59th
Annual Brucellosis
Research Conference
Marriott Downtown Magnificent Mile
Program and Abstracts
December 2-3, 2006
59th Annual Brucellosis Research Conference
December 2-3, 2006
Chicago Marriott Hotel -
Saturday
7:00am Registration
8:30am Welcome and Announcements
8:45am Introduction to the Cooperative Biological Research Program
Phil Elzer
9:00am USDA Status Report
Debbi Donch
9:30am Diagnostics and Taxonomy
Moderator: Adrian Whatmore
10:30am Break and Posters
11:15am Immunology and Host-Pathogen Interactions
Moderator: Bryan Bellaire
12:15 pm Lunch
1:45pm Immunology and Host-Pathogen Interactions cont.
2:30pm Virulence - Genes and Mechanisms
Moderator: David O’Callaghan
3:15pm Break and Posters
4:00pm Virulence - Genes and Mechanisms cont.
Sunday
8:00am Virulence - Genes and Mechanisms cont.
8:45am Keynote Speaker
Gabriel Nuñez
9:15am Brucella Genetics and Vaccines
Moderator: Ramesh Vemulapalli
10:15am Break
10:45am Brucella Genetics and Vaccines cont.
11:00am COST Report and Summary
David O’Callaghan
11:15am Business Meeting
12:00 pm Closing Remarks and Announcements
Front Cover image: “The Maltese Goat” by E. Caruana Dingli (1876-1950)
59th Annual Brucellosis Research Conference
December 2-3, 2006
Chicago Marriott Hotel -
Welcome to this year’s Brucellosis Research Conference. The officers hope the meeting offers you the opportunity to exchange data and ideas with your colleagues. As a satellite organization of CRWAD, we are in new surroundings; hopefully they will suit your needs and be conducive to a productive meeting environment.
Our guest speaker Dr. Gabriel Nunez is from the University of Michigan Health System Department of Pathology. His research program focuses on mechanistic studies to understand signaling pathways involved in apoptosis and innate immunity.
The organization is hosting a group of former Soviet Union scientists from
2006 Board of Directors and Organizing Committee
President – Betsy Bricker
Vice President - Renee Tsolis
Vice President-Elect – vacant
Past President – Francisco Suárez-Güemes
Secretary-Treasurer – Sue Hagius
Gabriel Nuñez, M.D.
Paul H. De Kruif Professor of Pathology
http://www.pathology.med.umich.edu/faculty/Nunez/index.html
Research Interests
Dr. Nunez research program focuses on mechanistic studies to understand signaling pathways involved in apoptosis and innate immunity. A family of cytosolic proteins, termed NODs, that are involved in recognition of microbes, has been identified and is being characterized. Two NOD proteins, Nod1 and Nod2, activate NF-kappaB and appear to be involved in the defense against bacterial pathogens. Mutations of the gene encoding Nod2 have been associated with susceptibility to Crohn’s disease, a common inflammatory disease of the bowel. Ongoing studies of Nod1 and Nod2 proteins include molecular studies to further define their mechanism of action and analyses of mutant mice deficient in Nod1 and Nod2. We are also studying Cryopyrin and Ipaf, two NOD proteins involved in caspase-1 activation and inflammation. Finally, the role of inflammatory pathways in cancer development is another interest of the laboratory.
Brief Biography
Dr. Nunez received his medical degree from the University of Seville Medical School in
Foundation and the Broad Medical Research Program.
Dr. Nunez is board certified in Anatomic Pathology.
Campus Address:
4219 CCGC
1500 East Medical Center Drive
Telephone: 734/764-8514
Fax: 734/647-9654
Gabriel_Nunez@umich.edu
Saturday
8:30 am
Welcome and Announcements- Betsy Bricker
8:45 am
Introduction to the Cooperative Biological Research - Defense Threat Reduction Agency Programs – Phil Elzer
9:00 am
USDA Report - Debbi Donch
Diagnostics and Taxonomy
Moderator: Adrian Whatmore
9:30 am
The development and applications of multilocus sequence analysis of the Brucella group. Adrian Whatmore, Julie Scott, Mike Stubberfield, Mark Koylass, and
9:45 am
Molecular epidemiology of marine mammal Brucella isolates based on multilocus sequence typing (MLST) and multiple locus VNTR analysis (MLVA). Pauline Groussaud, Stephen Shankster and Adrian Whatmore. Veterinary Laboratories Agency, Addlestone,
10:00 am
Isolation of a Brucella species from marine mammals in
10:15 am
DTRA Presentation 1 – Current Brucellosis Situation in
10:30 am
BREAK AND POSTERS
Immunology and Host-Pathogen Interactions
Moderator: Bryan Bellaire
11:15 am
BALB/c B cell deficient mice exhibit rapid control of Brucella abortus. Goenka R, Casey L. , Zou B. , CL Baldwin. Department of Veterinary and Animal Sciences,
11:30 am
Mammalian B lymphocytes act as an infection reservoir for Brucella abortus. Goenka R, Guirnalda P, Black SJ, CL Baldwin. Department of Veterinary and Animal Sciences,
11:45 am
Alteration of the Fc gamma Receptor I in murine macrophages during a Brucella spp. infection. A. Mathison, J. Harms, L. Eskra, G.A. Splitter. Department of Animal Health and Biomedical Sciences,
12:00 pm
Murine macrophage transcriptional responses following in vitro infections with virulent smooth and attenuated rough Brucella suis strains. Y. He1, X. Ding2, Y. Ding3, D. Ghosh3, Z. Fei4, G. G. Schurig5, N. Sriranganathan5, S. M. Boyle5. 1Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI. 2Dept of Vet. Pathobiology,
12:15 pm
LUNCH
Immunology and Host-Pathogen Interactions cont.
