50 years

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. The genus was considered to contain only three species, B. abortus, B. melitensis and B. suis. Since the early 1960’s, at least seven new species have been identified as belonging to the Brucell...

  3. Disposal of Industrial and Domestic Wastes: Land and Sea Alternatives.

    DTIC Science & Technology

    1984-01-01

    Bacillus anthracis Reovirua Ascaria lumbricoides Clostridiu perfringens Parvovirus Enterobius vermicularis Yersinia Hepatitis A Strongyloides sp...Texpratures (OC) Microorganisms 50 55 60 65 70 Cyst of Entamoeba 5 histolytica Eggs of Ascaris 60 7 lumbr icoides Brucella abortus 60 3 Corynebacterium

  4. An Aerosolized Brucella spp. Challenge Model for Laboratory Animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M) in a laboratory animal model of brucellosis, dosages of 10**3 to 10**10 CFU were nebulized to mice. Although tissue weights were minimally influenced, total colony-forming units (CFU) per tis...

  5. Recent Developments in Livestock and Wildlife Brucellosis Vaccination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Live attenuated brucellosis vaccines have been available for protecting domestic livestock against B. melitensis or B. abortus for more than 60 years. Current vaccines are effective in preventing abortion and transmission of brucellosis, but poor at preventing infection or seroconversion. In addit...

  6. Genotyping of Ochrobactrum spp. by AFLP Analysis

    PubMed Central

    Leal-Klevezas, Diana Sara; Martínez-de-la-Vega, Octavio; Ramírez-Barba, Ector Jaime; Osterman, Björn; Martínez-Soriano, Juan Pablo; Simpson, June

    2005-01-01

    AFLP was used to analyze the genetic diversity among Ochrobactrum strains. AFLP patterns showed a great genomic variability that separated the samples into three distinct clusters. Ochrobactrum intermedium was found to be closely related to Brucella abortus S99. PMID:15774899

  7. Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp.

    PubMed Central

    Gómez-Miguel, M J; Moriyón, I

    1986-01-01

    The sodium dodecyl sulfate (SDS) extraction-trypsin digestion protocol used by Braun and Sieglin (V. Braun and U. Sieglin, Eur. J. Biochem. 13:336-346, 1970) to show the peptidoglycan-linked lipoprotein of Escherichia coli was applied to both Brucella abortus and E. coli. Whereas a single polypeptide of 8,000 molecular weight was obtained from E. coli, several proteins of apparent molecular weight lower than 35,000 were demonstrated by SDS-polyacrylamide gel electrophoresis in B. abortus. These results did not change when the trypsin digestion conditions were modified. On the other hand, when the SDS extractions were performed under conditions more stringent than those used for other gram-negative bacteria, only a polypeptide fragment of apparent molecular weight of 8,000 was obtained from B. abortus. This polypeptide was similar to the trypsin fragment of the E. coli lipoprotein with respect to its behavior in SDS-polyacrylamide gels, isoelectric point in urea, molecular weight, and presence of both ester- and amide-linked fatty acids. Moreover, the amino acid analysis showed an overall similarity with respect to the amino acid composition of E. coli lipoprotein. A polypeptide of the same molecular weight, isoelectric point, and amino acid composition was also obtained from Brucella ovis by the same method. These results demonstrated that B. abortus and B. ovis cell envelopes contain a lipoprotein and strongly support the hypothesis that it is the only major protein covalently linked to the peptidoglycan. Images PMID:3744559

  8. Emerging cases of chlamydial abortion in sheep and goats in Croatia and Bosnia and Herzegovina.

    PubMed

    Spičic, Silvio; Račić Ivana; Andrijanić, Milan; Duvnjak, Sanja; Zdelar-Tuk, Maja; Stepanić, Maja; Cvetnić, Zeljko

    2015-01-01

    In a recent lambing season (2012/2013), the seroprevalence of ovine chlamydiosis was monitored in small ruminant abortion cases in Croatia. Blood samples of 93 sheep and 69 goats were examined. In addition, 50 sheep and 61 goat samples were tested using molecular methods. Furthermore, 14 sheep blood samples, one goat blood sample and one sheep placenta sample from Bosnia and Herzegovina (BIH) were also tested as a part of inter-laboratory cooperation. Overall high seroprevalence was detected in sheep, 19.6% with the ELISA IDEXX kit and 20.5% with the ClVTEST kit. Seroprevalence in goats was 11.4%. In BIH, four sheep and one goat blood sample were seropositive for chlamydiosis. The disease causing agent, Chlamydia abortus (C. abortus) was confirmed using molecular methods in two sheep flocks in continental Croatia and in one sheep flock in BIH. In this study, C. abortus infection in sheep was identified for the first time in Croatia using species specific molecular methods. Ovine chlamydiosis is present in national sheep and goat flocks in Croatia and BIH. Thus should be subject to ongoing controls in the case of abortion. A combination of serological and molecular methods should be used for optimal laboratory diagnostics of C. abortus.

  9. Prenatal diagnosis of 45,X/46,XY mosaicism in a fetus with asymmetric gonadal dysgenesis.

    PubMed

    Kirkilionis, A J; Rodney, P; Sergovich, F R; Armstrong, R

    1987-09-01

    An 18 week abortus had been prenatally diagnosed as a 45,X/46,XY mosaic. The fetus was a phenotypic male with glandular hypospadias, a horseshoe kidney and asymmetric gonadal dysgenesis. This case represents a rare instance of prenatally diagnosed 45,X/46,XY mosaicism with an abnormal phenotype.

  10. Parachlamydia spp. and Related Chlamydia-like Organisms and Bovine Abortion

    PubMed Central

    Borel, Nicole; Ruhl, Silke; Casson, Nicola; Kaiser, Carmen; Pospischil, Andreas

    2007-01-01

    Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion. PMID:18258043

  11. Ovine Enzootic Abortion (OEA): a comparison of antibody responses in vaccinated and naturally-infected swiss sheep over a two year period

    PubMed Central

    Gerber, Andrea; Thoma, Ruedi; Vretou, Evangelia; Psarrou, Evgenia; Kaiser, Carmen; Doherr, Marcus G; Zimmermann, Dieter R; Polkinghorne, Adam; Pospischil, Andreas; Borel, Nicole

    2007-01-01

    Background Prevention and control of ovine enzootic abortion (OEA) can be achieved by application of a live vaccine. In this study, five sheep flocks with different vaccination and infection status were serologically tested using a competitive enzyme-linked immunosorbent assay (cELISA) specific for Chlamydophila (Cp.) abortus over a two-year time period. Results Sheep in Flock A with recent OEA history had high antibody values after vaccination similar to Flock C with natural Cp. abortus infections. In contrast, OEA serology negative sheep (Flock E) showed individual animal-specific immunoreactions after vaccination. Antibody levels of vaccinated ewes in Flock B ranged from negative to positive two and three years after vaccination, respectively. Positive antibody values in the negative control Flock D (without OEA or vaccination) are probably due to asymptomatic intestinal infections with Cp. abortus. Excretion of the attenuated strain of Cp. abortus used in the live vaccine through the eye was not observed in vaccinated animals of Flock E. Conclusion The findings of our study indicate that, using serology, no distinction can be made between vaccinated and naturally infected sheep. As a result, confirmation of a negative OEA status in vaccinated animals by serology cannot be determined. PMID:17903243

  12. Immune Responses and Protection against Experimental Brucella suis biovar 1 Challenge in Non-vaccinated or RB51-Vaccinated Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty Hereford heifers, approximately 9 months of age, were vaccinated with saline (control) or 2 x 10**10 CFU of Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P<0.05) antibody and proliferative responses to RB51 antigens i...

