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Sample records for abortus ure2 region

  1. Response to different oxidants of Saccharomyces cerevisiae ure2Delta mutant.

    Todorova, Tatina T; Petrova, Ventsislava Y; Vuilleumier, Stéphane; Kujumdzieva, Anna V

    2009-11-01

    Growth of Saccharomyces cerevisiae ure2Delta mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H(2)O(2) was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Delta mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the gamma-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level. PMID:19777209

  2. Seroprevalence of Chlamydia abortus in camel in the western region of Libya

    Rabia Elzlitne

    2016-06-01

    Conclusion: The present findings signify the C. abortus as a potential agent to cause abortion in Libyan camel (C. dromedarius. Besides, the persons who handle camels in Libya are at risk of infecting with C. abortus. [J Adv Vet Anim Res 2016; 3(2.000: 178-183

  3. YEAST PRION PROTEIN Ure2p – A USEFUL MODEL FOR HUMAN PRION DISEASES.

    Tatina T. Todorova

    2015-03-01

    Full Text Available Mammalian transmissible spongiform encephalopathies are uncommon and irreversible diseases caused by prions. Prions lack nucleic acid and can self-propagate by converting normal cell protein to isomeric prion form. In the pathogenesis of these diseases a long variable incubation period occurs, followed by progressive appearance of severe clinical symptoms and death. A major knowledge in the field of prions comes from studies on a functionally unrelated protein of yeast Saccharomyces cerevisiae – [URE3], which in normal state (Ure2p possesses a variety of regulatory and enzymatic functions. Ure2p is a cytoplasmic homodimeric protein with structural homology to glutathione S-transferases and crucial role in nitrogen metabolism, oxidant protection and heavy metal resistance in yeast. In this work, we discuss the role of Ure2p to provide valuable information about protein infectivity, prion structure and functions.

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

    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. PMID:26302373

  5. Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic.

    Todorova, Tatina T; Kujumdzieva, Anna V; Vuilleumier, Stéphane

    2010-11-01

    The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2-2.5-fold upon addition of either arsenate (As(V)) or arsenite (As(III)). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3'-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation. PMID:20740275

  6. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly 13C, 15N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  7. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Wasmer, Christian [Harvard Medical School (United States); Bousset, Luc; Sourigues, Yannick [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Schuetz, Anne [ETH Zurich, Physical Chemistry (Switzerland); Loquet, Antoine [Max Planck Institute for Biophysical Chemistry (Germany); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Melki, Ronald, E-mail: melki@lebs.cnrs-gif.fr [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France)

    2011-11-15

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly {sup 13}C, {sup 15}N labeled protein sample, sequential chemical-shift information for 74% of the N, C{alpha}, C{beta} triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  8. Ure2 is involved in nitrogen catabolite repression and salt tolerance via Ca2+ homeostasis and calcineurin activation in the yeast Hansenula polymorpha.

    Rodríguez, Celia; Tejera, Paula; Medina, Braulio; Guillén, Rosa; Domínguez, Angel; Ramos, José; Siverio, José M

    2010-11-26

    Disruption of HpURE2 resulted in a low expression of genes encoding nitrate-assimilatory proteins; sensitivity to Li(+), Na(+), and Cd(2+); no induction of ENA1; low levels of the GATA-type transcription factor Gat1; and low intracellular Ca(2+) levels. Gat1 levels were also very low in a Δcnb1 mutant lacking the regulatory subunit of calcineurin. The strain Δure2 was very sensitive to the calcineurin inhibitor FK506 and displayed several phenotypes reminiscent of Δcnb1. The reporter 4xCDRE-lacZ, containing calcineurin-dependent response elements in its promoter, revealed that calcineurin activation was reduced in HpΔure2. Expression of ScURE2 in Δure2 rescued nitrogen catabolite repression and Cd(2+) tolerance but not those phenotypes depending on calcineurin activation, such as salt tolerance and nitrate assimilation gene derepression. HpΔure2 showed an increased expression of the gene PMR1 encoding the Golgi Ca(2+)-ATPase, whereas that of PMC1 encoding the vacuolar Ca(2+)-ATPase remained unaltered. PMR1 up-regulation was abolished by deletion of the GATA-type transcription factor GAT2 in a HpΔure2 genetic background, and normal Ca(2+) levels were recovered. Moreover, overexpression of GAT2 or PMR1 yielded strains mimicking the phenotype of the HpΔure2. This suggests that the low Ca(2+) levels in the HpΔure2 mutant are due to the high levels of Pmr1 that replenish the Golgi Ca(2+) content, thus acting as a negative signal for Ca(2+) entry into the cell. We conclude that HpUre2 is involved in salt tolerance and also in nitrate assimilation gene derepression via Ca(2+) homeostasis regulation and calcineurin activation, which control the levels of Gat1. PMID:20880842

  9. Genotipos de aislados de campo de Brucella abortus de distintas regiones geográficas de Chile Genotypes of Brucella abortus field isolates from different geographical regions of Chile

    M Mancilla

    2008-01-01

    Full Text Available La brucelosis bovina es una enfermedad zoonótica, endémica de alto impacto económico. La identificación genética de las cepas prevalentes de Brucella abortus, el patógeno, es clave para establecer estrategias epidemiológicas de control de la enfermedad. La secuencia de inserción IS711 ha sido utilizada como un marcador genético para diferenciar entre especies de Brucella, miembros de una misma especie y dentro de un mismo biovar. Hemos analizado los perfiles de IS711-RFLP de 46 aislados de B. abortus, recolectados durante el periodo 1997-2005, provenientes de 16 áreas geográficas diferentes de Chile. Todos los aislados fueron previamente identificados como B. abortus biovar 1, utilizando las técnicas convencionales. De estos, el 87% compartieron el mismo perfil de IS711-RFLP, mientras que el 8,7% correspondió al patrón de la cepa vacuna RB51. En este trabajo se informa el hallazgo de dos cepas indistinguibles por PCR AMOS con perfiles nuevos de IS711-RFLP, no reportados previamente.Bovine brucellosis is an endemic, zoonotic disease of high economic impact. The genetic identification of the prevalent Brucella abortus strains, the pathogen, is key to pursue further epidemiological strategies for disease control. The insertion sequence IS711 has been used as genetic marker to differentiate among Brucella species, members of the same specie and within the same biovar. We have analyzed the IS711-RFLP pattern for 46 B. abortus isolates, collected during the period of 1997-2005 from 16 different geographical areas of Chile. All isolates were previously identified by conventional techniques as B. abortus biovar 1. Of these, 87% sharedthesame IS711 DNA profile, while an 8.7 % corresponded to the pattern of RB51 vaccine strain. We report the finding of two new strains, not differentiated by AMOS PCR, which showed unreported patterns of IS711-RFLP.

  10. Crystallization and preliminary X-ray crystallographic analysis of yeast prion protein Ure2p with shortened N-terminal

    2001-01-01

    An orthorhombic crystal form of a recombinant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffusion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 ?, b = 74.7 ?, c = 131.0 ?. The crystals diffract to beyond 3.0 ? resolution at a synchrotron beamline.

  11. The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro.

    Kai Wang

    Full Text Available Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI+] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

  12. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo

    Vincenzo Caporale; Barbara Bonfini; Elisabetta Di Giannatale; Andrea Di Provvido; Simona Forcella; Armando Giovannini; Manuela Tittarelli; Massimo Scacchia

    2010-01-01

    Approximately 250 000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150 000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vacci...

  13. Conformational change of glutathione-S-transferase by its co-expression with prion domain of yeast Ure2p

    2001-01-01

    The Ure2 protein from Saccharomyces cerevisisae has a changeable structure similar to that ofrnammalian prion protein. Its N-terminal is the prion domain (PrD) consisting of 65 amino acids which plays a critical role in yeast prion development. In this study, PrD gene was recombinated with glutathione-S-transferase(GST) gene, and a soluble GST-PrD(sGST-PrD) fusion protein was expressed in E. coli. sGST-PrD could spontaneously polymerize into amyloid fibrils in vitro, displaying typical β-sheet-type structure; it had increased resistance to proteinase K and exhibited amvloid-like optical properties. Moreover, the aggregated GST-PrD(aGST-PrD) could induce sGST-PrD to aggregate into fibrils. These results indicate that PrD could change the conformation of GST moiety in a recombinant protein with PrD to form a prion-like chimeric protein, which proves that PrD has the ability to mediate a prion-like conversion of other proteins fused with it.

  14. Immunological response to the Brucella abortus GroEL homolog.

    Lin, J.; Adams, L G; Ficht, T A

    1996-01-01

    Western blot (immunoblot) analysis of sera from cattle vaccinated with Brucella abortus S19 exhibit an elevated serologic response to Hsp62, the GroEL homolog (BaGroEL). Serologic screening of individual cows vaccinated with B. abortus S19 revealed no correlation between the immune response to BaGroEL and protection against a challenge with virulent organisms. The humoral immune response to BaGroEL was restricted to a region of the mature protein which mapped to amino acids 317 to 355 and may...

  15. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo

    Vincenzo Caporale

    2010-03-01

    Full Text Available Approximately 250 000 water buffalo (Bubalus bubalis live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150 000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51 has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19. The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

  16. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo.

    Caporale, Vincenzo; Bonfini, Barbara; Di Giannatale, Elisabetta; Di Provvido, Andrea; Forcella, Simona; Giovannini, Armando; Tittarelli, Manuela; Scacchia, Massimo

    2010-01-01

    Approximately 250,000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150,000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group. PMID:20391363

  17. Biotyping and genotyping (MLVA16 of Brucella abortus isolated from cattle in Brazil, 1977 to 2008.

    Sílvia Minharro

    Full Text Available 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.

  18. Biotyping and genotyping (MLVA16) of Brucella abortus isolated from cattle in Brazil, 1977 to 2008.

    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

  19. An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

    Shao-Man YIN; Man-Sun SY; Po TIEN

    2004-01-01

    Production of the pathogenic prion isoform prpsc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentβ-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors ofPrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

  20. An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

    Shao-ManYIN; Man-SunSY; PoTIEN

    2004-01-01

    Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentB-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPse and will be helpful for further understanding the mechanism of conformational conversion process.

  1. Recent advances in Brucella abortus vaccines.

    Dorneles, Elaine M S; Sriranganathan, Nammalwar; Lage, Andrey P

    2015-01-01

    Brucella abortus vaccines play a central role in bovine brucellosis control/eradication programs and have been successfully used worldwide for decades. Strain 19 and RB51 are the approved B. abortus vaccines strains most commonly used to protect cattle against infection and abortion. However, due to some drawbacks shown by these vaccines much effort has been undertaken for the development of new vaccines, safer and more effective, that could also be used in other susceptible species of animals. In this paper, we present a review of the main aspects of the vaccines that have been used in the brucellosis control over the years and the current research advances in the development of new B. abortus vaccines. PMID:26155935

  2. Chlamydophila abortus em animais de produção Chlamydophila abortus in production animals

    Francielle Gibson da Silva

    2006-02-01

    Full Text Available A Chlamydophila abortus (anteriormente classificada como Chlamydia psittaci sorotipo 1 tem sido descrita em muitos países, associada principalmente com distúrbios reprodutivos em ovinos, bovinos e caprinos. O aborto enzoótico dos ovinos e caprinos e o aborto epizoótico dos bovinos são as doenças mais importantes causadas por esta bactéria. No Brasil, as pesquisas com C. abortus são praticamente inexistentes. O objetivo desta revisão é apresentar informações sobre modificações taxonômicas, ciclo de vida, epidemiologia, patogenia, sinais clínicos e diagnóstico da infecção por C. abortus principalmente em ovinos, bovinos e caprinos.Chlamydophila abortus (previously known as Chlamydia psittaci serovar 1 has been reported in many countries, associated with reproductive disorders in sheep, cattle, and goats. The enzootic abortion of sheep and goats and the epizootic bovine abortion are the most important diseases produced by this bacterium. In Brazil, there is scarce information about C. abortus. The objective of this review is to show information about taxonomic changes, life cycle, epidemiology, pathogenesis, clinical signs and diagnosis of C. abortus in sheep, cattle and goats.

  3. Valutazione dell’efficacia del vaccino Brucella abortus ceppo RB51 rispetto al vaccino di referenza Brucella abortus ceppo 19 nel bufalo

    Massimo Scacchia; Armando Giovannini; Manuela Tittarelli; Simona Forcella; Andrea Di Provvido; Elisabetta Di Giannatale; Barbara Bonfini; Vincenzo Caporale

    2010-01-01

    Il patrimonio zootecnico della specie bufalina (Bubalus bubalis) della regione Campania, è di 250 000 capi, di questi 150 000 allevati in aziende zootecniche della provincia di Caserta. In queste aziende, nel 2007, l’infezione da Brucella abortus ha avuto la prevalenza media, per allevamento, del 20%. Complessivamente, i 2/3 degli allevamenti positivi hanno evidenziato una prevalenza superiore al 10% e, di questi, i 3/4 una prevalenza superiore al 20%. Prendendo il 20% come valore di riferime...

  4. Prevalence of antibodies to Chlamydophila abortus in ovines in the Londrina area of Paraná state, Brazil

    Bruna Azevedo de Carvalho Lima

    2014-10-01

    Full Text Available The aim of this paper was to estimate the prevalence of antibodies to Chlamydophila abortus in ewes from the Londrina region of Paraná state. Blood samples from 267 adult ewes from eight herds were submitted to complement fixation testing. Tests were considered positive when the titers ? 32. Nineteen animals (7.11% in 7 (87.50% herds tested positive for antibodies against C. abortus. The titers were 32 in 17 animals and were 64 and 128 in the other two animals. The clinical signs recorded were abortion, stillbirth, weak newborns and heat repetition. The high frequency of herds with at least one positive animal and a seroprevalence of 7.11% suggest the presence of Chlamydophila abortus in the animals of studied herds.

  5. Prevalence of antibodies to Chlamydophila abortus in ovines in the Londrina area of Paraná state, Brazil

    Bruna Azevedo de Carvalho Lima; Kledir Anderson Hofstaetter Spohr; Francielle Gibson da Silva Zacarias; Sergio Mangano de Almeida Santos; Mario de Luca Neto; Carlo Turilli; Julio Cesar de Freitas

    2014-01-01

    The aim of this paper was to estimate the prevalence of antibodies to Chlamydophila abortus in ewes from the Londrina region of Paraná state. Blood samples from 267 adult ewes from eight herds were submitted to complement fixation testing. Tests were considered positive when the titers ? 32. Nineteen animals (7.11%) in 7 (87.50%) herds tested positive for antibodies against C. abortus. The titers were 32 in 17 animals and were 64 and 128 in the other two animals. The clinical signs recorded w...

  6. Brucella abortus Infection Acquired in Microbiology Laboratories

    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.

  7. Coombs Antiglobulin Test Using Brucella abortus 99 as Antigen To Detect Incomplete Antibodies Induced by B. abortus RB51 Vaccine in Cattle

    Ciuchini, Franco; Adone, Rosanna; Pasquali, Paolo

    2002-01-01

    This study showed that vaccination of cattle with Brucella abortus rough strain RB51 induces incomplete antibodies that can be detectable by a Coombs antiglobulin test using the B. abortus 99 smooth strain.