1:45 pm
Host and Brucella gene expression profiles in an in vitro model of infection. C. A. Rossetti1, K. Drake2, C. L. Galindo3, S. A. Johnston4, H. R. Garner3 and L. G. Adams1. 1Dept. of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, 77843-4467, 2Seralogix, Austin, TX, 3UT-SWMC, Dallas, TX, 4ASU, Phoenix, AZ.
2:00 pm
Nramp1 3’UTR polymorphisms are not associated with natural resistance to Brucella abortus in cattle. Paixão, T.A., Poester, F.P., Carvalho Neta, A.V., Borges, A.M., Lage, A.P., Santos, R.L. Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
2:15 pm
Drosophila S2 cells as a model system for studying host-Brucella interactions. Qingming Qin1, Jianwu Pei2, Brian D. Shaw1, Thomas A. Ficht2,3, and Paul de Figueiredo1,3,4. 1 Department of Plant Pathology and Microbiology. 2 Department of Veterinary Pathobiology; 3 Faculty of Genetics; 4 Program in Biotechnology;
Virulence - Genes and Mechanisms
Moderator: David O’Callaghan
2:30 pm
Erythritol regulates several virulence systems in Brucella. Sangari, F. J., M.C. Rodriguez, C. Viadas, I. López Goñi y J.M. García Lobo. Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain, and Departamento de Microbiología y Parasitología, Universidad de Navarra, Pamplona, Spain.
2:45 pm
Structure Function analysis of the B. suis VirB8 protein. David O'Callaghan. INSERM U431, UFR Medecine, 30908
david.ocallaghan@univ-montp1.fr
3:00 pm
DTRA Presentation 2 - Brucellosis in
3:15 pm
BREAK AND POSTERS
Virulence - Genes and Mechanisms cont.
4:00 pm
Evaluating the virulence of a putative Brucella melitensis hemagglutinin in the caprine model. Q.L. Perry1, S.D. Hagius2, J.V. Walker2, and P.H. Elzer1, 2. 1Department of Pathobiological Sciences,
4:15 pm
A conserved hypothetical protein of Brucella spp is essential for their virulence in animals. Mariela Carrica and Silvio L. Cravero. Instituto de Biotecnologia-Instituto Nacional de Tecnologia Agropecuaria (INTA) Castelar
4:30 pm
The role of the alkyl hydroperoxide reductase complex in Brucella abortus resistance to oxidative stress. Kendra Hitz, Michelle Wright-Valderas, John Baumgartner, Tim Brown, and R. M. Roop II. Department of Microbiology and Immunology,
4:45 pm
The Brucella abortus xthA-1 and xthA-2 gene products play overlapping roles in base excision repair and resistance to oxidative stress. Michael L. Hornback and R. Martin Roop II. Department of Microbiology and Immunology,
5:00 pm
DTRA Presentation 3 - Status of Brucellosis in
Sunday
Virulence - Genes and Mechanisms cont.
8:00 am
Hemin utilization by Brucella abortus 2308 is dependent on the ChrSA two component regulatory system. James T. Paulley, Eric S. Anderson, J. E. Baumgartner, and R. M. Roop II. East Carolina University Department of Microbiology and Immunology,
8:15 am
Creation of a rough Brucella mutant bank and elucidation of cytotoxic mechanisms. Jianwu Pei1, Qingmin Wu2, Melissa Kahl-McDonagh1, and Thomas A. Ficht1. 1Department of Veterinary Pathobiology,
8:30 am
Nod-like Receptors: Role of in Bacterial Recognition and Host Defense. Gabriel Nuñez. Department of Pathology and
Brucella Genetics and Vaccines
Moderator: Ramesh Vemulapalli
9:15 am
Determination of the genetic basis for the lack of expression of Cu/Zn superoxide dismutase in Brucella neotomae”. Dina Moustafa and Ramesh Vemulapalli. Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
9:30 am
Use of cre-lox technology to create an auxotrophic mutant of Brucella abortus strain RB51 as a vector for expressing heterologous antigens. Parthiban Rajasekaran1, Mohamed N. Seleem1, Andrea Contreras1, Raju Lathigra2, Nammalwar Sriranganathan1 and Stephen M. Boyle1. Department of Biomedical Sciences and Pathobiology, 1Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, 2 Walter Reed Army Institute of Research, Silver Spring, MD.