  13. DNA Vaccination of the American Crow (Corvus brachyrhynchos) Provides Partial Protection Against Lethal Challenge with West Nile Virus

    DTIC Science & Technology

    2007-01-01

    protección parcial contra el desafı́o letal con el virus del Oeste del Nilo. La cepa del virus del Oeste del Nilo aislada en Nueva York en el año 1999 es...murieron. La administración parenteral de la vacuna de ADN del virus del Oeste del Nilo estuvo asociada con una reducción de la mortalidad pero no

  14. Brucella and Osteoarticular Cell Activation: Partners in Crime.

    PubMed

    Giambartolomei, Guillermo H; Arriola Benitez, Paula C; Delpino, M Victoria

    2017-01-01

    Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4(+) T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the

  15. Molecular study of Brucellosis in camels by the use of TaqMan® real-time PCR.

    PubMed

    Khamesipour, Faham; Doosti, Abbas; Rahimi, Ebrahim

    2015-12-01

    Brucellosis is a zoonosis of economic importance that reduces productivity in livestock enterprises as it induces abortion in infected animals. A study was designed aimed at detecting Brucella in blood and lymph node specimens from camels by the use of real-time PCR in Iran. Sample collection and DNA extraction were done on blood (n = 135) and lymph node (n = 135) samples collected from 135 camels (abattoir survey) from both sexes at various ages in different seasons. The real-time PCR for species differentiation was based on unique genetic loci of B. melitensis and B. abortus. The regions were chosen for the construction of primers and TaqMan® probes for species differentiation: BMEII0466 gene for B. melitensis and Bru-Ab2_0168 gene for B. abortus. Brucella spp. were identified in 18 (13.33%) blood samples and 4 (2.97%) lymph node samples. This method showed to be effective in detecting B. abortus and B. melitensis in blood and lymph samples respectively. Brucella abortus was detected in 3 (2.22%) blood samples but was however, not detected in the lymph node samples. Brucella melitensis was only observed in 4 (2.97%) lymph node samples. Significant differences were observed on the blood prevalence of unknown Brucella spp. in different age groups and seasons (P < 0.05). However, there were no significant differences observed on the prevalence of B. abortus, B. melitensis, unknown Brucella spp. in different age groups, sex and seasons (P > 0.05). Therefore, Brucella was detected in apparent healthy camels slaughtered at an abattoir in Iran and this recommends the significance of the detection of Brucella in camels, since the infected camels appear to be healthy.

  16. Whole-genome analyses of speciation events in pathogenic Brucellae

    SciTech Connect

    Chain, Patrick S. G.; Comerci, Diego J.; Tolmasky, Marcelo E.; Larimer, Frank W; Malfatti, Stephanie; Vergez, Lisa; Aguero, Fernan; Land, Miriam L; Ugalde, Rodolfo A.; Garcia, Emilio

    2005-12-01

    Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

  17. Whole-genome analyses of the speciation events in the pathogenic Brucellae

    SciTech Connect

    Chain, P; Comerci, D; Tolmasky, M; Larimer, F; Malfatti, S; Vergez, L; Aguero, F; Land, M; Ugalde, R; Garcia, E

    2005-07-14

    Despite their high DNA identity and a proposal to group classical Brucella species as biovars of B. melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters as well as by a marked host range (e.g. B. suis for swine, B. melitensis for sheep and goats, B. abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucellae species and to the B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions and insertions support previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complement of both B. abortus strains is identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggest that these inactivations may play an important role in the establishment of host-specificity and may have been a primary driver of speciation in the Brucellae. Despite being non-motile, the Brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures, and may contribute to differences in host-specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g. glycogen, biotin, NAD, and choline) are consistent with adaptation of Brucellae to an intracellular lifestyle.

  18. Brucella and Osteoarticular Cell Activation: Partners in Crime

    PubMed Central

    Giambartolomei, Guillermo H.; Arriola Benitez, Paula C.; Delpino, M. Victoria

    2017-01-01

    Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the

  19. Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus▿

    PubMed Central

    Céspedes, Sandra; Salgado, Paulina; Valenzuela, Patricio; Vidal, Roberto; Oñate, Angel A.

    2011-01-01

    One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains. PMID:21543580

  20. Integrating ecology with management to control wildlife brucellosis.

    PubMed

    Treanor, J J

    2013-04-01

    Bison (Bison bison) and elk (Cervus elaphus) in the greater Yellowstone ecosystem have long been infected with Brucella abortus. The continued culling of large numbers of Yellowstone bison to reduce the risk of brucellosis transmission to cattle could negatively affect long-term conservation. A desirable management objective is to reduce the level of B. abortus infection while conserving wildlife populations. Identifying the ecological factors that influence immune suppression and vulnerabilityto infection will help initiate effective control measures. Seasonal food restriction during pregnancy has the potential to limit resources available for immune defence and may be an important factor sustaining brucellosis in wild ungulate populations. Consequently, effective management practices will need to include a diverse range of integrated methods, which include maintaining separation of livestock and wildlife, managing habitat to reduce brucellosis transmission, and reducing disease prevalence in wildlife. The long-term success of these management practices will depend on sound science and support of the stakeholders involved.

  1. Pathogens in water deer (Hydropotes inermis) in South Korea, 2010-12.

    PubMed

    Kim, Seol-Hee; Choi, Heelack; Yoon, Junheon; Woo, Chanjin; Chung, Hyen-Mi; Kim, Jong-Taek; Shin, Jeong-Hwa

    2014-07-01

    Water deer (Hydropotes inermis) are among the most common wildlife to approach farmhouses and livestock barns in Korea. We collected 305 water deer from Gangwon (n=168), South Chungcheong (n=89), and Gyeongsang (n=48) provinces in 2010-12 and used PCR and serologic tests to screen the deer for pathogens. In 2010, tests for bovine viral diarrhea virus (BVDV), rotavirus, and Brucella abortus were positive in 8% (5/60), 2% (1/60), and 59% (33/56) of the animals, respectively. In 2010, the water deer were negative for foot-and-mouth disease virus, coronaviruses, and Mycobacterium bovis. All samples collected in 2011 and 2012 were negative for all pathogens analyzed. These results suggest that at least two of the investigated pathogens, BVDV and B. abortus, circulate among water deer in South Korea.