  8. The Effect of Irradiation on the Immunogenity of Brucella Abortus

    An experiment was carried out to study the effect of irradiation on the immunogenity of B. abortus. The B. abortus were irradiated by Gamma Cells (60Co). An experiment were divided into four groups. The first group (V1) was inoculated by irradiated B. abortus with the dose of 0.25 kGy. The second group (V2) was inoculated by irradiated B. abortus with the dose of 0.50 kGy. The third group (V3) was inoculated by irradiated B. abortus with the dose of 0.75 kGy. The fourth group (V4) was inoculated by Brucella vaccine 8.19. The observation respectively were included purely test, safety test, RBT serological test, diffusion test, development the colony of B. abortus in lien, and pathology anatomic inspection. The results obtained showed that 0.25 kGy was the expectantly dose of irradiation which could not only decreasing the infectivity of B. abortus but also has the ability to become a good immunogen for stimulating the immune response in the experiment animals. (author)

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

    R. Villagra-Blanco

    2015-09-01

    Full Text Available 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.

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

    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

  11. Brucella abortus S19 genome sequenced, points toward virulence genes

    Whyte, Barry James

    2008-01-01

    Researchers at the Virginia Bioinformatics Institute at Virginia Tech; the National Animal Disease Center in Ames, Iowa; and collaborators at 454 Life Sciences, Branford, Conn., have sequenced the genome of Brucella abortus strain S19.

  12. A combined vaccine against Brucella abortus and infectious bovine rhinotracheitis

    Kamaraj, Govindasamy; Chinchkar, Shankar R.; Rajendra, Lingala; Srinivasan, Villuppanoor Alwar

    2009-01-01

    The present study was undertaken to study the immune response in calves vaccinated with Brucella abortus strain 19, infectious bovine rhinotracheitis (IBR) vaccines in monovalent form and combined vaccine containing both antigen. The seroconversion of monovalent and combined vaccines was tested in seronegative cattle calves. IBR vaccine alone and combination with live Brucella abortus S19 vaccine elicited an anamnestic response on day 60 post booster but started declining from day 90 onwards ...

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

    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. PMID:25131740

  14. Recombinant Brucella abortus gene expressing immunogenic protein

    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.

  15. Valutazione dell’efficacia del vaccino Brucella abortus ceppo RB51 rispetto al vaccino di referenza Brucella abortus ceppo 19 nel bufalo

    Massimo Scacchia

    2010-03-01

    Full Text Available Il patrimonio zootecnico della specie bufalina (Bubalus bubalis della regione Campania, è di 250 000 capi, di questi 150 000 allevati in aziende zootecniche della provincia di Caserta. In queste aziende, nel 2007, l’infezione da Brucella abortus ha avuto la prevalenza media, per allevamento, del 20%. Complessivamente, i 2/3 degli allevamenti positivi hanno evidenziato una prevalenza superiore al 10% e, di questi, i 3/4 una prevalenza superiore al 20%. Prendendo il 20% come valore di riferimento, la metà degli allevamenti infetti (22% degli allevamenti casertani ha evidenziato prevalenze inferiori o uguali al 20%, la restante metà (un altro 22% del totale prevalenze comprese tra il 20 e il 56%. In questo contesto epidemiologico è stato adottato un piano di eradicazione della brucellosi che prevedeva l’abbattimento dei capi infetti e la vaccinazione del restante patrimonio bufalino delle zone con più alta incidenza. Per la profilassi vaccinale della brucellosi, il Manual of diagnostic tests and vaccines for terrestrial animals (OIE prevede l’utilizzo del vaccino B. abortus S19 (S19. Purtroppo, l’utilizzo del vaccino negli animali adulti non è privo di possibili effetti indesiderati. Per superare questo aspetto negativo è stato ipotizzato l’impiego del vaccino di B. abortus RB51 (RB51 anche se in letteratura scientifica, sono risultati disponibili pochi dati relativi alla corretta dose vaccinale, all’efficacia e all’innocuità del vaccino nel bufalo. A tale scopo è stato condotto uno studio comparativo tra i due vaccini. Sono state utilizzate 13 femmine di bufalo di 5 mesi di età provenienti da un allevamento ufficialmente indenne da brucellosi. Un gruppo di 5 animali è stato vaccinato due volte, a distanza di un mese, con una dose di RB51 tre volte superiore a quella prevista per i bovini; un secondo gruppo di 5 bufale con S19 rispettando il dosaggio raccomandato per i bovini e un terzo gruppo, di 3 animali di controllo

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

    R. Villagra-Blanco; 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...

  17. Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea

    Jung Suk-Chan; Heo Young-Ran; Hwang In-Yeong; Cho Yun-Sang; Cho Dong-Hee; Kang Sung-Il; Her Moon; Yoo Han-Sang

    2009-01-01

    Abstract Background A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates. Results A total of 177 isolates originating from 105 cattle farms for the period 1...

  18. A combined vaccine against Brucella abortus and infectious bovine rhinotracheitis.

    Kamaraj, Govindasamy; Chinchkar, Shankar R; Rajendra, Lingala; Srinivasan, Villuppanoor Alwar

    2009-06-01

    The present study was undertaken to study the immune response in calves vaccinated with Brucella abortus strain 19, infectious bovine rhinotracheitis (IBR) vaccines in monovalent form and combined vaccine containing both antigen. The seroconversion of monovalent and combined vaccines was tested in seronegative cattle calves. IBR vaccine alone and combination with live Brucella abortus S19 vaccine elicited an anamnestic response on day 60 post booster but started declining from day 90 onwards against IBR. B. abortus S19 alone and in combination with IBR vaccine gave more than 2 log protection in mice two weeks post challenge. Fluorescence polarization assay analysis with sera samples of calves vaccinated with B. abortus S19 monovalent vaccine alone and in combination with IBR vaccine revealed the presence of B. abortus antibodies. The components of the combined vaccine did not show any evidence of interference in the development of immunity. This combined vaccine may provide economical and affordable biological for the control of brucellosis and IBR. PMID:23100765

  19. Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

    Rigby, C E; Fraser, A.D.

    1989-01-01

    Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintaine...

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

    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 atten...

  1. DeltaznuADeltapurE Brucella abortus 2308 mutant as a live vaccine candidate.

    Yang, Xinghong; Thornburg, Theresa; Walters, Nancy; Pascual, David W

    2010-01-22

    To create a new, safe brucellosis live vaccine, a double mutant strain was constructed from Brucella abortus 2308. Using the DeltaznuA B. abortus 2308 mutant, a second mutation was introduced by deleting purE gene. The DeltaznuA DeltapurE B. abortus 2308 strain was less capable of surviving in macrophages. When evaluated in vivo, it was cleared within 8 weeks (wks) from mice, causing significantly less inflammation than spleens obtained from wild-type B. abortus 2308-infected mice. Furthermore, two doses of DeltaznuA DeltapurE B. abortus 2308 conferred 0.79 log protection, similar to S19 as did a single dose of DeltaznuA B. abortus 2308. Thus, this study shows the DeltaznuA DeltapurE B. abortus 2308 strain to be a potential livestock vaccine candidate. PMID:19914192

  2. Detection of Chlamydophila abortus in Sheep (Ovis aries in Mexico

    Juan M. Jiménez-Estrada

    2008-01-01

    Full Text Available Chlamydophila abortus is one of the pathogens which induce abortion in small ruminants; this pathogen has a tropism for ruminant placenta and causes the disease commonly referred to as Ovine Enzootic Abortion (OEA. In Europe are estimated economic losses of around 20 million pounds a year by OEA. In the American Continent the disease has been reported only in Canada, the United States, Colombia and Chile while in Mexico it is unknown whether OEA is common and it is causing abortions in flocks of sheep from “Estado de Mexico”. The objective of this study was investigating the prevalence of anti-Chlamydophila abortus IgG antibodies and detection of C. abortus DNA in sheep with clinical abort history by mean of ELISA assay (C. abortus ELISA, Institute Pourquier, Montpellier, France and molecular identification of the principal outer membrane protein (POMP 90-91B gene by PCR, respectively. A cross-sectional study was carried out to enroll and random sample of ewes from november 2003 until march 2005. A total of 349 sera and vaginal swabs samples were collected from 35 flocks of sheep from Xalatlaco. The results showed that the seropositive rate was 31.1% (14/45 for healthy and 21.3% (65/304 for sheep with history clinical of abort. In vaginal swabs, the PCR showed 0% (0/45 for healthy animals and 0.65% (2/304 for aborted sheep. Samples from the lungs and liver of the fetus of one of these animals were also positive for C. abortus. In conclusion, these results confirmed that infection with C. abortus is common and is affecting sheep flocks in the Mexican highlands. Therefore, is necessary that the authorities responsible for animal welfare in Mexico (SAGARPA to set up appropriate epidemiological surveillance and control programs to eradicate this disease.

  3. Identification of new IS711 insertion sites in Brucella abortus field isolates

    Moriyón Ignacio

    2011-08-01

    Full Text Available Abstract Background Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated. Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. Results We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Conclusions Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  4. Seroprevalence of Brucella abortus and Leptospira hardjo in cattle

    S. Jegaveera Pandian

    2015-02-01

    Full Text Available Aim: The aim was to assess the seroprevalence of B. abortus and Leptospira hardjo in the cattle population of Bihar, this work was carried out. Materials and Methods: Randomly selected 450 cattle from nine districts of Bihar were serologically screened for antibodies against L. hardjo and B. abortus. DAS-ELISA for leptospira and AB-ELISA for brucella were carried out. Based on the results prevalence in each district and the state are reported herewith. Results: In this study, it was found that the seroprevalence of L. hardjo was 9.11% and that of B. abortus was 12.2% in Bihar. Indigenous cattle were found to be less susceptible to leptospirosis and brucellosis even though they accounted for 83.11% of the study population. Conclusion: Although there was no acute disease, antibodies detected against L. hardjo and B. abortus in the cattle population indicated the presence of chronic and subclinical infection, which could challenge the fertility of the animals.

  5. 9 CFR 113.65 - Brucella Abortus Vaccine.

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Brucella Abortus Vaccine. 113.65 Section 113.65 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Bacterial Vaccines § 113.65 Brucella...

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

    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. PMID:27143220

  7. Endocarditis por Brucella abortus: Reporte del primer caso en C.R Brucella abortus Endocarditis

    Manuel Antonio Villalobos-Zúñiga; Edith Barrantes-Valverde; Patricia Monge-Ortega

    2011-01-01

    Paciente masculino de 36 años de edad, proveniente de la zona rural de Costa Rica, con un cuadro clínico de 8 meses de evolución de fiebre, mialgias, artralgias, pérdida de peso y lumbalgia; referido por la detección de un soplo de insuficiencia aórtica. El ecocardiograma reveló endocarditis de la válvula aórtica, y se obtuvieron 4 hemocultivos positivos por Brucella abortus biotipo 3, con serologías negativas por brucelosis. Se inició tratamiento con antibióticos y luego se le realizó un ree...

  8. Can Chlamydia abortus be transmitted by embryo transfer in goats?

    Oseikria, M; Pellerin, J L; Rodolakis, A; Vorimore, F; Laroucau, K; Bruyas, J F; Roux, C; Michaud, S; Larrat, M; Fieni, F

    2016-10-01

    The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from

  9. Endocarditis por Brucella abortus: Reporte del primer caso en C.R Brucella abortus Endocarditis

    Manuel Antonio Villalobos-Zúñiga

    2011-09-01

    Full Text Available Paciente masculino de 36 años de edad, proveniente de la zona rural de Costa Rica, con un cuadro clínico de 8 meses de evolución de fiebre, mialgias, artralgias, pérdida de peso y lumbalgia; referido por la detección de un soplo de insuficiencia aórtica. El ecocardiograma reveló endocarditis de la válvula aórtica, y se obtuvieron 4 hemocultivos positivos por Brucella abortus biotipo 3, con serologías negativas por brucelosis. Se inició tratamiento con antibióticos y luego se le realizó un reemplazo valvular aórtico; 4 meses después ingresó con dolor torácico que se atribuyó a una oclusión de la arteria descendente anterior, demostrada angiográficamente, por posible embolismo. En la actualidad cursa clínicamente estable con manejo médico para su cardiopatía, sin recaída infecciosa.The case of a 36-year-old patient from a rural area is presented. He came with an 8 month history of fever, myalgias, arthralgias, weight loss and lower back pain; who also had an aortic insufficiency murmur detected. The diagnosis of aortic valve endocarditis was made by echocardiography, and had 4 positive blood cultures for Brucella abortus biotype 3, and negative serologic test for brucellosis. He was started on antibiotics and later on underwent aortic valve replacement, with a late coronary cardioembolism as a complication.

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

    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 prope...

  11. Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9.

    Baldi, P C; Giambartolomei, G H; Goldbaum, F A; Abdón, L F; C.A. Velikovsky; Kittelberger, R; Fossati, C A

    1996-01-01

    The humoral immune responses against three different antigens of Brucella abortus were monitored by enzyme-linked immunosorbent assay in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9. Immunoglobulin G (IgG) and IgM responses against (i) B. abortus lipopolysaccharide (LPS), (ii) total cytoplasmic proteins depleted of LPS (LPS-free CYT), and (iii) B. abortus 18-kDa cytoplasmic protein were measured. Vaccinated animals and Yersinia-inf...

  12. Investigation of the possible role of Chlamydophila abortus in reproductive failures in nrazilian herds of domestic ruminants

    Francielle Gibson da Silva-Zacarias; Amauri Alcindo Alfieri; Kledir Anderson Hofstaetter Spohr; Bruna Azevedo de Carvalho Lima; Rosângela Claret de Oliveira; Carlo Turilli; Michele Lunardi; Rodrigo Alejandro Arellano Otonel; Julio Cesar de Freitas

    2009-01-01

    Chlamydophila abortus (C. abortus) infection is related to reproductive failure in domestic ruminants. Although it has not been well characterized worldwide, this pathogen has already been identified in some European countries and in the USA. In Brazil, preliminary studies have shown serological evidence of C. abortus infection in herds with low antibody prevalence. Until now, the identification of C. abortus in biological samples from females presenting reproductive failures has not been des...

  13. Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

    Francielle Gibson da Silva-Zacarias; Amauri Alcindo Alfieri; Kledir Anderson Hofstaetter Spohr; Bruna Azevedo de Carvalho Lima; Fábio Juliano Negrão; Michele Lunardi; Julio Cesar de Freitas

    2009-01-01

    Chlamydophila abortus (C. abortus) is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR) assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragmen...

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

    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.

  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

    Pauline eNol

    2016-02-01

    Full Text Available 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 glycosytransferase 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

  16. Brucella abortus 1119-3 O-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion.

    Cherwonogrodzky, J W; Nielsen, K H

    1988-01-01

    Purified Brucella abortus 1119-3 and Brucella melitensis 16M lipopolysaccharide O-chain polysaccharides were not precipitated in agar gel immunodiffusion by any of 24 sera from vaccinated cattle but were precipitated by 18 of 24 sera from infected cattle. This difference can be used to differentiate sera of cattle vaccinated with B. abortus S-19 from sera of some field-strain-infected cattle.

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

    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.

  18. Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea

    Jung Suk-Chan

    2009-10-01

    Full Text Available Abstract Background A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates. Results A total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the Brucella isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via in-vitro and in-vivo passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign Brucella isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates. Conclusion The MLVA assay has enough discrimination power in the Brucella species level and can be utilized as a tool for the epidemiological trace-back of the B. abortus isolates. But it is important to consider that Brucella isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.