9:45 am
Enhanced immunogenicity and protective efficacy using live microencapsulated vaccines against brucellosis. A. M. Arenas1, T. A. Ficht1, M. Kahl1, A. C. Rice-Ficht1,2. 1Dept. of Veterinary Pathobiology, College of Veterinary Medicine,
10:00 am
Preliminary results of studying immunogenic properties of the vaccinal strain “Nevvsky-13” Brucella melitensis. H.A.Hamdamov1, R.G. Yaraev1, M.K. Butaev1, P.H. Elzer2. 1UzSRIV,
10:15 am
BREAK
10:45 am
DTRA Presentation 4 - Brucellosis in
11:00 am
COST Report and Summary – David O’Callaghan
11:15 am
Business Meeting
12:00 pm
Concluding Remarks and Announcements
Poster Presentations
Immunology and Host-Pathogen Interactions
P1. Persistence of Brucella abortus in Gamma-Interferon Stimulated Human Monocytes. Bryan H. Bellaire3*,
P2. Type I and II Interferon responses to Brucella abortus in mice depend on the presence of an intact Type IV secretion system. Christelle M. Roux, Hortensia G. Rolán and Renée M. Tsolis. Department of Medical Microbiology and Immunology,
P3. Effects of TLR4-directed RNA interference on cell-mediated immune response to Brucella infection. T. E. Todd1, Y. He1,2. 1Unit for Laboratory Animal Medicine, 2Dept. of Microbiology and Immunology, University of
P4. Pharmacological studies support the use of Drosophila S2 cells as a model system for studying Brucella infection of host cells. Qingming Qin *1,
P5. Pathogenesis of the experimental infection with a Brucella melitensis 16M mutant in the goat model. Maria Ceron Cucchi1, Sandra Conde1,
Virulence and Genetics
P6. Role of the outer membrane proteins of the Omp25/Omp31 family in the virulence of Brucella ovis in mice. Paola Caro-Hernández1, Luís Fernández-Lago1, María-Jesús Grilló2, María-Jesús de Miguel2, Ana-Isabel Martín-Martín1, Axel Cloeckaert3, and Nieves Vizcaíno1. 1Dpto. Microbiología y Genética, Universidad de Salamanca, Spain, 2Centro de Investigación y Tecnología Agroalimentaria, Gobierno de Aragón, Spain, 3Infectiologie Animale Santé Publique, INRA Centre de Tours, France.
P7. Immunogenicity and antigenic relationships of the Omp25/Omp31 family of Brucella spp. outer membrane proteins. Ana-Isabel Martín-Martín1, Paola Caro-Hernández1, Luís Fernández-Lago1, Clara M. Marín2, Axel Cloeckaert3 and Nieves Vizcaíno1. 1Dpto. Microbiología y Genética, Universidad de Salamanca, Spain, 2Centro de Investigación y Tecnología Agroalimentaria, Gobierno de Aragón, Spain, 3Infectiologie Animale Santé Publique, INRA Centre de Tours, France
P8. Structural characterization and lipid binding of the virulence factor IivA of Brucella abortus. Mariela Carrica1, Patricio Craig2, Julia Sabio y Garcia1, Osvaldo Rossetti1, Fernando Golbaum2 and Silvio Cravero1. 1Instituto de Biotecnología-INTA, Castelar. 2 Fundación Instituto Leloir, Buenos Aires, Argentina.
P9. Brucella abortus strain S19 as an expression vector for Babesia bovis Rhoptry-Associated Protein 1 (RAP-1). Julia Sabio y García1, Eleonora Campos1, Marisa Farber2, M. Carrica, Silvio L. Cravero1, F. Bigi and Osvaldo Rossetti1. 1 Instituto de Biotecnología, INTA
P10. Purification and biochemical characterization of Brucella suis urease. Araceli Contreras-Rodriguez1,2, Ahide Lopez-Merino1, Jose Quiroz-Limon1, Eric Avila-Calderon1, Victor Flores-Lopez1, Guadalupe Guerra1, Nammalwar Sriranganathan2, and Stephen M. Boyle2. 1Escuela Nacional de Ciencias Biológicas, I.P.N. México. 2Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Rd., Blacksburg, VA 24061-0342, USA.