  2. Molecular detection and characterization of Brucella species in raw informally marketed milk from Uganda

    PubMed Central

    Hoffman, Tove; Rock, Kim; Mugizi, Denis Rwabiita; Muradrasoli, Shaman; Lindahl-Rajala, Elisabeth; Erume, Joseph; Magnusson, Ulf; Lundkvist, Åke; Boqvist, Sofia

    2016-01-01

    This study identified and characterized Brucella species in the informal milk chain in Uganda. A total of 324 cattle bulk milk samples were screened for the genus Brucella by real-time PCR with primers targeting the bcsp31 gene and further characterized by the omp25 gene. Of the samples tested, 6.5% were positive for Brucella species. In the omp25 phylogeny, the study sequences were found to form a separate clade within the branch containing B. abortus sequences. The study shows that informally marketed cattle milk in Uganda is a likely risk factor for human brucellosis and confirms that B. abortus is present in the cattle population. This information is important for potential future control measures, such as vaccination of cattle. PMID:27839533

  3. Should foetuses or infants be utilized as organ donors?

    PubMed

    Caplan, Arthur L

    1987-04-01

    The shortage of organs and tissues for transplantation in infants is particularly severe. Caplan considers the moral and public policy implications of utilizing abortuses and brain dead or anencephalic infants as donors. Arguments favoring their use include the potential benefits for research, benefits to existing infants born with fatal conditions, the ethical cost of relying on primates as sources of organs, and the providing of solace to grieving parents. Arguments against their use include the potential for coercion or conflict of interest in parental decisions about donation, the possibility that abortion may be encouraged, the fact that brain death is difficult to diagnose in infants while organ procurement from anencephalics may be considered murder, and the charge that an increase in infant transplants would be too costly. Caplan concludes that the arguments for using abortuses, anencephalics, and brain dead infants as organ and tissue donors outweigh the arguments against.

  4. Enzyme-linked immunosorbent assay with major outer membrane proteins of Brucella melitensis to measure immune response to Brucella species.

    PubMed Central

    Hunter, S B; Bibb, W F; Shih, C N; Kaufmann, A F; Mitchell, J R; McKinney, R M

    1986-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) system to measure human immunoglobulin G (IgG) and IgM response to the major outer membrane proteins of Brucella melitensis. The ELISA was more sensitive in detecting antibody than a standard microagglutination (MA) test with B. abortus antigen. Of 101 sera from persons with suspected brucellosis, 79 (78.2%) gave ELISA IgM titers greater than or equal to the B. abortus MA titer without 2-mercaptoethanol (2ME), which measures both IgM and IgG. Of the 101 sera, 97% gave ELISA IgG titers greater than or equal to the MA with 2ME titer. A total of 58 sera, drawn from 11 human patients from 1 to 29 weeks after onset of brucellosis, gave higher geometric mean titers for the ELISA IgG test than for the MA with 2ME test. These 58 sera also gave ELISA IgM geometric mean titers that were greater than or within one doubling dilution of the geometric mean titers of MA without 2ME. In addition to detecting antibody response to B. abortus, B. melitensis, and B. suis, the ELISA was sensitive to antibody response to human and canine infections with B. canis. The B. canis antibody response is not detected by the MA test with B. abortus antigen. The ELISA, with a standard preparation of major outer membrane proteins of B. melitensis as antigen, appears to be useful in measuring antibody response in humans to infections by all species of Brucella known to infect humans. PMID:3095364

  5. Assessment of a DNA Vaccine Encoding Burkholderia pseudomallei Bacterioferritin

    DTIC Science & Technology

    2007-08-01

    bacterioferritin gene from Brucella abortus, when delivered to mice as a DNA vaccine, evokes a potent Th1 immune response, including strong IFN-γ...blocking buffer containing goat anti-mouse IgG alkaline phosphatase conjugate (Sigma) at a dilution of 1:30000 for 1hr at room temperature. Following...Walravens, and J. J. Letesson. 2001. Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella

  6. Waddlia chondrophila Infects and Multiplies in Ovine Trophoblast Cells Stimulating an Inflammatory Immune Response

    PubMed Central

    Wheelhouse, Nick; Coyle, Christopher; Barlow, Peter G.; Mitchell, Stephen; Greub, Gilbert; Baszler, Tim; Rae, Mick T.; Longbottom, David

    2014-01-01

    Background Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity. Methodology/Principal Findings Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways. Conclusions/Significance W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms. PMID:25010668

  7. The Brucella genome at the beginning of the post-genomic era.

    PubMed

    Michaux-Charachon, Sylvie; Jumas-Bilak, Estelle; Allardet-Servent, Annick; Bourg, Gisele; Boschiroli, Maria Laura; Ramuz, Michel; O'Callaghan, David

    2002-12-20

    The year 2002 began with the publication of the first complete genome sequence for a Brucella species, that of the two replicons of B. melitensis 16M. Hopefully in 2002, the complete genome of B. suis 1330, and, perhaps, a B. abortus strain will be published. This is the culmination of over 30 years investigation of the composition, structure, organisation and evolution of the Brucella genome. Brucella research must now adapt to the new challenges of the post-genomic era.

  8. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

    PubMed Central

    Cloeckaert, A; de Wergifosse, P; Dubray, G; Limet, J N

    1990-01-01

    A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development. Images PMID:1701417

  9. Epizoological Survey of Certain Endemic Diseases in the Southern Part of the Great Salt Lake Desert.

    DTIC Science & Technology

    1959-06-30

    wildlife specimens were examined for plague, Pasteurella pestis; tulareinia, P. tularen~~s~ anthrax, Bacillus anthracis; brucellosis, Brucella species...Q fever, American (Nine Mi~Le)strai~, (~) Rocky Mountain spotted fever, (3) psittacosis, (4) Febri].e ~~~~. abortus , and (5) P. tularensie. Another...No isolations of Brucella species were made during this report period, nor were there any agglutination. at 1:40 or higher. Sane

  10. Medical Surveillance Monthly Report (MSMR). Volume 10, Number 4, July/August 2004

    DTIC Science & Technology

    2004-08-01

    1,280) to B. abortus and a blood isolate of Brucella spp (later identified as B. melitensis ). The patient’s clinical course and laboratory test...workers, butchers, veterinarians) who have frequent close contact with animals (particularly cattle, pigs, goats , and sheep). Isolated cases and outbreaks...region. Vet Microbiol 2002 Dec 20;90(1-4):81-110. 3. Karim MA, Penjouian EK, Dessouky FI. The prevalence of brucellosis among sheep and goats in

  11. Force Protection Technologies for the 2010-2020 Timeframe

    DTIC Science & Technology

    2003-11-01

    Rickettsia prowasecki Rickettsiae R4. Rickettsia rickettsii B1. Bacillus anthracis B2. Brucella abortus B3. Brucella melitensis B4. Brucella suis B6...reduction Sensor systems that detect levels of chemical agents, radiological agents, pathogenic bacteria , viruses, or toxins in the environment will be...virus Viruses V20. Japanese encephalitis virus R1. Coxiella burnetti R2. Bartonella Quintana (Rochlimea quintana, Rickettsia quintana) R3

  12. Vaccines, Pharmaceutical Products, and Bioterrorism: Challenges for the U.S. Food and Drug Administration

    DTIC Science & Technology

    1999-08-01

    plague (Yersinia pestis), tularemia (Francisella tularensis), brucellosis ( Brucella abortus, B. melitensis , B. suis, B. canis), Q fever (Coxiella...Special Issue 20011029 090 Vaccines, Pharmaceutical Products, and Bioterrorism: Challenges for the U.S. Food and Drug Administration Kathryn C...Zoon U.S. Food and Drug Administration, Rockville, Maryland, USA In regards to bioterrorism, the goal of the U.S. Food and Drug Administration (FDA

  13. Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus▿

    PubMed Central

    Bukata, Lucas; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.; Comerci, Diego J.