  19. Circulating strains of Brucella abortus in cattle in the province of Santo Domingo de los Tsáchilas - Ecuador.

    Richar Ivan Rodríguez Hidalgo

    2015-03-01

    Full Text Available In Ecuador, the Province of Santo Domingo de los Tsáchilas represents the largest informal market of livestock due to its strategic position in the country; thus, given the high mobility of cattle in this region, the aim of this study was to determine the strain variation of Brucella sp.. Part of the study was the isolation, biotyping and genotyping of Brucella species isolated from milk and supra-mammary lymph nodes of sero-positive bovines. Protocols used for isolation, biotyping and genotyping of Brucella species were selective Farrell medium, biochemical assays and IS711-PCR, AMOS-PCR and HOOF-Prints techniques, respectively. In total, 656 animals from sero-positive dairy herds and from the slaughterhouse of the province were diagnosed by Rose Bengal and Wright’s Slow Agglutination test with EDTA. From these animals, 50 animals were found sero-positive for brucellosis. Twenty-five lymph nodes and 25 milk samples from each group of positive reactors were cultured and growth. Isolation was possible in 4 (16% and 9 (36%, respectively; and of these, 10 isolates were diagnosed as Brucella sp. All 4 isolates of lymphatic tissue corresponded to Brucella abortus biotype 1, confirmed as field strains by molecular analysis. Milk isolations, biochemically showed 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%.

  20. Isolation, purification, and partial characterization of Brucella abortus matrix protein.

    Moriyon, I; Berman, D T

    1983-01-01

    Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along wit...

  1. Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

    Nielsen, K.

    1985-01-01

    Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunog...

  2. Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina.

    Wallach, J C; Ferrero, M C; Victoria Delpino, M; Fossati, C A; Baldi, P C

    2008-08-01

    The pathological consequences of exposure to the vaccine strain Brucella abortus S19 were evaluated in 30 employees from vaccine-manufacturing plants. Active brucellosis was diagnosed in 21 subjects, of whom only five recalled an accidental exposure. Clinical manifestations were mild, and only one patient presented a complication. After antimicrobial therapy, initially symptomatic patients either experienced clinical remission or had mild persistent symptoms. This is the first study reporting infection by B. abortus S19 among workers from vaccine-manufacturing plants, which in many cases was acquired from unnoticed exposures. Measures to improve the safety of B. abortus S19 handling should be implemented. PMID:18727806

  3. Expression and validation of D-erythrulose 1-phosphate dehydrogenase from Brucella abortus: a diagnostic reagent for bovine brucellosis.

    Eoh, Hyungjin; Jeon, Bo-Young; Kim, Zhiyeol; Kim, Seung-Cheol; Cho, Sang-Nae

    2010-07-01

    Brucella abortus is a bacterium of brucellosis causing abortion in cattle. The diagnosis of bovine brucellosis mainly relies on serologic tests using smooth lipopolysaccharide (S-LPS) from B. abortus. However, the usefulness of this method is limited by false-positive reactions due to cross-reaction with other Gram-negative bacteria. In the present study, the eryC gene encoding B. abortus d-erythrulose 1-phosphate dehydrogenase, which is involved in the erythritol metabolism in virulent B. abortus strain but is absent from a B. abortus vaccine strain (S19), was cloned. Recombinant EryC was expressed and purified for the evaluation as a diagnostic reagent for bovine brucellosis. Other B. abortus proteins, Omp16, PP26, and CP39 were also purified and their seroreactivities were compared. Recombinant EryC, Omp16, PP26, and PP39 were all reactive to B. abortus-positive serum. The specificity of recombinant Omp16, PP26, CP39, and EryC, were shown to be approximately 98%, whereas that of B. abortus whole cell lysates was shown to be 95%. The sensitivity of Omp16, PP26, CP39, and EryC were 10%, 51%, 64%, and 43%, respectively, whereas that of B. abortus whole cell lysates was 53%. These results suggested that B. abortus EryC would be a potential reagent for diagnosis for bovine brucellosis as a single protein antigen. PMID:20622221

  4. Detection of Brucella abortus in Chiredzi district in Zimbabwe

    Calvin Gomo

    2012-02-01

    Full Text Available Brucellosis is an endemic disease in Zimbabwe caused by the genus Brucella. Brucella seroprevalence was recently reported to be high in the wildlife-livestock interface in the Chiredzi district and the neighbouring Gonarezhou National Park (GNP in Zimbabwe, and higher amongst communal cattle with an abortion history and access to grazing in GNP than amongst communal cattle with no abortion history or access to grazing in GNP. The aim of this study was to investigate Brucella species in brucellosis seropositive cattle in the Chiredzi district with access to GNP using isolation and identification. Isolation of Brucella species from whole blood (n = 18 and milk samples (n = 10 from seropositive animals with an abortion history was based on the rose Bengal test (RBT and enzyme-linked immunoassays (enzyme- linked immunosorbent assay [ELISA]; indirect ELISA and complement ELISA, using microbiology and polymerase chain reaction (PCR methods. Brucella abortus was cultured and identified from blood and milk collected from seropositive cows in both communal areas. The Brucella-specific 16-23S intergenic spacer (ITS PCR and multiplex AMOS-PCR assays verified the identification of the cultures. Our results confirmed that B. abortus is present in cattle on communal farms in the Chiredzi district in Zimbabwe and might cause cattle abortions. The need for implementing control measures and raising public awareness on zoonotic transmission of brucellosis are recommended.

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

    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. PMID:25704292

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

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

    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 ...

  7. Transplacentally transmitted congenital brucellosis due to brucella abortus biotype 1 in sprague-dawley rats

    In the investigation on the transplacentally transmitted congenital brucellosis due to Brucella abortus biotype 1 in Sprague- Dawley rats, neither any stillbirth, abortion or premature birth nor any abnormality of fetus was observed in the infected group or in the control group. B. abortus biotype was isolated from the fetus of infected rats only. Only one band of 498 base pair DNA was obtained in polymerase chain reaction products from DNA of the fetuses of infected SD rats. (author)

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

    Ayman Al-Mariri; George Saour; Razan Hamoud

    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 ext...

  9. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes

    Pizarro-Cerda, J. (Javier); Meresse, S. (Stéphane); Parton, R G; van der Goot, G; Sola-Landa, A.; Lopez-Goñi, I. (Ignacio); Moreno, E.; Gorvel, J P

    1998-01-01

    Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At approximately 1 h postinoculation, bacteria are loc...

  10. Isolation of Brucella abortus ssb and uvrA genes from a genomic library by use of lymphocytes as probes.

    Zhu, Y.; S.C. Oliveira; Splitter, G A

    1993-01-01

    Brucella abortus proteins from virulent S2308 expressed from a pBluescript II SK- genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total...

  11. Protein profile of Chlamydophila abortus isolates from Kerala, India

    Binu K Mani

    Full Text Available Chlamydiae are of microbiological interest because of their mode of interaction with eukaryotic host cells and their specialized life cycle with unique features of parasitism. Reports regarding prevalence of infections of Chlamydophila abortus, the causative organism for chlamydial abortions in livestock, was the basis of the study. Two isolates, one each from cattle and goat abortion along with a reference isolate, were used for characterization with Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE. Elementary bodies infected Mc Coy cells, harvested from bottle cultures were disrupted by Teflon coated magnetic pellet. Urografin-76 diluted with Tris-Potassium hydrochloride was used for purification of Elementary bodies of Chlamydophila abortus organism. On protein estimation of Elementary bodies by Biuret method, all the three isolates revealed protein concentration between 500-1000 mg/100ml, which were sufficient for electrophoresis. Ten percent of resolving gel and five percent of stacking gel of polyacrylamide in which 10g of processed isolate samples along with standard protein marker and Mc Coy cell protein (control were electrophoresed. Using Alpha Imager Gel Documentation System, the protein bands were analyzed. Twelve bands each for local bovine isolate and reference isolate were noticed while only 10 bands were there in the caprine isolate. Additional bands of 148 kDa and 135 kDa were present in bovine isolate, compared to the reference isolate, while 152 kDa and 137 kDa bands were unique for caprine isolate. [Vet. World 2011; 4(10.000: 470-472

  12. 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

    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...

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

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

    2016-06-01

    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

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

    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. PMID:18319583

  15. Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19.

    Sabio y García, Julia V; Farber, Marisa; Carrica, Mariela; Cravero, Silvio; Macedo, Gilson C; Bigi, Fabiana; Oliveira, Sergio C; Rossetti, Osvaldo; Campos, Eleonora

    2008-05-01

    Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis. PMID:18462974

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

    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

  17. In vitro and in vivo characterization of smooth small colony variants of Brucella abortus S19.

    Jacob, J; Hort, G M; Overhoff, P; Mielke, M E A

    2006-02-01

    Brucella abortus is known to produce chronic infections in both humans and a variety of animal species. However, the mechanisms underlying the persistence of the bacteria in the presence of an ongoing immune response are still unknown. In this respect we made use of the observation that in vitro grown B. abortus S19 exhibits heterogenicity in colony size when plated onto TS agar, while experimental infection of mice uniformly results in the in vivo selection of the small colony variant. We demonstrate that the spontaneous smooth small colony variant is characterized not only by a slower growth rate in vitro but also by an increased tolerance to hyperosmotic medium and, most importantly, a less effective clearance from spleens and livers of experimentally infected mice. On a molecular level, a gene with homology to a formerly described galactoside transport ATP binding protein (mglA) was differentially expressed in small versus large colonies of B. abortus S19. PMID:16239117

  18. Experimental infection of Richardson's ground squirrels (Spermophilus richardsonii) with attenuated and virulent strains of Brucella abortus.

    Nol, Pauline; Olsen, Steven C; Rhyan, Jack C

    2009-01-01

    A previous investigation of the safety of Brucella abortus strain RB51 (sRB51) in various nontarget species suggested that Richardson's ground squirrels (Spermophilus richardsonii) may develop persistent infections when orally inoculated with the vaccine. In the present study, sRB51, B. abortus strain 19 (s19), and virulent B. abortus strain 9941 (s9941) were administered orally to Richardson's ground squirrels to further characterize B. abortus infection in this species. Six groups of nongravid ground squirrels were orally inoculated with 6 x 10(8) colony forming units (cfu) sRB51 (n = 10), 2.5 x 10(4) cfu s19 (n = 10), 2.5 x 10(7) cfu s19 (n = 6), 1.3 x 10(6) cfu s9941 (n = 5), 2.1 x 10(8) cfu s9941 (n = 5), or vaccine diluent (control; n = 4). One of five animals in the lower-dose s19 group and two of three animals in the higher-dose s19 group showed persistence of bacteria in various tissues at 14 wk postinoculation (PI). At 18 wk PI, one of five animals in the sRB51 group and one of five animals in the high-dose s9941 group were culture positive. Although we did detect some persistence of B. abortus strains at 18 wk, we found no evidence of pathology caused by B. abortus strains in nonpregnant Richardson's ground squirrels based on clinical signs, gross lesions, and microscopic lesions. PMID:19204348

  19. A rare cause of native tricuspid valve endocarditis: Abortus

    M.Sıddık Evsen

    2011-03-01

    Full Text Available A 28-year-old female patient, who at her 7 weeks ofpregnancy, admitted to hospital with abdominal painand vaginal bleeding. She had been hospitalized in anothercenter with the diagnosis of spontaneous completeabortion. After discharge, her clinical process, deteriorateddue to fever, chills and fatigue therefore she hadbeen admitted to emergency unit of that hospital onceagain, and received non-specific antibiotics. She was referredto our clinic because of persistant complaints.Transthoracic echocardiography showed vegetations onthe tricuspid valve leading to diagnosis of infective endocarditis,so treatment was started at our clinic. No microorganismisolated in blood cultures. Following 15-days antibiotic therapy no reduction was seen in the diameterof the vegetation, therefore surgical operationwas planned and a bioprosthetic tricuspid valve was putinto place. In this article we aimed to report the developmentof spontaneous abortus at 7 weeks of pregnancy,in order to emphasize that tricuspid valve endocarditiscan be developed secondary to very rare causes.J Clin Exp Invest 2011; 2(1: 102-105

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

    Jerod A Skyberg

    Full Text Available 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.

  1. Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge.

    Yang, Xinghong; Becker, Todd; Walters, Nancy; Pascual, David W

    2006-07-01

    znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain. PMID:16790759

  2. Medición de respuesta inmune humoral y celular frente a antígenos de Brucella abortus RB51 en bovinos (Evaluation of) Humoral and cellular immune response evaluation against Brucella abortus strain RB51 antigens in bovine

    N.I. Montaña S.; O.E. Rueda L.; C.P. Calderon P.; Ortega, A.; A .R. Puentes; M.I. Gallego M.; O.C. Mariño J.

    1998-01-01

    Con el objeto de evaluar comparativamente el tipo de respuesta inmune inducido por los antígenos estructurales purificados y vacunas vivas de Brucella abortus, 14 bovinos criollos hembras de 19 meses de edad fueron distribuidos al azar, en cinco grupos experimentales e inmunizados vía subcutánea de la siguiente manera: OMP-II (proteínas de membrana externa), OMP-II-Cadena-O, OMP II acoplado a polisacárido-O, B. abortus cepa RB51, B. abortus cepa 19 (C19) y solución salina para el grupo contro...

  3. 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.

    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. PMID:26231838

  4. 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

    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 o...

  5. Antibody Production From Immunized Rabbits By Brucella Abortus

    In this research Brucella abortus was used as antigen which was made by killing the bacteria in boiling water for 1 hour and then add 0.5% phenol. The suspension of bacteria of 6x108 cells/mm3 was used as antigen. Rabbits of about 3 months old were injected with 0.50 mI of the antigen by intradermal route with an interval of two weeks. The animals were divided in three groups i.e. A (control group), B (immunization group) and C (immunization and irradiation group). In C group, the animals were first immunized by the antigen and then 2 days later were irradiated by a low dose of 0.50 Gy of gamma rays. Each group consisted of 3 animals. Parameters were observed by weighing the animals, counting leucocyte and lymphocyte cells, and anaIysing the antisera. The research were done two times, included immunization I x, boostered 4 x and analysed 5x. The results obtained were as follows: A (control group) yielded 2.34 g/dl of non specific antibody, B (immunization group) yielded 3.22 g/dI of specific antibody, C (immunization and irradiation group) yielded 3.50 g/dl of spesific antibody. The leucocyte cells of A, B , and C group were 8.240, 7.887, and 8.120 cells/mm3, respectively. The lymphocyte cells of A, B, and C group were 69%, respectively. The weigh of A, B, and C group were 1.44; 1.53; and l.41 kg, respectively. The purpose of this research was prepared to produce the diagnostic reagen (RIA Kit) for a rapid detection of animals disease especially brucellosis. It seemed that C group (the combination of immunization and irradiation treatments) yielded the highest value of antibody production compared to another group

  6. Identification of biovars of Brucella abortus in aborted cattle and buffaloes herd in Sri Lanka

    M. A. R. Priyantha

    Full Text Available Bovine brucellosis is an endemic disease in Sri Lanka, caused by Brucella abortus and had been reported all part of the country for last six decades. Since available biovar is still unknown, the objective of the study was to identify the biovar of B.abortus from sporadically aborted cattle and buffaloes. Samples were collected from 18 aborted herds out of 19 herds of Cattle and Buffaloes in the year 2010. Rose Bengal plate test and Compliment Fixation test were carried out. Milk, vaginal swabs, placental contents and aborted fetus were collected and cultured by conventional bacteriological methods. The detection of biovars were based on growth on Thionin and Bacto fuschin,CO2 requirement, H2S production, serum agglutination with Brucella negative, A,M and R reference antiserum. Eighteen herds investigated out of 19 herds reported, 11 herds were serologically positive for brucellosis (61.11% and only Brucella abortus were isolated from 8 individuals from six herds. All were identified as Biovar 3 of Brucella abortus. [Vet. World 2011; 4(12.000: 542-545

  7. Molecular Epidemiology of Brucella abortus in Northern Ireland-1991 to 2012.

    Adrian Allen

    Full Text Available Brucellosis is the most common bacterial zoonoses worldwide. Bovine brucellosis caused by Brucella abortus has far reaching animal health and economic impacts at both the local and national levels. Alongside traditional veterinary epidemiology, the use of molecular typing has recently been applied to inform on bacterial population structure and identify epidemiologically-linked cases of infection. Multi-locus variable number tandem repeat VNTR analysis (MLVA was used to investigate the molecular epidemiology of a well-characterised Brucella abortus epidemic in Northern Ireland involving 387 herds between 1991 and 2012.MLVA identified 98 unique B. abortus genotypes from disclosing isolates in the 387 herds involved in the epidemic. Clustering algorithms revealed the relatedness of many of these genotypes. Combined with epidemiological information on chronology of infection and geographic location, these genotype data helped to identify 7 clonal complexes which underpinned the outbreak over the defined period. Hyper-variability of some VNTR loci both within herds and individual animals led to detection of multiple genotypes associated with single outbreaks. However with dense sampling, these genotypes could still be associated with specific clonal complexes thereby permitting inference of epidemiological links. MLVA- based epidemiological monitoring data were congruent with an independent classical veterinary epidemiology study carried out in the same territory.MLVA is a useful tool in ongoing disease surveillance of B. abortus outbreaks, especially when combined with accurate epidemiological information on disease tracings, geographical clustering of cases and chronology of infection.