P11. DhbR, an AraC-like transcriptional activator of the 2,3-Dihydroxybenzoic acid (DHBA) biosynthetic operon in Brucella abortus. E. S. Anderson, Paulley, J. T. and R. M. Roop II. Department of Microbiology and Immunology,
P12. Identification of a small regulatory RNA in Brucella abortus.
Brook E. Ragle, Eric S. Anderson, J. T. Paulley, and R. Martin Roop II. Department of Microbiology and Immunology,
Vaccines and Inhibitors
P13. Evaluation of protective efficacy against aerosol challenge infection with Brucella melitensis and Brucella abortus. Kahl-McDonagh, M.M., A. M. Arenas-Gamboa, and T.A. Ficht. Department of Pathobiology, College of Veterinary Medicine,
P14. Co-trimoxazole plus Lactobacillus for treatment of experimental brucellosis. Grushina T.1, Gavrilova N.2, Ratnikova I.2 1M. Aikimbayev's Kazakh Scientific Center for Quarantine and Zoonotic Diseases, 2Institute of Microbiology and
P15. Drug susceptibility testing of Brucella abortus-infected Mono Mac 6 human macrophage cell line. M.W. Valderas, R.A. Duncan, J.H. Wyckoff, and W.W. Barrow. Department of Veterinary Pathobiology,
P16. Brucella melitensis survival in fresh and ripe goat cheese. Méndez-González Yuliett1, Monroy-López Francisco1, Suárez-Güemes Francisco1, López-Merino Ahidé2, Hernández-Castro Rigoberto1, Guerrero Isabel3. 1Facultad de Medicina Veterinaria y Zootecnia de
Abstracts of Oral Presentations
Keynote Presentation
1. Nod-like Receptors: Role of in Bacterial Recognition and Host Defense.
Gabriel Nuñez. Department of Pathology and
NOD-like receptors (NLRs) are members of a family of cytosolic proteins with structural homology to the apoptosis activator Apaf-1 and plant disease resistance (R) gene products. NLRs contain variable N-terminal effector domains, a centrally located nucleotide-binding oligomerization domain (NOD) and C-terminal leucine-rich repeats (LRRs). NLRs mediate recognition of conserved microbial structures through their LRRs and upon activation induce multiple defense signaling pathways. Nod1 and Nod2 sense conserved, but distinct structural motifs, in bacterial peptidoglycan while Ipaf and Cryopyrin sense cytosolic flagellin and microbial RNA, respectively. Cryopyrin/Nalp3 and Ipaf are critical for the activation of inflammasomes, molecular platforms that mediate the activation of caspase-1 and processing of pro- IL-1/IL-18 into mature IL-1 and IL-18 in response to intracellular bacteria. Mutations in Nod2 are associated with Crohn’s disease whereas Cryopyrin/Nalp3 are linked to several autoinflammatory syndromes that are characterized by inappropriate l secretion of IL-1. Genetic and biochemical analyses revealed that cytosolic NLR proteins activate host signaling pathways independently of TLR signaling, although both NLRs and TLRs cooperate for optimal immune responses to bacterial pathogens. The results available so far suggest that NLRs are critical mediators of innate immune responses by linking intracellular recognition of bacteria to host defense pathways and their deregulation play an important role in inflammatory disease.
Diagnostics and Taxonomy
2. The development and applications of multilocus sequence analysis of the Brucella group. Adrian Whatmore, Julie Scott, Mike Stubberfield, Mark Koylass, and
In order to investigate genetic relationships within the Brucella group we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus. Nine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 161 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03-2.45% while overall genetic diversity equated to 1.5%. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure.Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences. The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified.
3. Molecular epidemiology of marine mammal Brucella isolates based on multilocus sequence typing (MLST) and multiple locus VNTR analysis (MLVA). Pauline Groussaud, Stephen Shankster and Adrian Whatmore. Veterinary Laboratories Agency, Addlestone,
Research into marine mammal Brucella has been carried at the Veterinary Laboratories Agency since 1994 when the first isolation was reported by Ross et al. Since then there have been ongoing international discussions concerning the phylogeny and taxonomy of these new organisms. In 1997, Jahans et al. proposed the name Brucella maris, whilst Cloeckaert et al. (2001) proposed the names B. pinnipediae and B. cetaceae, based on two groups observed using omp2 typing. Many typing techniques are currently available to classify marine mammal isolates, including multilocus sequence typing (MLST) and multiple locus variable-number-tandem-repeats analysis (MLVA), which are sequence-based typing methods useful for epidemiology. MLST involves the amplification and sequencing of housekeeping gene fragments that accumulate change slowly and are therefore useful for global epidemiology. MLVA is based on short sequence repeats, which accumulate changes at a higher rate and are useful for local epidemiology. When applied to marine mammal isolates, both methods show that marine mammal strains form groups distinct from all other known Brucella species. Within the marine mammal strains, these typing methods reveal further subdivisions that are congruent between the methods and largely correlate with apparent host preference. One group contains strains predominantly found in pinnipeds (seals) and two groups are predominantly found in cetaceans (porpoises and dolphins). These typing methods should therefore assist in the clarification of controversies surrounding the phylogeny and taxonomy of the marine mammal Brucella.