    2008-01-01

    The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell. PMID:18931122

  14. [Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions].

    PubMed

    Chanton-Greutmann, H; Thoma, R; Corboz, L; Borel, N; Pospischil, A

    2002-09-01

    Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).

  15. Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro, Brazil.

    PubMed

    Martins, Gabriel; Penna, Bruno; Hamond, Camila; Leite, Rachel Cosendey-Kezen; Silva, Andressa; Ferreira, Ana; Brandão, Felipe; Oliveira, Francisco; Lilenbaum, Walter

    2012-04-01

    Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.

  16. Comparison of the brucellin skin test with the lymphocyte transformation test in bovine brucellosis.

    PubMed Central

    Chukwu, C. C.

    1986-01-01

    The brucellin skin test and the lymphocyte transformation test were compared in heifers infected with virulent Brucella abortus strain 544, heifers vaccinated against brucellosis and unexposed cattle. Results of the in vitro lymphocyte transformation test were consistently positive for all 9 Brucella-infected heifers while the skin test was consistently positive for 6 of the 9 heifers. In 7 heifers repeatedly vaccinated with B. abortus strain-19 vaccine the in vitro test classified 3 animals as positive whereas the skin test identified all the animals as infected during most of the experimental period. Four heifers injected with a single dose of B. abortus strain 19 were consistently negative to the lymphocyte transformation test while the skin test classified all the animals as infected during most of the experimental period. The skin test gave strong reactions indicative of Brucella infection in heifers vaccinated with 'Duphavac' and 'Abortox' vaccines whereas the lymphocyte transformation test was consistently negative with these vaccines. The two tests were negative in unexposed cattle. It was concluded that the in vitro test correlated better with Brucella isolation than the in vivo test did and that the lack of agreement between the results of the two tests is likely to be due to the different antigens used in the assays. PMID:3734426

  17. Importance of unusually modified lipid A in Sinorhizobium stress resistance and legume symbiosis.

    PubMed

    Ferguson, Gail P; Datta, Anup; Carlson, Russ W; Walker, Graham C

    2005-04-01

    Sinorhizobium meliloti, a legume symbiont and Brucella abortus, a phylogenetically related mammalian pathogen, both require their BacA proteins to establish chronic intracellular infections in their respective hosts. The lipid A molecules of S. meliloti and B. abortus are unusually modified with a very-long-chain fatty acid (VLCFA; C > or = 28) and we discovered that BacA is involved in this unusual modification. This observation raised the possibility that the unusual lipid A modification could be crucial for the chronic infection of both S. meliloti and B. abortus. We investigated this by constructing and characterizing S. meliloti mutants in the lpxXL and acpXL genes, which encode an acyl transferase and acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A. Our analysis revealed that the unusually modified lipid A is important, but not crucial, for S. meliloti chronic infection and that BacA must have an additional function, which in combination with its observed effect on the lipid A in the free-living form of S. meliloti, is essential for the chronic infection. Additionally, we discovered that in the absence of VLCFAs, S. meliloti produces novel pentaacylated lipid A species, modified with unhydroxylated fatty acids, which are important for stress resistance.

  18. Yersinia enterocolitica: an unlikely cause of positive brucellosis tests in greater yellowstone ecosystem bison (Bison bison).

    PubMed

    See, Wade; Edwards, William H; Dauwalter, Stacey; Almendra, Claudia; Kardos, Martin D; Lowell, Jennifer L; Wallen, Rick; Cain, Steven L; Holben, William E; Luikart, Gordon

    2012-07-01

    Yersinia enterocolitica serotype O:9 has identical O-antigens to those of Brucella abortus and has apparently caused false-positive reactions in numerous brucellosis serologic tests in elk (Cervus canadensis) from southwest Montana. We investigated whether a similar phenomenon was occurring in brucellosis antibody-positive bison (Bison bison) using Y. enterocolitica culturing techniques and multiplex PCR of four diagnostic loci. Feces from 53 Yellowstone bison culled from the population and 113 free-roaming bison from throughout the Greater Yellowstone Ecosystem (GYE) were tested. Yersinia enterocolitica O:9 was not detected in any of 53 the bison samples collected at slaughter facilities or in any of the 113 fecal samples from free-ranging bison. One other Y. enterocolitica serotype was isolated; however, it is not known to cause cross-reaction on B. abortus serologic assays because it lacks the perosamine synthetase gene and thus the O-antigens. These findings suggest that Y. enterocolitica O:9 cross-reactivity with B. abortus antigens is unlikely to have been a cause of false-positive serology tests in GYE bison and that Y. enterocolitica prevalence was low in bison in the GYE during this study.

  19. Serological trail of Brucella infection in an urban slum population in Brazil

    PubMed Central

    Angel, Martha Olivera; Ristow, Paula; Ko, Albert I.; Di-Lorenzo, Cecilia

    2013-01-01

    Introduction Brucellosis is a re-emerging zoonosis with new cases reported each year in many Latin American countries, but it is mostly under-recognized. This study presents a serological investigation of infection with Brucella abortus and Brucella canis in a poor urban community in the city of Salvador, Brazil. Methodology Human sera (n = 180) were randomly selected from 3,171 samples taken from healthy individuals during 2003-2004 and tested with C-ELISA for B. abortus and I-ELISA for B. canis. Results Thirteen percent (24/180) of the individuals were positive for B. abortus and 4.6 % (8/174) were positive for B. canis. Among the variables studied only age (older than 45 years) appeared to be a risk factor for the detection of Brucella antibodies. Conclusion These results indicate the presence of Brucella infection in this settlement and highlight the need to understand the epidemiology of infection under these circumstances to establish the necessary measures for surveillance and control. PMID:23000868

  20. Brucellosis in Venezuela.

    PubMed

    Francisco, J; Vargas, O

    2002-12-20

    Brucellosis is a public health problem in Venezuela and affects large numbers of animals. The most important biovar in the country is Brucella abortus. In cattle and buffalo it causes high rates of abortions in females and infertility in males; it is transmissible to occupationally exposed humans. In 1968, an official program was set up for the control and eradication of the disease and it is still in place. Amongst the control provisions, this program provides for the vaccination of female calves with strain 19 and the slaughtering of positive reactors following the official diagnosis (rapid agglutination in plate test). According to the official reports, the positive reactors ranged from 0.8 to 1.2% in the past few years. These values do not corroborate reports showing an average positive rate of 10.5% and even higher values in some areas of the country. The government is working to approve a new resolution that will replace the rapid agglutination in plate test with the Card Test, the use of 2-Mercaptoetanol, fixation of complement and competitive ELISA as confirmatory tests. In addition, there will be an obligatory vaccination with B. abortus strain 19 or B. abortus RB51 of all female calves between 3- and 8-month-old and a recommended revaccination at 10-15-month-old and adult cows in high prevalence areas. These measures should allow help to reduce the prevalence of the disease in cattle herds and thus minimize the risk for human populations.

  1. Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen.

    PubMed

    Golshani, Maryam; Rafati, Sima; Siadat, Seyed Davar; Nejati-Moheimani, Mehdi; Shahcheraghi, Fereshteh; Arsang, Amin; Bouzari, Saeid

    2015-08-01

    Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.

  2. Serologic evidence of Brucella infection in pinnipeds along the coast of Hokkaido, the northernmost main island of Japan.