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

    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...

  9. Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence.

    Almirón, M; Martínez, M; Sanjuan, N; Ugalde, R A

    2001-10-01

    Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well. PMID:11553564

  10. SAFETY OF REVACCINATION OF PREGNANT BISON WITH BRUCELLA ABORTUS STRAIN RB51 DURING PREGNANCY

    From December of 1998 through February of 1999, a study was conducted in a Brucella-infected bison herd to evaluate the safety of booster vaccination of adult bison with 6 x 10**9 CFU of Brucella abortus strain RB51 (SRB51), in bison which had previously been vaccinated as yearlings with 1 x 10**10 ...

  11. Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse.

    Gao, Jianpeng; Tian, Mingxing; Bao, Yanqing; Li, Peng; Liu, Jiameng; Ding, Chan; Wang, Shaohui; Li, Tao; Yu, Shengqing

    2016-01-01

    Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism. PMID:27561260

  12. A combined DNA vaccine provides protective immunity against Mycobacterium bovis and Brucella abortus in cattle.

    Hu, Xi-Dan; Yu, Da-Hai; Chen, Su-Ting; Li, Shu-Xia; Cai, Hong

    2009-04-01

    We evaluated the immunogenicity and protective efficacy of a combined DNA vaccine containing six genes encoding immunodominant antigens from Mycobacterium bovis and Brucella abortus. The number of lymph node and spleen cultures positive for M. bovis and B. abortus from calves immunized with the combined DNA vaccine was significantly reduced (p abortus 544. The combined DNA vaccine group displayed stronger antigen-specific interferon-gamma (IFN-gamma) responses and antigen-specific IFN-gamma ELISPOT activities 2 months after final immunization and after challenge. Antigen-specific CD4(+) and CD8(+) T cell responses in the combined DNA vaccine group were higher than either the Bacillus Calmette-Guerin (BCG)-positive or S19-positive control group. Likewise, more calves in the DNA vaccine group exhibited antigen-specific IgG titers and had higher IgG titers than those in the BCG- or S19-immunized groups 2 months after the final immunization. Moreover, two antigens in the combined DNA vaccine induced significant antigen-specific IFN-gamma responses 6 months after challenge (p S19 against B. abortus. This is the first report to demonstrate that a single combined DNA vaccine protects cattle against two infectious diseases. PMID:19364278

  13. Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers

    Kaissar Tabynov

    2016-01-01

    Full Text Available ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein 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 B. abortus S19 (n=10 or B. abortus RB51 (n=10 and a negative (PBS+Montanide Gel01; n=10 control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

  14. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR

    Reza Mirnejad; Reza Hosseini Doust; Reza Kachuei; Seied Mojtaba Mortazavi; Mehdi Khoobdel; Ali Ahamadi

    2012-01-01

    Objective:To evaluate simultaneous detection and differentiates of Brucella abortus(B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method. Methods:This study was designed using three primers that could simultaneously identify and differentiate two major species of pathogenic Brucella in humans and animals. Identification and differentiation of each species using the size of the PCR product were determined. To determine the specificity of the method, bacteria close to the genus Brucella were used. Finally, to confirm PCR products, In addition to the products sequence, RFLP was performed on PCR products using restriction enzymes. Results:The method of optimized combinatorial PCR in this study could simultaneously detect and differentiate B. abortus and B. melitensis with high specificity and sensitivity in clinical samples. Differentiation of species is based on the resulting bands;therefore, the band 494 bp for B. abortus and 733 bp for B. melitensis were obtained. RFLP and sequencing results confirmed PCR results. Conclusions:The results of this study shows that without routine diagnostic methods such as culture and serology tests, using the molecular method of combinatorial PCR, important species of Brucella can be simultaneously identified and differentiated in clinical samples.

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

    Ayman Al-Mariri

    2012-06-01

    Full Text Available 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.

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

    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 (...

  17. Antigens of Brucella abortus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting.

    Brooks-Worrell, B M; Splitter, G A

    1992-01-01

    Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced...

  18. Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes

    Crasta, Oswald R.; Folkerts, Otto; Fei, Zhangjun; Mane, Shrinivasrao P.; Evans, Clive; Martino-Catt, Susan; Bricker, Betsy; Yu, GongXin; Du, Lei; Sobral, Bruno W.

    2008-01-01

    The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this st...

  19. Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis.

    Rossetti, O L; Arese, A I; Boschiroli, M L; Cravero, S L

    1996-01-01

    Brucella spp. are the causative agents of brucellosis in many different hosts, including humans. Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult. By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome lambda gt11 library. This protein is in the periplasm of B. abortus and ...

  20. The Brucella abortus Cyclic β-1,2-Glucan Virulence Factor Is Substituted with O-Ester-Linked Succinyl Residues

    Roset, Mara S.; Ciocchini, Andrés E.; Ugalde, Rodolfo A.; Iñón de Iannino, Nora

    2006-01-01

    Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic β-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic β-1,2-glucan. The oligosaccharide backbone is substituted at C-...

  1. Genome Sequence of Brucella abortus Vaccine Strain S19 Compared to Virulent Strains Yields Candidate Virulence Genes

    Crasta, Oswald R.; Otto Folkerts; Zhangjun Fei; Mane, Shrinivasrao P.; Clive Evans; Susan Martino-Catt; Betsy Bricker; GongXin Yu; Lei Du; Sobral, Bruno W.

    2008-01-01

    The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this st...

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

    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. PMID:24718931

  3. Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

    Francielle Gibson da Silva-Zacarias

    2009-11-01

    Full Text Available Chlamydophila abortus (C. abortus is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragment of the rRNA gene of the Chlamydiaceae family. The PCR assay was evaluated using organs from 15 mice experimentally infected with the S26/3 reference strain of C. abortus. The results of the rRNA PCR were compared to the results from another PCR system (Omp2 PCR that has been previously described for the Omp2 (outer major protein gene from the Chlamydiaceae family. From the 15 C. abortus-inoculated mice, 13 (K=0.84, standard error =0.20 tested positive using the rRNA PCR assay and 9 (K=0.55, standard error=0.18 tested positive using the Omp2 PCR assay. The detection limit, measured using inclusion-forming units (IFU, for C. abortus with the rRNA PCR (1.05 IFU was 100-fold lower than for the Omp2 PCR (105 IFU. The higher sensitivity of the rRNA PCR, as compared to the previously described PCR assay, and the specificity of the assay, demonstrated using different pathogenic microorganisms of the bovine reproductive system, suggest that the new PCR assay developed in this study can be used for the molecular diagnosis of C. abortus in abortion and other reproductive failures in bovines, caprines, and ovines.Chlamydophila abortus (C. abortus é frequentemente associada a distúrbios reprodutivos em bovinos, ovinos e caprinos. Para o diagnóstico, os métodos de cultivo em ovo embrionado de galinha e em células de linhagem contínua apresentam baixa sensibilidade. A reação em cadeia da polimerase (PCR tem sido utilizada em placenta, órgãos fetais, secre

  4. Recombinant bovine interleukin 2 enhances immunity and protection induced by Brucella abortus vaccines in cattle.

    Wyckoff, John H; Howland, Jeri L; Scott, Catherine M O'Connell; Smith, Robert A; Confer, Anthony W

    2005-11-30

    Augmentation of immunization of cattle Brucella abortus S19 or a B. abortus soluble protein extract (SPEBA) vaccine through administration of recombinant bovine IL 2 (rBoIL 2) was evaluated. Seventy-five heifers were divided among 6 groups that were treated with the following: Group 1, no treatment; Group 2, rBoIL 2 (1microg/kg) on day 0; Group 3, SPEBA (2 mg) on day 0 and week 9; Group 4, SPEBA + rBoIL 2 on day 0, SPEBA on week 9; Group 5, S19 (10(7) CFU) on day 0 and week 9; Group 6, S19 + rBoIL 2 on day 0, S19 only on week 9. Approximately, 6 months after vaccination, cattle were bred by natural service, and at mid-gestation pregnant cattle were challenged intraconjunctivally with 9.1 x 10(5) CFU of virulent B. abortus S2308. Pre- and post-challenge antibody responses were measured by an enzyme-linked immunosorbent assay, a particle concentration fluorescence assay, and the card test. Lymphoproliferation (LP) responses to gamma-irradiated B. abortus and SPEBA antigens were measured in peripheral blood mononuclear cells. After vaccination, antibody responses to B. abortus elevated rapidly in SPEBA- and S19-vaccinates with and without rBoIL 2, however, these responses were significantly (P S19 resulted in significant (P abortus antigens following challenge. Characterization of the cytokine response of bovine monocyte-derived macrophages by real-time polymerase chain reaction indicated that in vitro stimulation of these cells with rBoIL 2 resulted in a profound up-regulation of genes encoding tumor necrosis factor-alpha, IL 12p40, and interferon-gamma reflecting activation of the cells. Overall, rBoIL 2-treatment was associated with fewer infections, sero-conversions and a significant (P = 0.02) level of protection against abortion as compared to vaccination alone or no treatment. PMID:16242273

  5. Brucella abortus: inmunidad, vacunas y estrategias de prevención basadas en ácidos nucleicos Brucella abortus: immunity, vaccines and prevention strategies based on nucleic acids

    R Rivers

    2006-01-01

    Full Text Available Brucelosis, enfermedad causada por la bacteria intracelular facultativa Brucella abortus, es una zoonosis ampliamente distribuida a nivel mundial que afecta principalmente al ganado bovino, causando esterilidad en machos y abortos en hembras en gestación. La resistencia depende del desarrollo de una inmunidad mediada por células, con la participación de células T CD4+ de tipo Th1, que secreten interferón gama (-INF, citosina que estimula la actividad bactericida por macrófagos y la actividad citotóxica de linfocitos T CD8+, que son capaces de matar células infectadas con Brucella. Brucella posee como componentes antigénicos importantes el lipopolisacárido (LPS y las proteínas, entre las que se destaca por su demostrada capacidad inmune la superóxido dismutasa (SOD. La prevención de la diseminación de la brucelosis se fundamenta en el desarrollo de vacunas eficientes contra B. abortus, utilizándose cepas atenuadas de Brucella abortus como la cepa 19, cepa 45/20 y la cepa RB51; vacunas subcelulares en base a antígenos que forman parte de la estructura de la bacteria y vacunas basadas en moléculas de ácidos nucleicos, como las vacunas ADN y las vacunas ARN. En la presente revisión se pretende dar una visión actualizada sobre la brucelosis, su patogenia y cuadro clínico. Se hace un análisis de las características genéticas, antigénicas e inmunológicas de Brucella. Luego, una exposición de las vacunas actualmente en uso para su prevención y los estudios con vacunas subcelulares para finalizar con las nuevas tendencias en la generación de vacunas, como las vacunas ADN y ARN para Brucella.Brucellosis, a disease caused by the intracellular facultative bacteria Brucella abortus, is a widely distributed zoonosis throughout the world, affecting mainly cattle and causing sterility in males and abortion in pregnant females. Protection against B. abortus relies on the activation of cellular immunity involving lymphocytes T CD4

  6. Seroprevalence of Brucella abortus and B. canis in household dogs in southwestern Nigeria: a preliminary report

    S. I. B. Cadmus

    2011-04-01

    Full Text Available A preliminary serological study of 366 household dogs in Lagos and Ibadan, southwestern Nigeria, was carried out to determine antibodies due to exposure to Brucella abortus and B. canis, using the rose bengal test (RBT and the rapid slide agglutination (RSA test, respectively. Results showed that 5.46 % (20/366 and 0.27 % (1/366 of the dogs screened were seropositive to B. abortus and B. canis, respectively.Of all dogs, 36 had a history of being fed foetuses from cows and 11 (30.6 % of these tested positive in the RBT. Our findings, although based on a limited sample size and a dearth of clinical details, revealed that dogs in Nigeria may be infected with Brucella spp. given the wide range of risk factors. Further studies are recommended to elucidate the epidemiology of brucellosis in dogs and its possible zoonotic consequences in the country.

  7. Characterisation of Brucella abortus strain 19 cultures isolated from vaccinated cattle.

    Thomas, E L; Bracewell, C D; Corbel, M J

    1981-01-31

    Thirty-four cultures recovered from material of bovine origin in England, Scotland and Wales were identified unequivocally as Brucella abortus strain 19 (S19). All had the properties of carbon dioxide-independent B abortus biotype 1 strains, were inhibited by penicillin G and thionin blue at standard concentrations and behaved in oxidative metabolism and guinea pig virulence tests as typical S19. Their sensitivity to i-erythritol varied somewhat between cultures as did reference subcultures of S19. Of the total number of isolates, 11 were recovered from abortion material or cyetic products, 10 were from calves which died from a hypersensitivity reaction within 24 hours of S19 vaccination and the remainder were from milk or internal organs. From the evidence available, there is little to suggest that calfhood vaccination with S19 has resulted in persistent systemic infection in other than a very small proportion of the animals inoculated. PMID:6789543

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

    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. PMID:24247358

  9. EXPRESSION OF BACILLUS ANTHRACIS PROTECTIVE ANTIGEN IN VACCINE STRAIN BRUCELLA ABORTUS RB51

    Poff, Sherry Ann

    1997-01-01

    Bacillus anthracis is a facultative intracellular bacterial pathogen that can cause cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are of limited duration and do not protect against the respiratory form of the disease. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated im...

  10. Comparison of B.melitensis and B.abortus Bacteremias with Respect to Diagnostic Laboratory Tests

    Hikmet Aliskan

    2016-04-01

    Full Text Available Aim: Brucellosis are most commonly caused by the Brucella species Brucella melitensis and Brucella abortus. This study was aimed to determine the differences in the routine diagnostic tests (serological tests and blood culture positivity that differentiate bacteremias caused by B. melitensis and B. abortus. Material and Method: This study included a total of 42 patients from whose blood cultures Brucella sp. were isolated between January 2010 and April 2014. A 8-10 ml blood sample was put into BACTEC plus/Aerobic F culture bottles after being drawn from patients (n:42 with suspected brucellosis. The obtained samples were incubated in BACTEC 9240 device (BD Diagnostic, Maryland, USA for 21 days. Sera of the blood samples taken simultaneously with the blood culture were studied with the Rose Bengal and Standard Tube Agglutination (STA tests. Results: In patients with acute brucellosis, B. melitensis and B. abortus species showed no significant differences with respect to time to positive signal in blood cultures (for hours p=0.850; for days p=0.696 and the mean time to positivity. The earliest signal in the device was delivered at day 2., 44th hour and the latest at day 6., 123rd hour. No significant difference was noted between the two species with respect to the mean time to positivity. Discussion: This study did not show any significant differences between B. melitensis (n=22 and B. abortus (n=20 bacteremias with respect to age, sex, time to blood culture positivity, and STA test titer level.