4. Isolation of a Brucella species from Marine Mammals in
Tissues, swabs or isolates from various marine mammals have been submitted to the National Veterinary Services Laboratories for the recovery and identification of a new species of Brucella. The serological history of these animals varied from negative for antibodies to Brucella to high titers by several of the conventional Brucella serological tests used for cattle. The rivanol provided the best sensitivity and specificity results. The complement fixation test was anti-complementary in 10 out of 11 tests. A variety of tissues were received for bacteriological examination but identical and complete sets were not received from all animals. Data from this study indicate the tissues of choice are pulmonary, hepatic, mesenteric, inguinal and mediastinal lymph nodes and the lungs. Isolates of Brucella were recovered from bottlenose dolphins (Tursiops truncates), Pacific harbor seals (Phoca vitulina richardsi), ringed seals (Phoca hispida), harp seals (Phoca groenlandica), and beluga whale (Delphinapteus leucas). The classical biochemical tests for Brucella and other tests including phage typing, DNA analysis, and oxidative metabolic studies, identified marine mammal isolates as a new Brucella species. The seal and dolphin isolates had several similarities but there were a few differences. The seal isolates required carbon dioxide for growth and had the dominant A antigen. The bottlenose dolphin isolates differed from the seal isolates by not requiring carbon dioxide for growth and having the dominant M antigen.
Test |
Bottlenose dolphin ( |
Bottlenose dolphin ( |
Harbor, Ringed, & Canadian Harp seals & Beluga whale |
Harp seals (US) |
CO2 |
Not required |
Not required |
Required |
Required |
H2S |
Negative |
Trace |
Negative |
Negative |
Urease |
> 1 hr |
> 1 hr |
> 1 hr |
> 1 hr |
Basic Fucshin |
Growth 1:25,000 |
Growth 1:100,000 |
Growth 1:25,000 |
Growth 1:25,000 |
Thionin |
Growth 1:25,000 |
Growth 1:25,000 |
Growth 1:25,000 |
Growth 1:25,000 |
Thionin blue |
Growth |
No Growth |
Growth |
Growth |
Penicillin |
Growth |
No Growth |
Growth |
Growth |
Erythritol |
Growth |
Growth |
Growth |
Growth |
Dominant antigen |
M |
A |
A |
A |
No lysis |
No lysis |
No lysis |
Lysis |
Immunology and Host-Pathogen Interactions
5. BALB/c B cell deficient mice exhibit rapid control of Brucella abortus. Goenka R, Casey L., Zou B., CL Baldwin. Department of Veterinary and Animal Sciences,
In mice infected with Brucella abortus protective effects of antibodies have been demonstrated. We tested the protective role of B cells and antibodies during brucellosis by infecting BALB/c B cell deficient Jh-/- mice. We demonstrated that despite the similar splenic colonization of brucellae at 1 week and 10 days p.i. in wild type (wt) and Jh-/- mice, by 2 weeks p.i. Jh-/- mice had 90% fewer bacteria and cleared 99% of the infection by 3 wks p.i.. Passive transfer of 8 week p.i. immune serum did not reverse this rapid clearance at 2 weeks p.i. despite the presence of anti-Brucella antibodies in recipient mice. In early stages of the infection, 50% of the bacteria were present in the acellular fraction of the infected wt and Jh-/- spleens and there was a significant increase in neutrophil recruitment to the Jh-/- spleens between 2 and 3 weeks p.i.. Hence, we hypothesize that this rapid clearance in Jh-/- spleen is due to neutrophil-mediated brucellicidal activity. Thus, B cells and/or their secreted products, other than antibodies, may facilitate colonization of infection by acting as regulatory cells by limiting neutrophil recruitment and decreasing protective IFNγ production, a characteristic of infection BALB/c mice as shown previously.