    PubMed

    Abe, Erika; Ohishi, Kazue; Ishinazaka, Tsuyoshi; Fujii, Ke; Maruyama, Tadashi

    2017-03-06

    Brucella infection in Hokkaido was serologically surveyed in four species of pinnipeds inhabiting Cape Erimo during 2008-2013 and the Shiretoko Peninsula in 1999, by enzyme-linked immunosorbent assay using Brucella abortus and B. canis as antigens. Anti-Brucella positive sera showed higher absorbance to B. abortus than B. canis in almost all samples. Anti-B. abortus antibodies were detected in serum samples from 24% (n=55) of Western Pacific harbor seals (Phoca vitulina stejnegeri) in Cape Erimo, and from 66% (n=41) of spotted seals (P. largha), 15% (n=20) of ribbon seals (Histriophoca fasciata) and 18% (n=17) of Western Steller's sea lions (Eumetopias jubatus jubatus) in the Shiretoko Peninsula. In the harbor seals, anti-Brucella antibodies were detected at higher absorbance in 1- to 4-year-old individuals than in pups and mature animals, suggesting either that the Brucella infection mainly occurred after weaning, or that it is maternally transmitted to the pups with premature or suppressed immunity. In the spotted seals and ribbon seals, anti-Brucella antibodies were detected in both immature and mature animals, with higher absorbance in the former. In the Western Steller's sea lions, the antibodies were detected only in mature animals. Western blot analysis of the serum samples showed some differences in band appearances, i.e. discrete or smeary, and in the number of bands. These indicate that multiple different Brucella may be prevalent in pinnipeds in Hokkaido. Or the Brucella of pinnipeds may have some intra-species diversity.

  3. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    PubMed

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  4. Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

    PubMed

    Golshani, Maryam; Rafati, Sima; Dashti, Amir; Gholami, Elham; Siadat, Seyed Davar; Oloomi, Mana; Jafari, Anis; Bouzari, Saeid

    2015-06-01

    Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.

  5. The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

    PubMed

    Van Lent, Sarah; Creasy, Heather Huot; Myers, Garry S A; Vanrompay, Daisy

    2016-01-01

    Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species.

  6. [Adverse effects of seasonal flu vaccine and new influenza A (H1N1) vaccine in health care workers].

    PubMed

    Torruella, Joan Inglés; Soto, Rosa Gil; Valls, Rosa Carreras; Lozano, Judit Valverde; Carreras, Dolors Benito; Cunillera, Arnau Besora

    2013-01-01

    OBJETIVOS: Valorar y comparar los efectos indeseados de la vacuna de la gripe estacional (VGE) y vacuna de la gripe A H1N1 (VGA) en trabajadores sanitarios. MÉTODOS: Estudio transversal multicéntrico en trabajadores sanitarios de hospitales de agudos, centros de asistencia primaria, centros sociosanitarios, centros de salud mental y un hospital geriátrico participantes en la campaña de vacunación antigripal del 2009. Se enviaron encuestas autocumplimentadas a todos los vacunados con VGE y/o VGA. RESULTADOS: De los 1123 vacunados con VGE se obtienen 527 encuestas válidas (46,9%) y de 461 vacunados con VGA se obtienen 242 encuestas (52,5%). De los trabajadores participantes 527 estaban vacunados sólo con VGE, 117 vacunados previamente con VGE y después VGA (VGE+VGA) y 125 sólo vacunados sólo con VGA. El 18,4% (IC 95% 15,1-21,7) del grupo VGE presentaron algún efecto adverso a la vacuna VGE; en el grupo VGE+VGA el 45,3% (IC 95% 36,3-54,3) presentó una reacción adversa al recibir la VGA, y en el grupo VGA fue el 46,4% (IC 95% 37,7-55,1). En todos los participantes el problema más frecuente fue una reacción local. Las mujeres presentan mayor reacción a VGA y VGE que los hombres. Para todas las edades la VGE es menos reactógena que VGA y que la combinación de ambas vacunas, con la excepción de los trabajadores menores de 29 años. CONCLUSIONES: La VGA es más reactógena que la VGE, sin diferencias por orden de administración. Se observan variaciones por sexo y edad, pero siempre con mayor reactogenicidad para la VGA.

  7. A public-professional web-bridge for vaccines and vaccination: user concerns about vaccine safety.

    PubMed

    García-Basteiro, Alberto L; Alvarez-Pasquín, María-José; Mena, Guillermo; Llupià, Anna; Aldea, Marta; Sequera, Victor-Guillermo; Sanz, Sergi; Tuells, Jose; Navarro-Alonso, José-Antonio; de Arísteguí, Javier; Bayas, José-María

    2012-05-28

    Vacunas.org (http://www.vacunas.org), a website founded by the Spanish Association of Vaccinology offers a personalized service called Ask the Expert, which answers any questions posed by the public or health professionals about vaccines and vaccination. The aim of this study was to analyze the factors associated with questions on vaccination safety and determine the characteristics of questioners and the type of question asked during the period 2008-2010. A total of 1341 questions were finally included in the analysis. Of those, 30% were related to vaccine safety. Questions about pregnant women had 5.01 higher odds of asking about safety (95% CI 2.82-8.93) than people not belonging to any risk group. Older questioners (>50 years) were less likely to ask about vaccine safety compared to younger questioners (OR: 0.44, 95% CI 0.25-0.76). Questions made after vaccination or related to influenza (including H1N1) or travel vaccines were also associated with a higher likelihood of asking about vaccine safety. These results identify risk groups (pregnant women), population groups (older people) and some vaccines (travel and influenza vaccines, including H1N1) where greater efforts to provide improved, more-tailored vaccine information in general and on the Internet are required.

  8. Chlamydia-related abortions in cattle from Graubunden, Switzerland.

    PubMed

    Borel, N; Thoma, R; Spaeni, P; Weilenmann, R; Teankum, K; Brugnera, E; Zimmermann, D R; Vaughan, L; Pospischil, A

    2006-09-01

    In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.

  9. Evaluation of lipopolysaccharides and polysaccharides of different epitopic structures in the indirect enzyme-linked immunosorbent assay for diagnosis of brucellosis in small ruminants and cattle.

    PubMed

    Alonso-Urmeneta, B; Marín, C; Aragón, V; Blasco, J M; Díaz, R; Moriyón, I

    1998-11-01

    Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939-1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.

  10. Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice.