  11. Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis

    Langella Philippe

    2006-03-01

    Full Text Available Abstract Background Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. Results Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. Conclusion We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization.

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

    Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 x 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

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

    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. PMID:25946018

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

    Elías Barquero-Calvo

    2015-05-01

    Full Text Available 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.

  15. Virulent Brucella abortus Prevents Lysosome Fusion and Is Distributed within Autophagosome-Like Compartments

    Pizarro-Cerdá, Javier; Moreno, Edgardo; Sanguedolce, Veronique; Mege, Jean-Louis; Gorvel, Jean-Pierre

    1998-01-01

    Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinucl...

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

    Elzer, P.H.; Rowe, G.E.; Enright, F.M.; Winter, A.J. (Department of Veterinary Microbiology, Immunology and Parasitology, College of Veterinary Medicine, Cornell University, Ithaca, NY (United States))

    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.

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

    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

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

    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. PMID:27075847

  19. Protection against infection in mice vaccinated with a Brucella abortus mutant.

    Boschiroli, M L; Cravero, S L; Arese, A I; Campos, E.; Rossetti, O L

    1997-01-01

    This study determines whether a genetically engineered mutant of Brucella abortus, strain M-1, possesses differences in protective properties compared to the parental strain, vaccine S19. M-1 is a mutant unable to express BP26, a periplasmic protein with potential use in diagnosis. Mice vaccinated with S19 developed antibodies against BP26, while those vaccinated with M-1 did not. However, mice vaccinated with S19 or M-1 were similarly protected against challenge with pathogenic strain 2308, ...

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

    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 th...

  1. Outer Membrane Proteins of Brucella abortus Vaccinal and Field Strains and their Immune Response in Buffaloes

    Rukhshanda Munir*, M. Afzal1, M. Hussain2, S. M. S. Naqvi3 and A. Khanum3

    2010-04-01

    Full Text Available Outer membrane proteins (OMPs of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, and 17.7 kDa, while field isolate had OMPs of 151.3, 89.0, 75.8, 67.6, 37.0, 27.0, 24.0 and 19.0 kDa. B. abortus RB51 yielded 11 OMP bands ranging from 12.5 to 107.1 kDa, with 34.2, 15.8 and 12.5 kDa as additional OMPs. Western immunoblot analysis using antisera raised against all three strains in buffaloes indicated an almost similar pattern of immuno-reactive OMPs in S19 and field strain. Two OMPs of molecular weight 37-38 and 19 kDa were immuno-reactive in all strains in buffaloes. There is possibility of use of these OMPs in a recombinant vaccine for B. abortus. A distinct protein of molecular weight of 151.3 kDa was identified in field strain but not in both vaccine strains of B. abortus. Use of this OMP in a diagnostic assay may differentiate between vaccinated and infected animals.

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

    Tabatabai, L.B. (ARS-USDA, Ames, IA (United States))

    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.

  3. Comparative Brucella abortus antibody prevalence in cattle under contrasting husbandry practices in Uganda

    Gerald Nizeyimana

    2013-02-01

    Full Text Available A study was conducted in the Luwero and Nakasongola districts in central Uganda to determine and compare the prevalence and distribution of antibodies against Brucella abortus in cattle under contrasting husbandry practices, using two serological tests. Three hundred and fifteen serum samples were systematically sampled from 29 farms and subsequently tested using the Rose Bengal plate test (RBPT and Indirect Antibody Enzyme Linked Immunosorbent Assay (I-ELISA. The overall prevalence of antibodies against Brucella abortus in the Nakasongola and Luwero districts was 2.4% and 4.7% on RBPT, compared with 1.2% and 3.34 % on I-ELISA. There was no significant difference between the results obtained by RBPT and indirect antibody ELISA (p > 0.05. It was noted that antibodies against Brucella abortus were widely spread over different farms regardless of the cattle grazing system (p > 0.05. Based on the findings, it is feasible to use RBPT as a cheaper screening alternative for brucellosis. A comprehensive national brucellosis study should be undertaken to study the epidemiology and prevalence of brucellosis in Uganda.

  4. Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle.

    Pajuaba, Ana C A M; Silva, Deise A O; Almeida, Karine C; Cunha-Junior, Jair P; Pirovani, Carlos P; Camillo, Luciana R; Mineo, José R

    2012-03-01

    Brucella abortus is a Gram-negative intracellular bacterium that causes infectious abortion in food-producing animals and chronic infection in humans. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Three groups of bovine sera were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. One-dimensional (1D) and two-dimensional electrophoretic profiles of TX-114 hydrophilic phase antigen revealed a broad spectrum of polypeptides (10-79 kDa). 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides abortus S19 proteins (Invasion protein B, Sod, Dps, Ndk, and Bfr), which were related with antigenicity in naturally infected cattle. In conclusion, immunoproteomics of this new antigen preparation enabled the characterization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis, with emphasis on differentiation between S19 vaccinated and infected cattle. PMID:22539433

  5. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

    Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

    2006-07-01

    Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans. PMID:16739129

  6. Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus.

    Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Nicoletti, P; Perez, B; Bermudez, R; Renteria, T

    2007-09-20

    A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA. PMID:17467200

  7. Brucella abortus Strain RB51 Vaccine: Immune Response after Calfhood Vaccination and Field Investigation in Italian Cattle Population

    Manuela Tittarelli

    2008-01-01

    Full Text Available Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv, the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv. Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI 76.2%–100%. The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals.

  8. Intranasal infection with Chlamydia abortus induces dose-dependent latency and abortion in sheep.

    David Longbottom

    Full Text Available 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 (5×10(3, 5×10(5 and 5×10(7 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 2×10(6 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

  9. 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).

    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 (PS19 or RB51). Similar results were observed on BALB/c groups. However, in Swiss mouse groups, S19 was more protective than RB51 (Pabortus strains 544 and 2308, can be used in immunogenicity tests of S19 and RB51 vaccines. PMID:25498211

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

    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

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

    Gamal Wareth

    2016-04-01

    Full Text Available 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.

  12. The Brucella abortus cyclic beta-1,2-glucan virulence factor is substituted with O-ester-linked succinyl residues.

    Roset, Mara S; Ciocchini, Andrés E; Ugalde, Rodolfo A; Iñón de Iannino, Nora

    2006-07-01

    Brucella periplasmic cyclic beta-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic beta-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic beta-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic beta-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-beta-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection. PMID:16816173

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

    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

  14. Identificación de la cepa vacunal Brucella abortus S19 en muestras de leche de vaca

    Luary Carolina Martínez Chavarría; Antonio Verdugo Rodríguez; Rigoberto Hernández Castro

    2006-01-01

    La brucelosis es una enfermedad infectocontagiosa de origen bacteriano que afecta tanto al humano como a diferentes especies animales domésticas y silvestres. En la década pasada, la vacuna que se usaba ampliamente en los bovinos era la cepa vacunal B. abortus S19, que tiene una deleción en dos de los genes del operón ery que participa en el catabolismo del eritritol. En México, desde 1997 se aprobó la cepa B. abortus RB51 como vacuna para el ganado. Esta cepa presenta una secuencia de inserc...

  15. Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

    Yang, Xinghong; Becker, Todd; Walters, Nancy; Pascual, David W.

    2006-01-01

    znuA is known to be an important factor for survival and normal growth under low Zn2+ concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortu...

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

    Elaine M S Dorneles; Lima, Graciela K.; Andréa Teixeira-Carvalho; Araújo, Márcio S S; Martins-Filho, Olindo A; Nammalwar Sriranganathan; Hamzeh Al Qublan; Heinemann, Marcos B; Andrey P. Lage

    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...

  17. Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51.

    Stevens, M G; S. C. Olsen; Pugh, G W; Brees, D

    1995-01-01

    Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following vaccination with B. abortus 19 (S19) or the lipopolysaccharide (LPS) O-antigen-deficient mutant, strain RB51 (SRB51). Live bacteria persisted for 8 weeks in spleens of mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51, whereas bacteria persisted for 12 weeks in mice vaccinated with 5 x 10(6) CFU of S19. Mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51 had increased re...

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

    Karina L. Miranda

    2015-03-01

    Full Text Available 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 CO2. 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.

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

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

    2015-01-01

    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. PMID:26221116

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

    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. PMID:26221116

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

    Diego J Comerci; 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 goo...

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

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

    2015-01-01

    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 ...

  3. Outer Membrane Proteins of Brucella abortus Vaccinal and Field Strains and their Immune Response in Buffaloes

    Rukhshanda Munir*, M. Afzal1, M. Hussain2, S. M. S. Naqvi3 and A. Khanum3

    2010-01-01

    Outer membrane proteins (OMPs) of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, a...

  4. Virulence Criteria for Brucella abortus Strains as Determined by Interferon Regulatory Factor 1-Deficient Mice

    Ko, Jinkyung; Gendron-Fitzpatrick, Annette; Thomas A Ficht; Gary A Splitter

    2002-01-01

    Interferon regulatory factor 1-deficient (IRF-1−/−) mice infected with virulent Brucella abortus 2308 at 5 × 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1−/− mice were unable to resolve infection and died. In contrast, IRF-1−/− mice survived when infected at 5 × 105 CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1−/− mice is likely a function of the level of virulence of each Brucella stra...

  5. Investigation of the possible role of Chlamydophila abortus in reproductive failures in nrazilian herds of domestic ruminants

    Francielle Gibson da Silva-Zacarias

    2009-11-01

    Full Text Available Chlamydophila abortus (C. abortus infection is related to reproductive failure in domestic ruminants. Although it has not been well characterized worldwide, this pathogen has already been identified in some European countries and in the USA. In Brazil, preliminary studies have shown serological evidence of C. abortus infection in herds with low antibody prevalence. Until now, the identification of C. abortus in biological samples from females presenting reproductive failures has not been described in Brazilian herds of domestic ruminants. The aim of this study was to evaluate the presence of the C. abortus in a collection of abortions from cattle (n=85, sheep (n=12, and goats (n=8, in samples of vaginal mucus from cows (n=13, sheep (n=90, and goats (n­=20, and in semen from sheep (n=10 and goats (n=5. The specimens (n=243 were evaluated using a PCR assay developed to amplify the 16S-23S rRNA intergenic space of C. abortus. A PCR assay with an internal control, which amplifies a fragment from the ND5 gene of bovine mitochondrial DNA, was used in order to evaluate the efficiency of the DNA extraction and of the PCR reaction. All biological samples (n=243 included in this study were negative for C. abortus in the PCR assay. The internal control enabled the amplification of a product from the bovine mitochondrial ND5 gene in all cattle abortion samples (n=85. Given the serological evidence indicating the presence of C. abortus infection in Brazilian herds of domestic ruminants, and considering the wide sampling evaluated, the failure to identify C. abortus in this survey suggests that the frequency of clinical signs in infected animals may be low or even absent.A infecção pela Chlamydophila abortus (C. abortus em ruminantes domésticos está relacionada com distúrbios reprodutivos. Apesar de ainda pouco estudada em todo o mundo, a infecção já foi identificada em alguns países europeus e também nos EUA. No Brasil, estudos preliminares

  6. Post-Abortion Syndrome and nursing approachesPost-Abortus Sendromu ve hemşirelik yaklaşımları

    Yılda Arzu Aba

    2016-02-01

    Full Text Available The relation between the life outputs, are associated especially with the mental health outputs at Abortus and the period that follows it. Post-Abortus Syndrome (PAS is the concept of abortus trauma management that show up with the chronic or delayed symptoms as a result of the hidden emotional reactions that are experienced because of the post-abortus physical and psychological trauma. The individual, the systemic and the social factors that are existing at post-Abortus women affect PAS development. The interventions, in general, that are oriented towards mental problems of the individual at the PAS period, focused on the  individual’s sufficiency and efficiency for coping with all the problems that are faced in her life. There is no any program that is developed for post-Abortus women or their family in our country. It is important for the individual who is diagnosed with PAS to receive consultancy from a health professional for her recovery from the period.   Özet Abortus ve bunu izleyen süreçte yaşam sonuçları arasındaki ilişki özellikle mental sağlık sonuçlarıyla ilişkilendirilmektedir. Post-Abortus Sendromu (PAS, abortus sonrası fiziksel ve psikolojik travma nedeniyle deneyimlenen gizlenmiş duygusal tepkiler sonucu, kronik ya da gecikmiş belirtilerle ortaya çıkan abortus travmasını yönetebilme kavramıdır. Abortus sonrası kadınlarda var olan bireysel, sistemik ve sosyal faktörler PAS gelişimini etkilemektedir. PAS dönemindeki bireyin ruhsal sorunlarına yönelik yapılan müdahaleler genel olarak, bireyin yaşamında karşılaştığı tüm sorunlarla başetmesinde yeterli ve etkili hale gelmesine odaklanır. Ülkemizde abortus sonrası kadınlara ya da ailelerine yönelik geliştirilen herhangi bir program bulunmamaktadır. PAS tanısı alan bireyin süreci atlatabilmesi için sağlık profesyoneli tarafından danışmanlık alması önemlidir.

  7. Genetic stability of Brucella abortus isolates from an outbreak by multiple-locus variable-number tandem repeat analysis (MLVA16)

    Dorneles, Elaine Maria S.; Santana, Jordana A; Telma M. Alves; Pauletti, Rebeca B; Mol, Juliana Pinto da Silva; Marcos B. Heinemann; Andrey P. Lage

    2014-01-01

    Abstract Background Brucellosis caused by Brucella abortus is one of the most important zoonoses in the world. Multiple-locus variable-number tandem repeat analysis (MLVA16) has been shown be a useful tool to epidemiological traceback studies in B. abortus infection. Thus, the present study aimed (i) to evaluate the genetic diversity of B. abortus isolates from a brucellosis outbreak, and (ii) to investigate the in vivo stability of the MLVA16 markers. Results Three-hundred and seventy-five c...

  8. Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice.

    Tatum, F M; Detilleux, P G; Sacks, J M; Halling, S. M.

    1992-01-01

    Cu-Zn superoxide dismutase (SOD) deletion mutants of Brucella abortus S2308, a virulent strain, and S19, a vaccine strain, were generated by gene replacement. A deletion plasmid, pBA delta sodknr, was constructed by excising the Cu-Zn SOD gene (Cu-Zn sod) from a 2.3-kb B. abortus DNA fragment of plasmid pBA20-1527 and inserting a 1.4-kb DNA fragment encoding kanamycin resistance into the Cu-Zn sod excision site. The deletion plasmid was introduced into B. abortus by electroporation, and South...

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

    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...

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

    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. PMID:26342452

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

    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...

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

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

    2012-06-01

    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. PMID:22521283

  13. [Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin].

    Serafino, J; Conde, S; Zabal, O; Samartino, L

    2007-01-01

    Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus. PMID:18390151

  14. 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.