6. Mammalian B lymphocytes act as an infection reservoir for Brucella abortus. Goenka R, Guirnalda P, Black SJ, CL Baldwin. Department of Veterinary and Animal Sciences,
Brucella abortus is known to infect trophoblastic epithelial cells and professional phagocytes such as macrophages and dendritic cells. We have shown that B cell deficient mice rapidly clear the infection and are unable to sustain the high level plateau associated with chronic infection. Passive transfer of immune serum did not inhibit rapid clearance of brucellae. Thus we hypothesized that the B cells may act as an infection target. Here, we demonstrate that B. abortus can infect and survive inside B lymphocytes both in vitro and in vivo. Immune serum and specifically, IgM antibodies were needed for internalization of brucellae into B lymphocytes as shown by in vitro experiments. This data brings to light the susceptibility of B cells to bacterial infection and of their ability to act as a reservoir for intracellular bacteria.
7. Alteration of the Fc gamma Receptor I in murine macrophages during a Brucella spp. infection. A. Mathison, J. Harms, L. Eskra, G.A. Splitter. Department of Animal Health and Biomedical Sciences,
Infection of macrophages with Brucella spp. initiates an intricately balanced host-pathogen relationship. Shifting the balance of this relationship in either direction may result in the clearance or persistence of Brucella spp. within the host. Murine macrophages (RAW264.7) were infected with B. melitensis, B. neotomate and B. ovis for four hours continuously and transcript level analysis indicated 163 genes with altered transcript levels in comparison to uninfected macrophages. Among the genes altered during the early stages of infection was the Fc gamma Receptor I (Fcgr1, CD64). Fcgr1, a high affinity receptor for IgG, initiates signal transduction in phagocytic cells that ultimately alters phagocytosis, cytokine secretion, and development of immune responses. Following a four-hour infection with Brucella spp. the transcript level of Fcgr1 was decreased 2-fold in the infected macrophages as compared to uninfected macrophages. The protein level of Fcgr1, in contrast, had time dependent expression that varied between B. melitensis and B. neotomae. During a B. melitensis infection the number of cells expressing Fcgr1 decreased over the timecourse of infection. Conversely, there is an increase in the number of cells expressing Fcgr1 upon infection with B. neotomae. Treatment of the macrophages with IFN-gamma prior to infection abrogated the altered expression of Fcgr1 during infection. Macrophages were engineered by transduction and screened for stable clones with constitutively increased or decreased levels of Fcgr1. B. melitensis had an altered survival and/or persistence within the macrophages expressing increased or decreased Fcgr1 levels. Studies with Fcgr1 have identified a host defense mechanism that is not uniform across the Brucella spp. during infection. Furthermore, shifting the host-pathogen balance by altering Fcgr1 expression has provided insight into the importance of Fcgr1 during the Brucella infection process.
8. Murine macrophage transcriptional responses following in vitro infections with virulent smooth and attenuated rough Brucella suis strains. Y. He1, X. Ding2, Y. Ding3, D. Ghosh3, Z. Fei4, G. G. Schurig5, N. Sriranganathan5, S. M. Boyle5. 1Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI. 2Dept of Vet. Pathobiology,
The interaction between Brucella and macrophages is critical for establishment of a chronic Brucella infection. Macrophages kill more than 90% of smooth virulent B. suis strain 1330 within the first 24 hours post infection; however, the surviving Brucella rapidly multiply afterwards. B. suis strain 1330 does not induce macrophage cell death. Rough attenuated B. suis strain VTRS1 cannot replicate or survive inside macrophages, and it also induces necrotic cell death. We have used mouse Affymetrix 430 2.0 microarrays to analyze and compare the transcriptional responses of murine macrophage-like J774.A1 cell line to a time series of infections with B. suis strains 1330 and VTRS1. The differential transcriptional responses in infected macrophages will be summarized and discussed.
9. Host and Brucella gene expression profiles in an in vitro model of infection. C. A. Rossetti1, K. Drake2, C. L. Galindo3, S. A. Johnston4, H. R. Garner3 and L. G. Adams1. 1Dept. of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, 77843-4467, 2Seralogix, Austin, TX, 3UT-SWMC,
Brucella natural infections occur primarily through mucosal membranes. HeLa cells, as non-professional phagocytic cells, have been used to investigate adhesion, internalization, intracellular trafficking and survival and replication of brucellae. Post-infection global gene expression profiles of both agent and host have yet to be analyzed. The goals of this study were to characterize the transcriptome of Brucella melitensis and B. melitensis-infected host cells during the acute infectious process for investigating the initial strategies employed for the pathogen to survive and replicate intracellularly and to identify perturbations of major gene(s) modulating critical cellular pathways during initial infection. Our preliminary results revealed the intracellular replication of B. melitensis in HeLa cells to begin after an initial adaptation period of 4h post-infection. Analysis of human and B. melitensis microarray data using classical statistical methods and dynamic Bayesian modeling revealed 161 (35 up- and 126 down-regulated) and 115 (86 up- and 29 down-regulated) genes differentially expressed in B. melitensis, and 152 (45 up- and 107 down-regulated) and 957 (733 up- and 224 down-) altered genes in infected cells compared to non-infected ones at 4 and 12 h post-infection, respectively. Brucella genes related with transcription/translation (transcriptional regulators, ribosomal proteins) and metabolic processes (carbohydrate, lipid and amino acid transporters, kinases, dehydrogenases) were down-regulated at 4 but were up-regulated at 12 h post-infection. Among others, principal candidate host mechanistic genes related to apoptosis (caspase 1 & 3), cell cycle (cyclin, histone deacetylase) and MAPK signaling (MAPK1, 6 & 8) pathways were differentially expressed at both time points.