    PubMed Central

    Doyle, A G; Halliday, W J; Barnett, C J; Dunn, T L; Hume, D A

    1992-01-01

    Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression. PMID:1548070

  11. Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders

    PubMed Central

    Tavernier, Paul; Sys, Stanislas U.; De Clercq, Kris; De Leeuw, Ilse; Caij, Anne Brigitte; De Baere, Miet; De Regge, Nick; Fretin, David; Roupie, Virginie; Govaerts, Marc; Heyman, Paul; Vanrompay, Daisy; Yin, Lizi; Kalmar, Isabelle; Suin, Vanessa; Brochier, Bernard; Dobly, Alexandre; De Craeye, Stéphane; Roelandt, Sophie; Goossens, Els; Roels, Stefan

    2015-01-01

    Introduction In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. Materials and methods Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. Results and discussion High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). Conclusions Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species. PMID:26609692

  12. High frequency of chlamydial co-infections in clinically healthy sheep flocks

    PubMed Central

    2011-01-01

    Background The epidemiological situation of ovine chlamydial infections in continental Europe, especially Germany is poorly characterised. Using the German state of Thuringia as a model example, the chlamydial sero- and antigen prevalence was estimated in thirty-two randomly selected sheep flocks with an average abortion rate lower than 1%. Seven vaccinated flocks were reviewed separately. Results A wide range of samples from 32 flocks were examined. Assumption of a seroprevalence of 10% (CI 95%) at flock level, revealed that 94% of the tested flocks were serologically positive with ongoing infection (i.e. animals with seroconversion) in nearly half (47%) of the flocks. On the basis of an estimated 25% antigen prevalence (CI 95%), PCR and DNA microarray testing, together with sequencing revealed the presence of chlamydiae in 78% of the flocks. The species most frequently found was Chlamydophila (C.) abortus (50%) followed by C. pecorum (47%) and C. psittaci genotype A (25%). Mixed infections occurred in 25% of the tested flocks. Samples obtained from the vaccinated flocks revealed the presence of C. abortus field samples in 4/7 flocks. C. pecorum was isolated from 2/7 flocks and the presence of seroconversion was determined in 3/7 flocks. Conclusions The results imply that chlamydial infections occur frequently in German sheep flocks, even in the absence of elevated abortion rates. The fact that C. pecorum and the potentially zoonotic C. psittaci were found alongside the classical abortifacient agent C. abortus, raise questions about the significance of this reservoir for animal and human health and underline the necessity for regular monitoring. Further studies are needed to identify the possible role of C. psittaci infections in sheep. PMID:21679409

  13. Ovine chlamydial abortion: characterization of the inflammatory immune response in placental tissues.

    PubMed

    Buxton, D; Anderson, I E; Longbottom, D; Livingstone, M; Wattegedera, S; Entrican, G

    2002-01-01

    Ovine chlamydial abortion is a serious cause of fetal mortality in several sheep-rearing countries. The causal agent, Chlamydophila abortus (Chlamydia psittaci), does not generally induce clinical signs in the ewe other than abortion; this is associated with macroscopically visible damage in the placenta, which may be inflamed and thickened. To investigate the nature of the placental inflammation, seven pregnant sheep were inoculated subcutaneously at 70 days' gestation with C. abortus (strain S 26/3). A further five pregnant sheep received control inoculum by the same route at the same stage of pregnancy. Three of the infected ewes produced stillborn lambs and four produced live lambs. Lesions characteristic of chlamydial infection were present in all placentas except for two from one ewe that gave birth to twins. Histopathological examination of placental tissues from aborted fetuses showed a mixed inflammatory cell infiltrate with vasculitis and thrombosis in the mesenchyme of the intercotyledonary membranes. Cells expressing the macrophage-associated molecule CD 14 were found to be numerous, as were cells expressing major histocompatibility complex class II (MHC II) molecules. Many cells expressing messenger RNA (mRNA) encoding for tumour necrosis factor-alpha (TNF-alpha) were demonstrated, but few cells expressing interferon gamma mRNA and none expressing interleukin-4 mRNA were detected. The fetal immune response included small numbers of CD4+ and CD8+ cells, gamma delta T cells and B cells. It is concluded that abortion is the result of several factors, including destruction of tissue by C. abortus, vascular thrombosis, and an inflammatory response by the fetus. Production of TNF-alpha by fetal macrophages expressing MHC II molecules may be of considerable significance in the pathogenesis of abortion.

  14. Serologic survey for selected infectious diseases in free-ranging Brazilian tapirs (Tapirus terrestris) in the cerrado of central Brazil.

    PubMed

    Furtado, Mariana Malzoni; Jácomo, Anah Tereza de Almeida; Kashivakura, Cyntia Kayo; Tôrres, Natália Mundim; Marvulo, Maria Fernanda Vianna; Ragozo, Alessandra Mara Alves; de Souza, Silvio Luis Pereira; Neto, José Soares Ferreira; Vasconcellos, Silvio Arruda; Morais, Zenaide Maria; Cortez, Adriana; Richtzenhain, Leonardo José; Silva, Jean Carlos Ramos; Silveira, Leandro

    2010-03-01

    From September 2000 to January 2002, a serologic survey was conducted in a population of free-ranging Brazilian tapirs (Tapirus terrestris) inhabiting Emas National Park and surrounding areas in Goiás state, central Brazil, as part of an ecologic study. Ten tapirs were immobilized with a tiletamine-zolazepam combination, and blood samples were collected. All sera were negative for Leptospira spp., Brucella abortus, and equine infectious anemia; and one of 10 animals was positive for Toxoplasma gondii. This report represents the first serologic survey for selected infectious diseases in a free-ranging population of Brazilians tapirs in central Brazil.

  15. Diagnostic and vaccine chapter.

    PubMed

    Wolfram, J H; Kokanov, S K; Verkhovsky, O A

    2010-10-01

    The first report in this chapter describes the development of a killed composite vaccine. This killed vaccine is non-infectious to humans, other animals, and the environment. The vaccine has low reactivity, is non-abortive, and does not induce pathomorphological alterations to the organs of vaccinated animals. The second report of this chapter describes the diagnostic value of a competitive enzyme-linked immunosorbent assay for detecting Brucella-specific antibodies and its ability to discriminate vaccinated cattle from infected cattle. The results indicated that the competitive enzyme-linked immunosorbent assay is more sensitive than traditional tests for detecting antibodies to Brucella abortus in naturally and experimentally infected cattle.

  16. Brucellosis mimicking Henoch-Schönlein purpura.

    PubMed

    Massasso, David; Gibson, Kathryn

    2007-06-04

    A young male immigrant from Syria with a vasculitic-appearing leg rash, asymmetrical polyarthritis, microscopic haematuria, and raised inflammatory markers was provisionally diagnosed with Henoch-Schönlein purpura. Skin biopsy showed leukocytoclastic vasculitis. Low-grade fevers persisted despite non-steroidal anti-inflammatory therapy, and Brucella sp. was subsequently grown from both blood and synovial fluid aspirates. Further tests gave positive results for B. abortus, and triple antibiotic therapy produced a rapid clinical response. Cutaneous vasculitis has rarely been described in brucellosis, and this is the first report in the English medical literature of brucellosis mimicking Henoch-Schönlein purpura.

  17. [Inactivated saponin vaccine against salmonellal abortion in sheep].

    PubMed

    Girginov, G; Vodas, K; Bozhilov, B

    1978-01-01

    An inactivated saponine vaccine is prepared from five highly immunogenic Salmonella abortus ovis strains, selected by means of a biological test on white rats. Saline was used as a diluent of the vaccine, with the addition of 30 per cent glycerine, 0.012 per cent saponine and 0.1 per cent propiolactone. The optimum immunization dose of 5 cm3 is injected singly subcutaneously behind the elbow, two and a half months after impregnation. The vaccine is applied on infected farms before the disease occurs. The cellular-humoral immunity, which forms 14 days after the injection, lasts 4--5 months and protects the sheep against salmonellosis abortion.