    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. PMID:27057678

  15. Low aneuploidy rate in early pregnancy loss abortuses from patients with polycystic ovary syndrome.

    Wang, Qiong; Luo, Lu; Lei, Qiong; Lin, Ming-Mei; Huang, Xuan; Chen, Ming-Hui; Zeng, Yan-Hong; Zhou, Can-Quan

    2016-07-01

    A prospective cohort study was conducted to determine whether chromosome aneuploidy increases the risk of early spontaneous abortions in patients with polycystic ovary syndrome (PCOS). A total of 1461 patients who conceived after IVF and embryo transfer were followed; 100 patients who had experienced clinical spontaneous abortion were recruited, 32 with PCOS and 68 without PCOS. Before 2013, genetic analysis comprised conventional cultured villus chromosome karyotyping and a multiplex ligation-dependent probe amplification subtelomere assay combined with fluorescence in-situ hybridization; since 2013, array-based comparative genomic hybridization technique combined with chromosome karyotyping has been used. Age, BMI, pregnancy history, gestational age and total gonadotrophin dosage did not differ significantly between the PCOS and non-PCOS groups. In the PCOS group, 28.1% of abortuses demonstrated aneuploidy, which was significantly lower (P = 0.001) than in the non-PCOS group (72.1%). Further statistical analyses controlling for maternal age demonstrated that abortuses of women with PCOS were significantly less (P = 0.001) likely to have chromosome aneuploidy. Embryonic aneuploidy does not play a vital role in early spontaneous abortion in women with PCOS. Maternal factors resulting in endometrial disorders are more likely to be responsible for the increased risk of early spontaneous abortion in patients with PCOS. PMID:27157933

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

    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. PMID:27012915

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

    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. PMID:26095034

  18. Optimization of production of Brucella abortus S19 culture in bioreactor using soyabean casein digest medium.

    Kamaraj, Govindasamy; Rajendra, Lingala; Shankar, Chinchkar Ramachandra; Srinivasan, Villuppanoor Alwar

    2010-10-01

    A method of cultivating Brucella abortus S19 culture in bioreactor was attempted using three different media. Culture conditions in bioreactor were optimized by varying agitation and aeration parameters. Varying the aeration ranging from 0.5 vvm to 0.8 vvm and agitation rate ranging from 250 rpm to 400 rpm during bacterial growth was found to yield highest viable count within 48 hours of culture period. A count of > 1 x 10(11) CFU per ml within 48 to 60 hours post seeding was obtained consistently in all five consecutive batches (P > 0.05) with 6 x 10(11) CFU per ml being the maximum yield when the organism is grown in soyabean casein digest medium. B. abortus S19 maintained its smooth characteristics throughout its growth in bioreactor. The vaccine prepared with soyabean casein digest medium was found to be potent and safe with a protective index of 3.33 in mice. The vaccine was tested in 10 cattle calves of 3 to 13 months age and all the vaccinated animals were seropositive on 28, 60, 90, 120 and 150 days post-vaccination when analyzed by fluorescence polarization assay (FPA). PMID:21213590

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

    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. PMID:26526957

  20. Infection of C57BL/6 mice by Trypanosoma musculi modulates host immune responses during Brucella abortus cocolonization.

    Lowry, Jake E; Leonhardt, Jack A; Yao, Chaoqun; Belden, E Lee; Andrews, Gerard P

    2014-01-01

    Brucellosis, which results in fetal abortions in domestic and wildlife animal populations, is of major concern in the US and throughout much of the world. The disease, caused by Brucella abortus, poses an economic threat to agriculture-based communities. A moderately efficacious live attenuated vaccine (B. abortus strain RB51) exists. However, even with vaccine use, outbreaks occur. Evidence suggests that elk (Cervus canadensis), a wild host reservoir, are the source of recent outbreaks in domestic cattle herds in Wyoming, USA. Brucella abortus establishes a chronic, persistent infection in elk. The molecular mechanisms allowing the establishment of this persistent infective state are currently unknown. A potential mechanism could be that concurrent pathogen burdens contribute to persistence. In Wyoming, elk are chronically infected with Trypanosoma cervi, which may modulate host responses in a similar manner to that documented for other trypanosomes. To identify any synergistic relationship between the two pathogens, we simulated coinfection in the well-established murine brucellosis model using Trypanosoma musculi and B. abortus S19. Groups of C57BL/6 mice (Mus musculus) were infected with either B. abortus strain 19 (S19) or T. musculi or both. Sera were collected weekly; spleens from euthanized mice were tested to determine bacterial load near the end of normal brucellosis infection. Although changes in bacterial load were observed during the later stages of brucellosis in those mice coinfected with T. musculi, the most significant finding was the suppression of gamma interferon early during the infection along with an increase in interleukin-10 secretion compared with mice infected with either pathogen alone. These results suggest that immune modulatory events occur in the mouse during coinfection and that further experiments are warranted to determine if T. cervi impacts Brucella infection in elk. PMID:24171573

  1. Evaluation and Selection of Multilocus Variable-Number Tandem-Repeat Analysis Primers for Genotyping Brucella abortus Biovar 1 Isolated from Human Patients

    Lee, Subok; Hwang, Kyu-Jam; Park, Mi-Yeoun; Hwang, Seon-Do; Chai, Hee-Youl; Chu, Hyuk; Park, Sang-Hee

    2013-01-01

    Objectives Brucellosis is the most common bacterial zoonosis in the world. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is a molecular method for genotyping bacterial species. Brucella abortus biovar I was isolated from most of the brucellosis-suspected patients in Korea. This study was conducted to investigate the ability of various MLVA primers that are used for molecular typing B. abortus isolates and for analyzing their epidemiological data. Methods A total of 80 human ...

  2. Granulocyte-Macrophage Colony-Stimulating Factor- and Tumor Necrosis Factor Alpha-Mediated Matrix Metalloproteinase Production by Human Osteoblasts and Monocytes after Infection with Brucella abortus

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.; Delpino, M. Victoria

    2010-01-01

    Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. Since matrix metalloproteinases (MMPs) are involved in joint and bone damage in inflammatory and infectious diseases, we investigated the production of MMPs by human osteoblasts and monocytes, either upon Brucella abortus infection or upon reciprocal stimulation with factors produced by each infected cell type. B. abortus infection of the normal human osteoblastic cell lin...

  3. Preparation by ultrafiltration and control by high-performance liquid chromatography of the native hapten of Brucella abortus for use in radial immunodiffusion diagnostic test.

    Zygmunt, M S; Dubray, G

    1987-01-01

    An effective method was developed for the preparation of Brucella abortus native hapten by use of an ultrafiltration system. The membranes PM30 and YM5 were used to separate the lipopolysaccharide and the native hapten. The yield of the native hapten was higher than that obtained by a more complex procedure reported previously. This method is economical for the large-scale preparation of B. abortus native hapten. The effects of ultrafiltration were evaluated by a quick and sensitive high-perf...

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

    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. PMID:26578650

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

    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. abort...

  6. Molecular Cloning and Characterization of cgt, the Brucella abortus Cyclic β-1,2-Glucan Transporter Gene, and Its Role in Virulence

    Roset, Mara S.; Ciocchini, Andrés E.; Ugalde, Rodolfo A.; Iñón de Iannino, Nora

    2004-01-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic β-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic β-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt cont...

  7. Identification and Characterization of a Brucella abortus ATP-Binding Cassette Transporter Homolog to Rhizobium meliloti ExsA and Its Role in Virulence and Protection in Mice

    G.M.S. Rosinha; Freitas, Daniela A.; Miyoshi, Anderson; Azevedo, Vasco; Campos, Eleonora; Cravero, Silvio L; Rossetti, Osvaldo; Splitter, Gary; S.C. Oliveira

    2002-01-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The ded...

  8. 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

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

    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 wer...

  9. FREQÜÊNCIA DE AGLUTININAS ANTI-Brucella abortus EM CAPRINOS E OVINOS DO SERTÃO DO ESTADO DE PERNAMBUCO, BRASIL FREQUENCY OF ANTI-Brucella abortus AGGLUTININS IN GOATS AND sheep OF THE “SERTÃO” (BACKLANDS OF THE STATE OF PERNAMBUCO, BRAZIL

    Vânia Lúcia de Assis Santana

    2008-12-01

    .936 and sex (p = 0.562. Of the 360 sheep samples, nine (2.5% were reactive. There also was no significant association between the analyzed variables and the seropositiveness for brucellosis: age group (p = 0.522; race (p = 0.576 and sex (p = 0.461. Significant association was observed (p = 0.042 among the studied species and seropositiveness for brucellosis in the investigated animals. The seropositiveness for Brucella abortus in goats and sheep was traced for the first time in the “Sertão” (dry interior region, backlands of Pernambuco, fact that can hinder the success of the National Program of Control and Erradication of Brucellosis, due to the fact that it is common to raise small ruminants with bovines in this area, besides representing risks to Public Health.
     
    KEY WORDS: Brucellosis, ovines, caprines, small ruminants, serodiagnosis.

  10. Medición de respuesta inmune humoral y celular frente a antígenos de Brucella abortus RB51 en bovinos (Evaluation of Humoral and cellular immune response evaluation against Brucella abortus strain RB51 antigens in bovine

    N.I. Montaña S.

    1998-01-01

    Full Text Available Con el objeto de evaluar comparativamente el tipo de respuesta inmune inducido por los antígenos estructurales purificados y vacunas vivas de Brucella abortus, 14 bovinos criollos hembras de 19 meses de edad fueron distribuidos al azar, en cinco grupos experimentales e inmunizados vía subcutánea de la siguiente manera: OMP-II (proteínas de membrana externa, OMP-II-Cadena-O, OMP II acoplado a polisacárido-O, B. abortus cepa RB51, B. abortus cepa 19 (C19 y solución salina para el grupo control. Dos meses posinmunización, los animales fueron desafiados, vía intramuscular, con cepa patógena de referencia B. abortus 2308 y evaluados en su nivel de protección 30 días posdesafío. La respuesta inmune humoral específica fue determinada mediante las pruebas convencionales, de aglutinación Rosa de Bengala, fijación de complemento, inmunodifusión radial, ELISA indirecta, a los 8, 15, 30, 60 y 90 días posinmunización. Adicionalmente se determinó la subclase de IgG por ELISA tipo doble sandwich. La evaluación de respuesta celular fue determinada mediante linfoproliferación, expresada en índices de estimulación (IS, obtenidos por los niveles de incorporación de 3H timidina, medición de Interferón gamma (IFN-g, igualmente expresado como IS y cuantificación de subpoblaciones linfoides CD4+ y CD8+ por citofluorometría. Animales vacunados con cepas vivas demostraron protección total al reto, mientras se observó un 66% de protección en los vacunados con antígenos purificados. Se observó ausencia de anticuerpos a lipopolisacárido, sLPS, por las pruebas convencionales para B. abortus en todos los grupos inmunizados, excepto el vacunado con cepa 19, confirmando la ventaja diagnóstica de la utilización de la cepa RB51 o sus antígenos purificados. La linfoproliferación como una de las medidas de respuesta celular no demostró IS significativos (p0.05 frente a los antígenos estructurales purificados, OMP-II, Cadena-O y sLPS en

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

    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. PMID:26156404

  12. Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7.

    Nielsen, K; Smith, P; Widdison, J; Gall, D; Kelly, L; Kelly, W; Nicoletti, P

    2004-05-20

    Sera from cattle naturally infected with Brucella abortus (n = 160), vaccinated with B. abortus S19 (n = 88) or immunized with Yersinia enterocolitica O:9 (n = 25) or Escherichia coli O157:H7 (n = 80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera (n = 523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA(Br)), CELISA and FPA (FPA(Br)) and the Y. enterocolitica IELISA (IELISA(Ye)) while the Y. enterocolitica FPA (FPA(Ye)) detected antibody in 93.8% and the E. coli IELISA (IELISA(Ec)) 86.9% and the E. coli FPA (FPA(Ec)) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA(Ec), 21.6% in the FPA(Br) and 5.7% in the FPA(Ye). Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA(Ec) and FPA(Ec) although surprisingly 87.5% reacted in the IELISA(Ye) and only 3.8% in the IELISA(Br). No reactions were observed with these sera in the FPA(Br) and FPA(Ye) but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA(Br) and IELISA(Ye) and 80% in the IELISA(Ec). In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA(Br), 28% in the FPA(Ye) and 12% in the FPA(Ec). Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA. PMID:15135510

  13. The use of ELISA to detect antibodies to Brucella abortus in naturally infected cattle

    An indirect ELISA using the immunodominant antigen, smooth lipopolysaccharide (SLPS) of Brucella abortus, was used to detect antibodies in a statistically selected dairy cattle population from the Sabana of Bogota (Colombia). Based on the FAO/IAEA Brucella ELISA kit, the test was standardized in terms of plates, reagents and reference sera. Quality control for routine testing was also established. Calculation of the appropriate cut-off optical density (O.D) value for negativity was made after discriminant analysis and examination of the frequency of the sera distributed as positive or negative. The sensitivity and specificity of the ELISA for diagnosis of Brucella infection were compared to the complement fixation (CF) test results and were 94.8% and 96.0% respectively. The use of ELISA for monitoring bovine populations undergoing an eradication of brucellosis is recommended. The application of an ELISA using B. bovis outer membrane proteins (OMP) as antigen is discussed. (author). 38 refs, 3 figs, 4 tabs

  14. Use of an indirect elisa for Brucella abortus diagnosis in Cuba

    Introducing immunoassays in Brucella diagnosis requires a comparative study with reference techniques such as the complement fixation reaction (CFR). Sensitivity and relative specificity studies allowed us to observe the behaviour of this immunoassay, using samples from free of disease, free by vaccination and affected areas. Sensitivity results for a cut-off point of 40PP and a confidence interval of 95% ranged from 94.8 to 99.5% and the specificity between 94.1 and 97.5%. For free of disease areas a cut-off point of 22 PP was calculated that reached a 99% specificity. This immunoassay for the detection of antibodies against Brucella abortus must be used with two different cut-off points, depending on the epidemiologic conditions of the country, with CFR in affected or vaccinated areas as a confirmative method. (author)

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

    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. PMID:22826490

  16. Endocarditis por Brucella abortus: Reporte del primer caso en C.R

    2011-01-01

    Paciente masculino de 36 años de edad, proveniente de la zona rural de Costa Rica, con un cuadro clínico de 8 meses de evolución de fiebre, mialgias, artralgias, pérdida de peso y lumbalgia; referido por la detección de un soplo de insuficiencia aórtica. El ecocardiograma reveló endocarditis de la válvula aórtica, y se obtuvieron 4 hemocultivos positivos por Brucella abortus biotipo 3, con serologías negativas por brucelosis. Se inició tratamiento con antibióticos y luego se le realizó un ree...

  17. Transcriptome analysis of the Brucella abortus BvrR/BvrS two-component regulatory system.

    Cristina Viadas

    Full Text Available BACKGROUND: The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. METHODOLOGY/PRINCIPAL FINDINGS: A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d, lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. CONCLUSIONS/SIGNIFICANCE: All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche.