10. Nramp1 3’UTR polymorphisms are not associated with natural resistance to Brucella abortus in cattle. Paixão, T.A., Poester, F.P., Carvalho Neta, A.V., Borges, A.M., Lage, A.P., Santos, R.L. Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Natural resistance against intracellular pathogens such as Brucella abortus has been associated with the Nramp1 gene in mice, man, and cattle. The Nramp1 gene encodes a divalent cation tranporter that is located in phagolysosome membrane in macrophages. In mice, Nramp1 plays an important role in innate immunity, preventing intracellular bacterial growth in macrophages. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3’untranslated region (3’UTR) of the Nramp1 gene, which are detectable by single strand conformational analysis (SSCA). This study aimed to evaluate the association between Nramp1 3’UTR polymorphisms and natural resistance against bovine brucellosis in experimental and natural infections with Brucella abortus. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCAr, n=19) and in 43.1% of those with a susceptible genotype (SSCAs, n=23). Furthermore, no association between intensity of pathologic changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis as determined by serology, clinical signs and bacterial isolation, the frequencies of the SSCAr genotype were 86 and 84% in serologically positive (n=64) and negative (n=36) cows, respectively. Therefore, no association was found between the Nramp1 resistant allele and the resistant phenotype neither in experimental nor in naturally-occurring brucellosis. To further confirm these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either resistant or susceptible genotypes. In agreement with our previous results, no difference in the B. abortus intracellular rate of survival was observed when comparing macrophages with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the Nramp1 3’UTR do not affect resistance against B. abortus in cattle, and therefore they are not suitable as makers of natural resistance against bovine brucellosis.
Virulence - Genes and Mechanisms
11. Drosophila S2 cells as a model system for studying host-Brucella interactions. Qingming Qin1, Jianwu Pei2, Brian D. Shaw1, Thomas A. Ficht2,3, and Paul de Figueiredo1,3,4 . 1 Department of Plant Pathology and Microbiology. 2 Department of Veterinary Pathobiology; 3 Faculty of Genetics; 4 Program in Biotechnology;
Brucella spp. are intracellular bacterial pathogens that cause brucellosis, a chronic and debilitating disease in humans and animals. Over the past decade, genetic studies have succeeded in uncovering many Brucella virulence determinants. In contrast, a systematic analysis of host factors that support Brucella infection has not been achieved. Recently, RNA interference (RNAi) technology and Drosophila S2 macrophage-like cells have been combined to form a powerful genetic system for elucidating host cell biology. In fact, significant advances in our understanding of several bacterial pathogens, including Listeria monocytogenes, have been achieved using this model system. Here, we provide evidence that Drosophila S2 cells support the uptake and replication of Brucella. In addition, we show that the intracellular trafficking of the pathogen in the insect cell system mimics that seen in mammalian cells. Finally, we employ RNAi-mediated gene knockdown experiments to demonstrate important similarities between these systems. Taken together, our results indicate that the insect cell system can be exploited to identify and characterize evolutionarily conserved host factors.
12. Erythritol regulates several virulence systems in Brucella. Sangari, F. J., M.C. Rodriguez, C. Viadas, I. López Goñi y J.M. García Lobo. Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain, and Departamento de Microbiología y Parasitología, Universidad de Navarra, Pamplona, Spain.
Erythritol is C4 sugar-alcohol molecule playing an important role in the biology of the pathogenic bacteria of the genus Brucella. It has been described that members of this genus metabolize this compound preferentially than other sugars and the presence of this molecule in the placenta of ungulates has been implicated in the tropism of this pathogen for the reproductive organs and its capability to induce abortions. The erythritol catabolic operon in Brucella contains at least four genes eryABCD, eryD codifying for a repressor that controls expression of the operon. Several lines of evidence suggest that this transcriptional regulator could be involved in the control of some other systems, apart from the control of the ery and virB operons already reported by our group. Taking advantage of the construction of the Brucella ORFeome, we have designed and constructed a DNA microarray containing PCR products with most of the ORFs of Brucella. The microarray was probed with RNA extracted from B. abortus 2308 grown in rich medium with or without erythritol. A set of genes significatively induced or repressed by erythritol was obtained that included all the systems allegedly regulated by erythritol that, as well as others previously associated with Brucella virulence. These results suggest that erythritol could be sensed by Brucella as a marker of intracellular environment.