  18. Chronic Bacterial Pathogens: Mechanisms of Persistence

    PubMed Central

    Byndloss, Mariana X.; Tsolis, Renee M

    2015-01-01

    Summary Many bacterial pathogens can cause acute infections that are cleared with onset of adaptive immunity, however a subset of these pathogens can establish persistent, and sometimes lifelong infections. While bacteria causing chronic infections are phylogenetically diverse, they share common features in their interactions with the host that enable a protracted period of colonization. This chapter will compare the persistence strategies of two chronic pathogens from the Proteobacteria, Brucella abortus, and Salmonella enterica serovar Typhi (S. Typhi) to consider how these two pathogens, which are very different at the genomic level, can utilize common strategies to evade immune clearance to cause chronic intracellular infections of the mononuclear phagocyte system. PMID:27227304

  19. Phenotypic characterization of Brucella strains isolated from livestock in Nigeria.

    PubMed

    Ocholi, R A; Kwaga, J K P; Ajogi, I; Bale, J O O

    2004-10-05

    Isolation of brucellae from aborted fetuses, hygroma fluids, milk and vaginal swabs obtained from aborting cattle, sheep, goats, pigs, and horses in Nigeria was carried out. A total of 25 isolates, obtained mainly from cattle, sheep and horses, were biotyped. All strains belonged to one species, Brucella abortus biovar 1. The epidemiological significance of this finding is discussed. Some preliminary observations on the zoonotic and public health implications of Brucella infection in Nigerian livestock are presented. A control programme involving improved management, animal movement restrictions, public health education and mass vaccination of animals is suggested.

  20. Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase

    PubMed Central

    Beaufay, François; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    ABSTRACT Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria. PMID:27066186

  1. Safety and Immunogenicity Testing of a Pilot Polysaccharide Vaccine Preparation to Pseudomonas aeruginosa.

    DTIC Science & Technology

    1982-09-01

    residues similar to that produced by strains of S. 4 yeast . Although yeast extract is not a component of the media, this mannan appears to be part of...nization wTth this vaccine. We have also shown that passive transfer to mice of rabbit antibody made to the IT-l PS vaccine was effective in...abortus by hyerimmune rabbit immunoglobulin A. J. InuunoT r.7IM ’ 8. Onderdonk, A.B., R.B. Markham, D. Zaleznik, R.C. Cisneros, and D.L. Kasper. 1982

  2. Significance of detection of extra metacentric microchromosome in amniotic cell culture.

    PubMed Central

    Bernstein, R; Hakim, C; Hardwick, B; Nurse, G T

    1978-01-01

    A metacentric bisatellited microchromosome was detected in all metaphases from an amniotic culture performed because of maternal age. A wide-ranging survey of the literature failed to disclose any consistent anomaly associated with such a marker, but did reveal that the clinical picture of patients manifesting it could range from complete normality through mental retardation to a variety of deformities. The parents elected for termination, and the only deformity detected in the abortus was fixed talipes equinovarus. The implications of the finding of this marker chromosome on amniocentesis, believed to be reported for the first time here, are discussed particularly in the context of genetic counselling. Images PMID:641948

  3. Maternal-age effect in aneuploidy: Does altered embryonic selection play a role?

    PubMed Central

    Aymé, Ségolène; Lippman-Hand, Abby

    1982-01-01

    The age of mothers of children with trisomy 21 (47,+21) is elevated no matter if the extra chromosome is of maternal or paternal origin, and it has been postulated that decreasing maternal selection against affected conceptuses with advancing age might explain this observation. Since the absence of sufficient data on 47,+21 abortuses precludes a direct test of this hypothesis, we have taken an indirect approach. Pooled data from spontaneous abortions and live births with autosomal trisomies, XXY and XXX, were examined to determine the natural history of these aneuploid conceptuses and its relation to maternal age. The results are consistent with decreasing embryonic selection in older women. PMID:6213153

  4. [Detection of cytomegalovirus by real-time PCR in HIV-positive plasm].

    PubMed

    Ruiz-Tachiquín, Martha Eugenia; Gómez-Delgado, Alejandro; Valdez-Salazar, Hilda Alicia; Aguilera, Penélope

    2014-01-01

    INTRODUCCIÓN: el citomegalovirus es responsable de infecciones persistentes, generalmente asintomáticas en personas sanas pero que en ausencia de una respuesta inmune efectiva puede causar enfermedad severa, por ello es muy importante su detección temprana en los individuos con trastornos de la inmunidad. El objetivo de esta investigación fue hacer un análisis del límite de detección, sensibilidad y concordancia de la reacción en cadena de la polimerasa (PCR) en punto final con los obtenidos con la PCR en tiempo real. MÉTODOS: se realizó un estudio transversal con 43 muestras de plasma humano positivas al virus de la inmunodeficiencia humano, provenientes de individuos de 18 o más años de edad, de uno u otro sexo. Todas las muestras tuvieron una carga viral-VIH mayor a 100 000 copias/mL. Para la PCR en punto final se empleó un método comercial para identificar UL54 (gen viral blanco) y para la PCR en tiempo real se amplificaron fragmentos de los genes UL54 (gen temprano) y UL83 (gen tardío) del citomegalovirus humano.

  5. In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA.

    PubMed

    Turkmani, Aun; Psaroulaki, Anna; Christidou, Athanasia; Chochlakis, Dimosthenis; Tabaa, Darem; Tselentis, Yannis

    2008-09-01

    In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91-->Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91-->Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87-->Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.

  6. Snapshots of Conformational Changes Shed Light into the NtrX Receiver Domain Signal Transduction Mechanism.

    PubMed

    Fernández, Ignacio; Otero, Lisandro H; Klinke, Sebastián; Carrica, Mariela del Carmen; Goldbaum, Fernando A

    2015-10-09

    Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the β5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.

  7. Assessment of a strain 19 brucellosis vaccination program in elk

    USGS Publications Warehouse

    Maichak, Eric J.; Scurlock, Brandon M.; Cross, Paul C.; Rogerson, Jared D.; Edwards, William H.; Wise, Benjamin; Smith, Scott G.; Kreeger, Terry J.

    2017-01-01

    Zoonotic diseases in wildlife present substantial challenges and risks to host populations, susceptible domestic livestock populations, and affected stakeholders. Brucellosis, a disease caused by the bacterium Brucella abortus, is endemic among elk (Cervus canadensis) attending winter feedgrounds and adjacent areas of western Wyoming, USA. To minimize transmission of brucellosis from elk to elk and elk to livestock, managers initiated a B. abortus strain 19 ballistic vaccination program in 1985. We used brucellosis prevalence (1971–2015) and reproductive outcome (2006–2015) data collected from female elk attending feedgrounds to assess efficacy of the strain 19 program while controlling for potentially confounding factors such as site and age. From our generalized linear models, we found that seroprevalence of brucellosis was 1) not lower following inception of vaccination; 2) not inversely associated with proportion of juveniles vaccinated over time; 3) not inversely associated with additional yearlings and adults vaccinated over time; and 4) associated more with feeding end-date than proportion of juveniles vaccinated. Using vaginal implant transmitters in adult females that were seropositive for brucellosis, we found little effect of vaccination coverage at reducing reproductive failures (i.e., abortion or stillbirth). Because we found limited support for efficacy of the strain 19 program, we support research to develop an oral vaccine and suggest that continuing other spatio-temporal management actions will be most effective to minimize transmission of brucellosis and reduce dependency of elk on supplemental winter feeding.