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

    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. PMID:22589715

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

    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. PMID:25644010

  20. Protection levels in vaccinated heifers with experimental vaccines Brucella abortus M1-luc and INTA 2.

    Fiorentino, M A; Campos, E; Cravero, S; Arese, A; Paolicchi, F; Campero, C; Rossetti, O

    2008-12-10

    Brucella abortus M1-luc is a mutant strain derived from S19 vaccine strain in which most of bp26 sequence has been replaced by the luciferase coding gene. Strain I2 is a double mutant derived from M1-luc in which most of omp19 has been deleted without introduction of any genetic markers. In BALB/c mice, M1-luc presented equivalent performance to S19 regarding persistence, splenomegaly and protection against challenge. Interestingly, I2 was more attenuated than S19, with no reduction of protection against challenge. In order to evaluate the potential for vaccine use of these strains in the natural host, four groups of 15 heifers, 6-month old, were either non-vaccinated or vaccinated with S19, M1-luc or I2. To at reached 17-month old, heifers were synchronized with two doses of PGF2alpha and received natural service during 60 days with two bulls. Pregnant heifers were challenged at approximately six gestation months with virulent B. abortus S2308. Blood samples post-challenge of heifers were collected for serologic test as well as specimens of aborted fetuses and premature calves for bacterial isolation and histopathological analyses. Protection levels against abortion were 78.6% for S19, 81.8% for M1-luc and 45.5% for I2, compared to the 25% that did not abort from the non-vaccinated group. These results indicate that in bovines BP26 had no influence in protective capacity of S19, correlating with the results obtained in mice. However, contrarily to what was previously observed in mice, lack of expression of Omp19 rendered in less protection capacity of S19 in the natural host. PMID:18565697

  1. Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

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

    2015-01-01

    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. PMID:25644010

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

    Monreal, D.; Grillo, M.J. (María Jesús); Gonzalez-fernandez, D.; Marin, C.M.; de Miguel, M. J.; Lopez-Goñi, I. (Ignacio); Blasco, J.M. (José); Cloeckaert, A.; Moriyon, I. (Ignacio)

    2003-01-01

    Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-po...

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

    Cora N Pollak

    Full Text Available 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.

  4. Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular Bacterium Chlamydophila abortus

    Everson, J. S.; Garner, S. A.; Fane, B.; Liu, B.-L.; Lambden, P R; Clarke, I N

    2002-01-01

    A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has prec...

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

    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. PMID:25812219

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

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

    2015-11-01

    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

  7. In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

    Martirosyan, Anna; von Bargen, Kristine; Arce Gorvel, Vilma; Zhao, Weidong; Hanniffy, Sean; Bonnardel, Johnny; Méresse, Stéphane; Gorvel, Jean-Pierre

    2013-01-01

    CD4+ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4+ T cells (CD4+ CTL) during Brucella abortus infection. These CD4+ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cel...

  8. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51.

    Bricker, B J; Halling, S. M.

    1995-01-01

    Because the brucellosis eradication program uses slaughter and quarantine as control measures, it would benefit from faster methods of bacterial identification. Distinguishing vaccine strains from strains that cause infections among vaccinated herds in the field is essential. To accomplish this, our PCR-based, species-specific assay (B. J. Bricker and S. M. Halling, J. Clin. Microbiol. 32:2660-2666, 1994) was updated to identify Brucella abortus vaccine strains S19 and RB51. Three new oligonu...

  9. Immune Responses of Elk to Initial and Booster Vaccinations with Brucella abortus Strain RB51 or 19

    S. C. Olsen; Fach, S. J.; Palmer, M. V.; Sacco, R. E.; Stoffregen, W. C.; Waters, W.R.

    2006-01-01

    Previous studies have suggested that currently available brucellosis vaccines induce poor or no protection in elk (Cervus elaphus nelsoni). In this study, we characterized the immunologic responses of elk after initial or booster vaccination with Brucella abortus strains RB51 (SRB51) and 19 (S19). Elk were vaccinated with saline or 1010 CFU of SRB51 or S19 (n = seven animals/treatment) and booster vaccinated with a similar dosage of the autologous vaccine at 65 weeks. Compared to nonvaccinate...

  10. Reaction of Native and Denatured Brucella abortus (S19) Proteins with Antibody Using Affinity Chromatography and Immunoblotting

    Karimi, R.; A Mostafaie; B. Tabaraie; Y. Bahrami; J. Abdolalizadeh

    2005-01-01

    Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (m...

  11. Increases of efficacy as vaccine against Brucella abortus infection in mice by simultaneous inoculation with avirulent smooth bvrS/bvrR and rough wbkA mutants.

    Grilló, María Jesús; Manterola, Lorea; de Miguel, María Jesús; Muñoz, Pilar María; Blasco, José María; Moriyón, Ignacio; López-Goñi, Ignacio

    2006-04-01

    The Brucella abortus S19 and RB51 strains are the most widely used live vaccines against bovine brucellosis. However, both can induce abortion and milk excretion, S19 vaccination interferes in serological tests, and RB51 is less effective. We have shown previously that a rough wbkAB. abortus mutant is attenuated and a better vaccine than RB51 in BALB/c mice, and that mutants in the two-component regulatory system bvrS/bvrR are markedly attenuated while keeping a smooth lipopolysaccharide (S-LPS). In this work, we tested whether simultaneous inoculation with live bvrS increases wbkA vaccine efficacy in mice. Even at high doses, the bvrS mutant was cleared much faster from spleens than the wbkA mutant. The splenic persistence of the wbkA mutant increased when inoculated along with the bvrS mutant, but also with inactivated bvrS cells or with purified B. abortus S-LPS, strongly suggesting that S-LPS in the bvrS mutant played a determinant role in the wbkA persistence. When inoculated alone, both mutants protected against virulent B. abortus but less than when inoculated simultaneously, and the protection afforded by the combination was better than that obtained with B. abortus S19. Increased protection was also obtained after simultaneous inoculation of the wbkA mutant and inactivated bvrS cells or purified S-LPS, showing again the role played by the S-LPS in the bvrS cells. In mice, the bvrS-wbkA combination induced an antibody response reduced with respect to B. abortus S19 vaccination. Thus, the simultaneous use of live bvrS and wbkA B. abortus mutants seems a promising approach to overcome the problems of the S19 andRB51 vaccines. PMID:16439039

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

    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. PMID:24941369

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

    Vera Forsbach-Birk

    Full Text Available 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.

  14. Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern.

    Leyre Palacios-Chaves

    Full Text Available 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.

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

    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

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

    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. PMID:23765549

  17. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms.

    Kim, Ji-Yeon; Kang, Sung-Il; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Jung, Suk Chan; Park, Yong Ho; Yoo, Han-Sang; Her, Moon

    2016-05-01

    To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy. PMID:26666176

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

    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. PMID:24994009

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

    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. PMID:26093199

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

    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 (19kDa) and P39 (39kDa) 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 30μg/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. PMID:26826463

  1. Mass vaccination as a complementary tool in the control of a severe outbreak of bovine brucellosis due to Brucella abortus in Extremadura, Spain.

    Sanz, Cristina; Sáez, José Luis; Alvarez, Julio; Cortés, María; Pereira, Gema; Reyes, Aurelia; Rubio, Félix; Martín, Javier; García, Nerea; Domínguez, Lucas; Hermoso-de-Mendoza, María; Hermoso-de-Mendoza, Javier

    2010-11-01

    We report the evolution of an outbreak of bovine brucellosis (Brucella abortus) in the region of Extremadura (Spain) involving more than 1000 herds and nearly 40,000 animals. S19 vaccination of young cattle combined with a test and slaughter strategy did not result in a rapid decrease in herd prevalence and animal incidence; these parameters showed a constant decreasing trend only when a combination of restriction of cattle movements, increased test frequency, S19 vaccination and mass RB51 vaccination (with yearly revaccinations) were applied to all susceptible populations. These measures were applied for 5 years; abortions following RB51 vaccination of pregnant cows were limited to the first inoculation and the involvement of the vaccine strain could only be demonstrated in 78 out of 897 abortions. Our results demonstrate the usefulness - and lack of significant side effects - of RB51 mass vaccination as a complementary tool to control bovine brucellosis outbreaks in areas where the disease cannot be contained using more conservative approaches. PMID:20833439

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

    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. PMID:25218295

  3. Anti-Brucella abortus antibodies in free-ranging equids from Mossoró, Rio Grande do Norte, BrazilAnticorpos anti-Brucella abortus em equídeos errantes do município de Mossoró, Rio Grande do Norte

    Elaine Maria Seles Dorneles

    2013-06-01

    Full Text Available The aims of the present study were (i to determine the occurrence and (ii to evaluate possible factors associated with infection by Brucella abortus in free-ranging equids from Mossoró, Rio Grande do Norte, Brazil. Sera from 227 free-ranging equids (178 donkeys, 43 horses and 6 mules, captured by the highway police and the prefecture agents, were screened by the rose bengal test (RBT and confirmed for B. abortus-antibodies by the standard tube agglutination (STAT and the 2-mercaptoethanol (2ME tests. Of the 227 equids tested, four (1.76% were positive for B. abortus antibodies. All were horses, which resulted in an observed frequency of infection for this species of 9.30% (4/43. No association was found among seropositivity for B. abortus and the age and sex. Thus, data from the present study showed that infection by B. abortus is present among horses in Mossoró, Rio Grande do Norte, Brazil. Os objetivos deste trabalho foram (i determinar a ocorrência da infecção por Brucella abortus em equídeos de vida livre no município de Mossoró, Rio Grande do Norte e (ii avaliar possíveis fatores associados a esta infecção. Soros de 227 equídeos (178 asininos, 43 equinos e 6 muares, capturados pela polícia rodoviária e funcionários da prefeitura, foram coletados por punção venosa. A pesquisa de anticorpos anti-Brucella abortus foi realizada empregando-se, como triagem, o teste do antígeno acidificado tamponado (AAT e como confirmatório o teste de redução pelo 2-Mercaptoetanol (2ME. Dos animais testados, quatro (1,76% foram positivos para anticorpos anti- B. abortus. Todos os positivos eram equinos (9,30%, 4/43. A análise das variáveis levantadas (sexo e idade como possíveis fatores associados à infecção por B. abortus não revelou a existência de associação entre estas e a soropositividade. Assim, o presente estudo permite concluir que a infecção por B. abortus está presente em equinos do município de Mossoró, Rio Grande

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

    Selma Samiko Miyazaki ONUMA

    2015-04-01

    Full Text Available 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.

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

    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. PMID:26297212

  6. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Jake E Lowry

    Full Text Available Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA. All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence

  7. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Lowry, Jake E; Isaak, Dale D; Leonhardt, Jack A; Vernati, Giulia; Pate, Jessie C; Andrews, Gerard P

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of

  8. Endocarditis por Brucella abortus: Reporte del primer caso en C.R

    Manuel Antonio Villalobos-Zúñiga

    2011-09-01

    Full Text Available Paciente masculino de 36 años de edad, proveniente de la zona rural de Costa Rica, con un cuadro clínico de 8 meses de evolución de fiebre, mialgias, artralgias, pérdida de peso y lumbalgia; referido por la detección de un soplo de insuficiencia aórtica. El ecocardiograma reveló endocarditis de la válvula aórtica, y se obtuvieron 4 hemocultivos positivos por Brucella abortus biotipo 3, con serologías negativas por brucelosis. Se inició tratamiento con antibióticos y luego se le realizó un reemplazo valvular aórtico; 4 meses después ingresó con dolor torácico que se atribuyó a una oclusión de la arteria descendente anterior, demostrada angiográficamente, por posible embolismo. En la actualidad cursa clínicamente estable con manejo médico para su cardiopatía, sin recaída infecciosa.

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

    María Inés Marchesini

    Full Text Available 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.

  10. Immune Modulation of Recombinant OmpA against Brucella abortus 544 Infection in Mice.

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

    2016-03-28

    Brucellosis affects a wide range of host species, including humans and many livestock animals. Chronic infections of the disease make antibiotic treatment costly, and the current vaccine used in livestock has not been approved for human use. This study investigated the possible use of the Brucella abortus outer membrane protein A (OmpA) as a candidate subunit vaccine in an infected mouse model. The ompA gene was cloned and overexpressed, and the recombinant OmpA (rOmpA) protein fused to maltose binding protein (MBP) was purified in Escherichia coli. Immunogenicity was verified through western blotting, and mice were immunized and challenged to evaluate its protective effect. Mice treated with rOmpA exhibited induced humoral and host cell-mediated responses, with a significant increase in immunoglobulin G (IgG1 and IgG2a) and cytokine levels, especially TNF-α and IL-12, compared with the control groups treated with either MBP or PBS. In conclusion, rOmpA should be highly considered as a future subunit vaccine for brucellosis, and further studies regarding rOmpA and its protective ability are suggested. PMID:26699748

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

    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. PMID:23458832

  12. Identification of Brucella abortus virulence proteins that modulate the host immune response.

    Wang, Yufei; Chen, Zeliang; Qiu, Yefeng; Ke, Yuehua; Xu, Jie; Yuan, Xitong; Li, Xianbo; Fu, Simei; Cui, Mingquan; Xie, Yongfei; Du, Xinying; Wang, Zhoujia; Huang, Liuyu

    2012-01-01

    Brucellosis is an important zoonotic disease of almost worldwide distribution. One significant immune phenomenon of this disease is the ability of the pathogen to hide and survive in the host, establishing long lasting chronic infections. Brucella was found to have the ability to actively modulate the host immune response in order to establish chronic infections, but the mechanism by which the pathogen achieves this remains largely unknown. In our screening for protective antigens of Brucella abortus, 3 proteins (BAB1_0597, BAB1_0917, and BAB2_0431) were found to induce significantly higher levels of gamma interferon (IFNγ) in splenocytes of PBS immunized mice than those immunized with S19. This finding strongly implied that these three proteins inhibit the production of IFNγ. Previous studies have shown that LPS, PrpA, and Btp1/TcpB are three important immunomodulatory molecules with the capacity to interfere with host immune response. They have been shown to have the ability to inhibit the secretion of IFNγ, or to increase the production of IL-10. Due to the role of these proteins in virulence and immunomodulation, they likely offer significant potential as live, attenuated Brucella vaccine candidates. Understanding the mechanisms by which these proteins modulate the host immune responses will deepen our knowledge of Brucella virulence and provide important information on the development of new vaccines against Brucellosis. PMID:22743689

  13. Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

    Jake E Lowry; Isaak, Dale D.; Leonhardt, Jack A.; Giulia Vernati; Jessie C Pate; Andrews, Gerard P.

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for ...

  14. Otpornost na antimikrobne tvari, fenotipska obilježja i fagotipovi sojeva vrste B. abortus izdvojenih iz goveda i azijskih bivola (Bubalus bubalis) u Trinidadu.

    Adesiyun, Abiodun A.; Baird, Kadian; Stewart-Johnson, Alva

    2011-01-01

    Određivana su fenotipska obilježja, fagotipovi i otpornost na antimikrobna sredstva sojeva bakterije Brucella abortus izdvojenih iz goveda i azijskog bivola (Bubalus bubalis) u Trinidadu i Tobagu. Ukupno je 86 izolata bilo izdvojeno iz limfnih čvorova 14 klinički zdravih serološki pozitivnih goveda i 17 indijskih bivola, iz ozljeda kože devet bivola te tkiva pobačenih plodova 16 bivola. Analizirana su bila i dva cjepna soja bakterije B. abortus, soj 19 (S19) i RB51 (SRB51). Svi analizirani so...

  15. Comparison of spleen cell proliferation in response to Brucella abortus 2308 lipopolysaccharide or proteins in mice vaccinated with strain 19 or RB51.

    Stevens, M G; S. C. Olsen; Pugh, G W

    1995-01-01

    Mice vaccinated with Brucella abortus 19 (S19) or RB51 (SRB51) had spleen cells which proliferated in response to proteins of 32, 27, 18, and < 18 kDa but not in response to proteins of 106, 80, and 49 kDa from B. abortus 2308 (S2308) following vaccination and challenge infection with S2308. Spleen cells from mice vaccinated with S19 but not with SRB51 had increased proliferation in response to S2308 lipopolysaccharide (LPS) following challenge infection with S2308. We previously reported tha...

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

    Neha eDabral

    2015-06-01

    Full Text Available 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.

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

    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. PMID:25187406

  18. Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection.

    Simei Fu

    Full Text Available Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB or BAB1_1688 (AsnC plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

  19. Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection.

    Fu, Simei; Xu, Jie; Li, Xianbo; Xie, Yongfei; Qiu, Yefeng; Du, Xinying; Yu, Shuang; Bai, Yaoxia; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Peng, Guangneng; Huang, Kehe; Huang, Liuyu; Wang, Yufei; Chen, Zeliang

    2012-01-01

    Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy. PMID:22383953

  20. Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes.

    Crasta, Oswald R; Folkerts, Otto; Fei, Zhangjun; Mane, Shrinivasrao P; Evans, Clive; Martino-Catt, Susan; Bricker, Betsy; Yu, GongXin; Du, Lei; Sobral, Bruno W

    2008-01-01

    The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism. PMID:18478107

  1. Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes.

    Oswald R Crasta

    Full Text Available The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.

  2. The mglA gene and its flanking regions in Brucella: The role of mglA in tolerance to hostile environments, Fe-metabolism and in vivo persistence

    Jacob, Jens; Finke, Antje; Mielke, Martin

    2012-01-01

    We previously demonstrated that a spontaneous smooth small-colony variant of Brucella abortus S19 is characterized by increased in vivo persistence and the differential expression of a gene predicted to encode a galactoside transport ATP binding protein (mglA). In order to further investigate the role of this gene in the context of its flanking regions, we analyzed the respective DNA sequences from the formerly described B. abortus S19 as well as from avirulent B. neotomae 5K33 and compared t...

  3. DNA vaccine encoding L7/L12-P39 of Brucella abortus induces protective immunity in BALB/c mice

    LUO De-yan; LI Peng; XING Li; ZHAO Guang-yu; SHI Wei; ZHANG Song-le; WANG Xi-liang

    2006-01-01

    @@ Brucella abortus is a gram-negative, facultative, intracellular bacterium that infects both cattle and humans, causing abortion and infertility in the former and undulant fever, endocarditis, arthritis, and osteomyelitis. Resistance to Brucella depends on acquired cell-mediated immunity (CMI).1 Live attenuated vaccines can stimulate strong CMI response, which are usually very effective against brucellosis and are used to control brucellosis in domestic animals. However, there is no safe and effective vaccine available for human because the vaccine strains used for animals are considered too virulent for humans. A vaccine that will be noninfectious to humans but effective in stimulating a broad protective immune response is needed.2

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

    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.

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

    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 (125I)-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

  6. Prediction of T cell epitopes of Brucella abortus and evaluation of their protective role in mice.

    Afley, Prachiti; Dohre, Sudhir K; Prasad, G B K S; Kumar, Subodh

    2015-09-01

    Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. The animal vaccines are available but have disadvantage of causing abortions in a proportion of pregnant animals. The animal vaccines are also pathogenic to humans. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, efforts were made to identify MHC-I- and MHC-II-restricted T cell epitopes of Brucella abortus and evaluate their vaccine potential in mice. The peptides were designed using online available immunoinformatics tools, and five MHC-I- and one MHC-II-restricted T cell peptides were selected on the basis of their ability to produce interferon gamma (IFN-γ) in in vivo studies. The selected peptides were co-administered with poly DL-lactide-co-glycolide (PLG) microparticles and evaluated for immunogenicity and protection in BALB/c mice. Mice immunized with peptides either entrapped in PLG microparticles (EPLG-Pep) or adsorbed on PLG particles (APLG-Pep) showed significantly higher splenocyte proliferation and IFN-γ generation to all selected peptides than the mice immunized with corresponding irrelevant peptides formulated PLG microparticles or phosphate-buffered saline (PBS). A significant protection compared to PBS control was also observed in EPLG-Pep and APLG-Pep groups. A plasmid DNA vaccine construct (pVaxPep) for peptides encoding DNA sequences was generated and injected to mice by in vivo electroporation. Significant protection was observed (1.66 protection units) when compared with PBS and empty vector control group animals. Overall, the MHC-I and MHC-II peptides identified in this study are immunogenic and protective in mouse model and support the feasibility of peptide-based vaccine for brucellosis. PMID:26150246

  7. The efficacy of the skin delayed-type hypersensitivity using a brucellin prepared from a mucoid strain of Brucella abortus to detect brucellosis

    Bercovich, Z.; Muskens, J.A.M.

    1998-01-01

    Eight-hundred-and-ninety-six cattle belonging to herds officially designated Brucella-free, and 190 cattle belonging to infected herds were tested with the skin delayed-type hypersensitivity (SDTH) test, using brucellin (273) prepared from a rnucoid strain of Brucella abortus. An increase in skinfol

  8. The Assessment of Cytokine and Antibody Responses to Recombinant 31kDa Brucella Cell-Surface Protein in Brucella Abortus Infected Mouse Model

    Nima Khoramabadi

    2014-06-01

    Conclusion: These findings suggest that specific humoral and cell-mediated responses to BCSP31 is formed during murine host infection with B. abortus. Based on these findings, rBCSP31 can be used in further design of immunogenic strategies for vaccination against brucellosis.

  9. The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

    Emmerzaal, A.; Wit, de J.J.; Dijkstra, T.; Bakker, D.; Ziiderveld, van F.G.

    2002-01-01

    The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the progra

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

    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...

  11. Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence.

    Roset, Mara S; Ciocchini, Andrés E; Ugalde, Rodolfo A; Iñón de Iannino, Nora

    2004-04-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor. PMID:15039351

  12. Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1β Production.

    Miraglia, M Cruz; Costa Franco, Miriam M; Rodriguez, Ana M; Bellozi, Paula M Q; Ferrari, Carina C; Farias, Maria I; Dennis, Vida A; Barrionuevo, Paula; de Oliveira, Antonio C P; Pitossi, Fernando; Kim, Kwang Sik; Delpino, M Victoria; Oliveira, Sergio Costa; Giambartolomei, Guillermo H

    2016-05-01

    Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1β and TNF-α, activation of HBMEC was dependent on IL-1β because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1β inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1β secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1β-induced activation of the brain microvasculature. Inflammasome-mediated IL-1β secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1β-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1β mediates

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

    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. PMID:27124329

  14. Summary of field trials using the direct and competitive enzyme immunoassays for detection of antibody to brucella abortus

    Two indirect and two competitive enzyme immunoassays for detection of antibody to Brucella abortus, validated elsewhere, were field tested in five different Latin American laboratories. Testing was performed according to standardised protocols using sera obtained in each area. Sera from B. abortus infected herds, from vaccinated (but serologically negative in a screening test) and non-vaccinated cattle were tested in each assay and compared to the results obtained with conventional diagnostic tests used for diagnosis of brucellosis in each country. Relative sensitivity and specificity values were calculated for each country as well as a weighted summary combining the data from all the participating laboratories. The result demonstrate that all ELISAs performed as well as, or better than, the conventional aerological tests. Given the inherent errors in the use of the latter in the diagnosis of brucellosis, it is recommended that the ELISAs described here be considered as replacements for the conventional tests. The CELISA using the lipopolysaccharide antigen with the competing monoclonal antibody M84, should be considered as the most useful because of cross-species and vaccination considerations. (author)

  15. In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

    Martirosyan, Anna; Von Bargen, Kristine; Arce Gorvel, Vilma; Zhao, Weidong; Hanniffy, Sean; Bonnardel, Johnny; Méresse, Stéphane; Gorvel, Jean-Pierre

    2013-01-01

    CD4+ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4+ T cells (CD4+ CTL) during Brucella abortus infection. These CD4+ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4+ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4+ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4+ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection. PMID:24367519

  16. In vivo identification and characterization of CD4⁺ cytotoxic T cells induced by virulent Brucella abortus infection.

    Martirosyan, Anna; Von Bargen, Kristine; Arce Gorvel, Vilma; Zhao, Weidong; Hanniffy, Sean; Bonnardel, Johnny; Méresse, Stéphane; Gorvel, Jean-Pierre

    2013-01-01

    CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus infection. These CD4(+) CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4(+) CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4(+) T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4(+) CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection. PMID:24367519

  17. Mouse cytokine profile skewed towards Th2 in pregnancy during infection with Brucella abortus S19 strain.

    Wamonje, Francis O; Waihenya, Rebecca K; Ng'ang'a, Zipporah; Ngeranwa, Joseph N

    2011-04-01

    The two classes of cytokines Th1 and Th2 determine the type of immune response elicited. The Th2 immune response is associated with successful pregnancy. Brucellosis is an intracellular bacterium that elicits the Th1 response and is known to cause spontaneous abortion in mammalian species. This study sought to determine if Brucella infection causes spontaneous abortion by causing the circulating cytokine profile be Th1 dominant during pregnancy. Forty-eight Swiss white mice were used in this murine model and the S19 strain of Brucella abortus was used in as the infective agent. Pregnant mice in the test group were injected intraperitoneally with 10(5-8) CFU of Brucella and cytokine profile evaluated over the three trimesters of pregnancy. Pregnant mice in the control group were left to go through normal pregnancy and their cytokine profile evaluated over the three trimesters of pregnancy. Cytokines in serum samples were analyzed by Cytometric Bead Array. The data was analyzed using the Paired T- test and p Brucella abortus cannot cause spontaneous abortion by altering the mouse cytokine profile towards Th1 in pregnancy. Elevated IL-4 levels with corresponding suppression of IFN-γ can be used as a marker for successful pregnancy in Brucellosis. PMID:25566611

  18. Early Transcriptional Responses of Bovine Chorioallantoic Membrane Explants to Wild Type, ΔvirB2 or ΔbtpB Brucella abortus Infection

    Mol, Juliana P. S.; Costa, Erica A.; Carvalho, Alex F.; Sun, Yao-Hui; Tsolis, Reneé M.; Paixão, Tatiane A.; Santos, Renato L.

    2014-01-01

    The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion. PMID:25259715

  19. Early transcriptional responses of bovine chorioallantoic membrane explants to wild type, ΔvirB2 or ΔbtpB Brucella abortus infection.

    Juliana P S Mol

    Full Text Available The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM explants inoculated with wild type (strain 2308, ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05 were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion.

  20. Serological response to an indirect and a competitive elisa in heifers vaccinated with Brucella abortus strain 19

    The different serologic techniques for bovine brucellosis diagnosis have different abilities to detect antibodies after vaccination with Brucella abortus strain 19. The humoral response in heifers vaccinated with Brucella abortus strain 19 was evaluated by using several serologic techniques. In the experimental field of INTA, Pilcaniyeu, Rio Negro province, sixteen 5 months old heifers were vaccinated subcutaneously with a standard dose (2ml, containing 20x109 to 10x109 living organisms) of Brucella abortus strain 19. Sera from all the heifers were obtained on 18 occasions (one 87 days before vaccination, one immediately before vaccination and on 16 occasions after vaccination, during 488 days) and analyzed by buffered plate antigen test, rose bengal test, standard tube agglutination test, 2-mercaptoetanol test, complement fixation test, indirect ELISA, and competitive ELISA. Prior vaccination, 100% of the heifers gave negative results in all the techniques used, while 100% of them gave positive reaction in the first sampling after vaccination to all the techniques, with the exception of standard tube agglutination test that showed agglutinating titters of 1/100 or higher (positive threshold) in only 71.4% of the heifers. The indirect ELISA technique showed a reducing percentage of positive animals up until 316 days after vaccination, after which positive results were obtained. The competitive ELISA gave positive results in a variable number of heifers up to 253 days after vaccination when 100% of the sera were negative to this technique. Buffered plate antigen test was the technique that gave positive results for a longest period, being 100% of the animals negative to this technique at 450 days after vaccination. The other serological techniques assayed gave positive results during variable periods of time, intermediate between standard tube agglutination test and buffered plate antigen test. Although the present results were obtained from a limited number of

  1. Diagnóstico sorológico da brucelose bovina em animais adultos vacinados com dose reduzida da cepa 19 de Brucella abortus Serological diagnosis of bovine brucellosis in adult herd vaccinated with Brucella abortus strain 19 reduced dose

    Gustavo Coelho Jardim

    2006-09-01

    Full Text Available Com o presente trabalho avaliou-se o uso de dose reduzida da vacina produzida com a amostra 19 de Brucella abortus, em rebanho adulto negativo para a enfermidade, por meio de técnicas de diagnóstico sorológico preconizadas pelo Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal e por um ensaio indireto de imunoadsorção enzimática (ELISA ID. A prova de fixação de complemento detectou 46,77% de positivos, o antígeno acidificado tamponado 67,74%, o 2-mercaptoetanol com soroaglutinação lenta 87,09% e o ELISA ID 100%. A dose reduzida interferiu no diagnóstico sorológico. Nenhuma das técnicas apresentou especificidade adequada para uso em rebanho nestas condições, até 3 meses após a vacinação.The study evaluated the use of a reduced dose of the Brucella abortus strain 19 vaccine, in an adult herd negative for the disease, by serological diagnostic techniques, advocated by the Brazilian Program for Animal Brucellosis and Tuberculosis Control and Eradication, and by an indirect ELISA. The complement fixation test detecteed 46.77% positives, the rose bengal test 67.74%, the mercaptoethanol with standard agglutination test 87.09% and the ELISA ID 100%. The reduced dose influenced the serological diagnosis. None of the techniques reached a suitable specificity for use in the herd under those conditions, up to 3 months after vaccination.

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

    Serer, María I.; Bonomi, Hernán R. [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina); Guimarães, Beatriz G. [Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin BP 48, 91192 Gif-sur-Yvette CEDEX (France); Rossi, Rolando C. [Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires (Argentina); Goldbaum, Fernando A.; Klinke, Sebastián, E-mail: sklinke@leloir.org.ar [IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina)

    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.

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

    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 C3 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

  4. Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.

    Lacerda, Thaís Lourdes Santos; Cardoso, Patrícia Gomes; Augusto de Almeida, Leonardo; Camargo, Ilana Lopes Baratella da Cunha; Afonso, Daniela Almeida Freitas; Trant, Cyntia Cardoso; Macedo, Gilson Costa; Campos, Eleonora; Cravero, Silvio L; Salcedo, Suzana P; Gorvel, Jean-Pierre; Oliveira, Sérgio Costa

    2010-08-01

    Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. PMID:20580469

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

    Neha Dabral

    Full Text Available 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.

  6. Oral Immunization of Mice with Gamma-Irradiated Brucella neotomae Induces Protection against Intraperitoneal and Intranasal Challenge with Virulent B. abortus 2308

    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 immuniz...

  7. Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases ▿

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; De Simone, Emilio A.; Silvia I. Vanzulli; 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 (SW9...

  8. Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins.

    Lin, J.; Adams, L G; Ficht, T A

    1992-01-01

    In an effort to define the heat shock response in the bovine intracellular pathogen Brucella abortus, a rough variant lacking extensive lipopolysaccharide was pulse-labeled with [35S]methionine following exposure to elevated temperatures. The major heat shock proteins observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography migrate at 70, 62, 18, and 10 kDa. The maximum response was observed between 42 and 46 degrees C and within 2 to 3 h of the shif in...

  9. Validation of the Abbreviated Brucella AMOS PCR as a Rapid Screening Method for Differentiation of Brucella abortus Field Strain Isolates and the Vaccine Strains, 19 and RB51

    Ewalt, Darla R; Bricker, Betsy J.

    2000-01-01

    The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isola...

  10. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    Mario Weinhold; Martin Eisenblätter; Edith Jasny; Michael Fehlings; Antje Finke; Hermine Gayum; Ursula Rüschendorf; Pablo Renner Viveros; Verena Moos; Kristina Allers; Thomas Schneider; Schaible, Ulrich E; Schumann, Ralf R.; Martin E Mielke; Ralf Ignatius

    2013-01-01

    Background: Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4+ and CD8+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-speci...