  8. Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

    PubMed Central

    Arnold, Markus F. F.; Caro-Hernandez, Paola; Tan, Karen; Runti, Giulia; Wehmeier, Silvia; Scocchi, Marco; Doerrler, William T.; Ferguson, Gail P.

    2014-01-01

    yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions. PMID:24214946

  9. Induced Disruption of the Iron-Regulatory Hormone Hepcidin Inhibits Acute Inflammatory Hypoferraemia

    PubMed Central

    Armitage, Andrew E.; Lim, Pei Jin; Frost, Joe N.; Pasricha, Sant-Rayn; Soilleux, Elizabeth J.; Evans, Emma; Morovat, Alireza; Santos, Ana; Diaz, Rebeca; Biggs, Daniel; Davies, Benjamin; Gileadi, Uzi; Robbins, Peter A.; Lakhal-Littleton, Samira; Drakesmith, Hal

    2016-01-01

    Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation. PMID:27423740

  10. Longitudinal prevalence and faecal shedding of Chlamydia pecorum in sheep.

    PubMed

    Yang, Rongchang; Jacobson, Caroline; Gardner, Graham; Carmichael, Ian; Campbell, Angus J D; Ryan, Una

    2014-09-01

    The prevalence and faecal shedding of Chlamydia spp. in sheep in Australia has not been well described. Two species-specific quantitative PCRs (qPCRs) targeting the chlamydial outer membrane protein cell surface antigen gene (ompA) were validated and used to determine the prevalence and faecal shedding of C. abortus and C. pecorum from faecal samples of lambs at three sampling times (weaning, post-weaning and pre-slaughter) from eight farms in South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected and screened from approximately 1189 lambs across the four states. C. abortus was not detected in any of the samples screened. The overall prevalence of C. pecorum was 1027/3412 (30.1%) and median bacterial concentrations at weaning, post-weaning and pre-slaughter were 1.8 × 10(7), 1.2 × 10(7) and 9.6 × 10(5)/g faeces, respectively. A subset of C. pecorum positive samples from each farm, (n = 48) was sequenced to confirm their identity. The present study demonstrates that C. pecorum is prevalent in Australian sheep, highlighting a need for further research on the impact of this bacterium on production.

  11. Outbreaks of brucellosis related to the consumption of unpasteurized camel milk.

    PubMed

    Garcell, Humberto G; Garcia, Elias G; Pueyo, Pedro V; Martín, Isis R; Arias, Ariadna V; Alfonso Serrano, Ramon N

    2016-01-01

    Brucellosis is the most frequent zoonosis reported in Qatar, mainly related to exposure to infected camels. An outbreak of human brucellosis in 14 members of a family living in a rural area in Qatar is reported herein. Clinical, epidemiological and laboratory results from all 14 patients with Brucella and 12 non-confirmed family members were collected from files. All patients reported fever for a maximum of 14 days, associated with arthralgia (6 patients), weakness (4 patients), headache (4 patients), diarrhea (2 patients) and abdominal pain (2 patients). The median age of the patients was 10 years and that of non-cases was 16 years, with a predominance of males (92.9%). Elevated levels of transaminases were observed in patients. A mixed infection caused by Brucella abortus and Brucella melitensis was identified by blood culture and serology. The source of the infection was the milk of an infected camel. The outbreak of brucellosis melitensis/abortus related to the consumption of camel milk constitutes a gap in the prevention and control of the potential sources of brucellosis in animal farms. Proper control and education of the population are required.

  12. Genome structure and phylogeny in the genus Brucella.

    PubMed Central

    Michaux-Charachon, S; Bourg, G; Jumas-Bilak, E; Guigue-Talet, P; Allardet-Servent, A; O'Callaghan, D; Ramuz, M

    1997-01-01

    PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus. PMID:9150220

  13. Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.

    PubMed

    Mohamad, Khalil Yousef; Rekiki, Abdessalem; Berri, Mustapha; Rodolakis, Annie

    2010-07-14

    Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection.

  14. Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum.

    PubMed

    Yousef Mohamad, Khalil; Rekiki, Abdessalem; Myers, Garry; Bavoil, Patrik M; Rodolakis, Annie

    2008-01-01

    Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.

  15. Abortion in small ruminants in the Netherlands between 2006 and 2011.

    PubMed

    van den Brom, R; Lievaart-Peterson, K; Luttikholt, S; Peperkamp, K; Wouda, W; Vellema, P

    2012-07-01

    During five successive lambing seasons between 2006 and 2011, 453 submissions of abortion material, 282 of ovine and 171 of caprine origin, were examined at the Animal Health Service in the Netherlands. Infectious agents as the most plausible cause of the abortion were found in 48 percent of the ovine submissions and in 34 percent of the caprine submissions. Submission of both aborted fetus and placental membranes increased the diagnostic yield of laboratory investigations (17 percent and 21 percent for ovine and caprine submissions, respectively). The main infectious causes of abortion in sheep were Chlamydia abortus, Campylobacter spp., Toxoplasma gondii, Listeria spp., and Yersinia pseudotuberculosis. The main infectious causes of abortion in goats were Coxiella burnetii, Chlamydia abortus, Listeria spp., Toxoplasma gondii, and Campylobacter spp. In 42 percent of the ovine and in 56 percent of the caprine submissions a causal agent was not identified. Furthermore, in 12 percent of the ovine and 10 percent of the caprine submissions evidence of placentitis, indicative of an infectious cause of the abortion, was found, but no infectious agent was identified. Most infectious causes of ovine and caprine abortion have zoonotic potential. Humans, especially pregnant women, who are in close contact with lambing sheep or goats should be aware of the importance of precautionary hygiene measures.

  16. Brucellosis in Yellowstone National Park bison: Quantitative serology and infection

    USGS Publications Warehouse

    Roffe, T.J.; Rhyan, Jack C.; Aune, K.; Philo, L.M.; Ewalt, D.R.; Gidlewski, T.; Hennager, S.G.

    1999-01-01

    We collected complete sets of tissues, fluids, and swabs (approx 30) from 37 Yellowstone National Park (YNP) female bison (Bison bison) killed as a result of management actions by the Montana Department of Livestock and YNP personnel. Our goal was to establish the relation between blood tests demonstrating an animal has antibody to Brucella and the potential of that animal to be infected during the second trimester of pregnancy, the time when most management actions are taken. Twenty-eight of the 37 bison were seropositive adults (27) or a seropositive calf (1). We cultured samples using macerated whole tissues plated onto 4 Brucella-selective media and incubated with added CO2 for 1 week. Specimens from 2 adult seropositive females were contaminated, thus eliminating them from our data. Twelve of the remaining 26 seropositive adult and calf female bison (46%) were culture positive for Brucella abortus from 1 or more tissues. Culture positive adult females had high serologic titers. All 11 adults measured 3+ at 1:40 for 10 of 11 (91%) animals. All culture positive female adults had either a PCFIA ???0.080 or a CF reaction ???4+ at 1:80. However 5 (36%) bison with high titers were culture negative for B. abortus. Our findings on the relation between Brucella serology and culture are similar to those reported from studies of chronically infected cattle herds.

  17. Small GTPases and Brucella entry into the endoplasmic reticulum.

    PubMed

    de Bolle, Xavier; Letesson, Jean-Jacques; Gorvel, Jean-Pierre

    2012-12-01

    A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.