13. Structure Function analysis of the B.suis VirB8 protein. David O'Callaghan. INSERM U431, UFR Medecine, 30908
The VirB Type IV secretion system is essential for Brucella virulence.T4SS are multi-protein assemblies spanning the bacterial envelope. VirB8 is a bitopic inner membrane protein with a short cytoplasmic N-terminus and a mainly periplasmic C-terminus. It is thought to form part of the core elements of the translocation machinery (VirB6, VirB8, VirB9 and VirB10), which span the cell envelope and presumably form the translocation channel.
A B. suis mutant with a non polar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Structure-based site directed mutagenesis was used to introduce changes into VirB8 at residues potentially involved in dimerization and protein-protein interactions. Variants were first characterized in vitro with biochemical methods, identifying residues required for interaction with VirB4, VirB10 and for dimerization. Subsequent functional analysis of VirB8 variants in B. suis demonstrated that these residues are important for protein function in vivo.
Heterologous complementation with TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence to B. suis, however virulence was partially restored by a chimeric protein containing the N-terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.
14. Evaluating the Virulence of a Putative Brucella Melitensis hemagglutinin in the caprine model. Q.L. Perry1, S.D. Hagius2, J.V. Walker2, and P.H. Elzer1, 2. 1Department of Pathobiological Sciences,
Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortions in goats and sheep and Malta fever in humans. The zoonotic disease brucellosis causes severe economic losses in the Mediterranean region and parts of Africa, Asia, and
The B. melitensis hemagglutinin gene was isolated, cloned into pBBR1MCS-4, and electroporated into B. abortus 2308 yielding the variant 2308-QAE. 2308-QAE was characterized biochemically to confirm its Brucella speciation and screened by antibiotic selective pressure.
Being the primary host for B. melitensis, a majority of pregnant goats are colonized and abort when infected with this strain. In contrast, pregnant goats being a secondary host for B. abortus, less than half of the animals are colonized or abort. B. abortus 2308, B. melitensis 16M, or 2308-QAE were introduced into the pregnant goat model and evaluated for pathogenicity. Pregnancy/delivery results revealed 27%, 78%, and 67% abortion rates in goats infected with 2308, 16M, and 2308-QAE, respectively. Bacterial culture of tissues from 2308, 16M, and 2308-QAE groups revealed 45 %, 79%, 75% colonization of dam/kid pairs, respectively. The expression of the B. melitensis hemagglutinin gene in trans in B. abortus (2308-QAE) revealed a significant (p<0.05) increase in colonization and the rate of abortions when compared to the B. abortus 2308 parental strain, thus mimicking the virulence of B. melitensis 16M in pregnant goats. A colonization study of non-pregnant goats revealed similar growth of B. abortus (2308-QAE) when compared to the B. abortus 2308 wildtype strain at 4, 7, and 21 days post inoculation.
The hemagglutinin gene was also cloned into the pUC19 vector to aid in making a potentially attenuated knockout strain. The gene was cut internally and the deleted portion replaced with an antibiotic cassette. A disrupted gene fragment was produced by enzyme digestion and electroporated into B. melitensis. The resulting mutants were characterized and screened for future evaluation in the pregnant goat model. A colonization study of non-pregnant goats revealed no attenuation of the 16MΔE mutant when compared to the B. melitensis 16M wildtype strain at 4, 7, and 21 days post inoculation.
15. A conserved hypothetical protein of Brucella spp is essential for their virulence in animals. Mariela Carrica and Silvio L. Cravero. Instituto de Biotecnologia-Instituto Nacional de Tecnologia Agropecuaria (INTA) Castelar
By gene inactivation and allelic exchange in Brucella abortus S2308, B. melitensis 16M and B. suis 1330, we identify the B. melitensis BMEI0489 ORF as being essential for their virulence, and we named this ORF as iivA. This ORF encodes a hypothetical conserved protein of 106 amino acids that belongs to the Cluster of Orthologous Group (COG) 2960 that belongs to a family of uncharacterized proteins currently contains 126 members.
IivA is trimeric in solution, with a coiled-coil domain in its carboxy-terminal and an unstructured amino-terminal. The B. abortus S2308 and its knock-out mutant grow at a similar rate in vitro. The mutant is protective in the mice model against a challenge with S2308. A Salmonella typhimurium knock-out mutant in the orthologous gene of BMEI0489 is attenuated in the mice model. We are trying to define the biochemical function of IivA through its protein structure.
16. The role of the alkyl hydroperoxide reductase complex in Brucella abortus resistance to oxidative stress. Kendra Hitz, Michelle Wright-Valderas, John Baumgartner, Tim Brown, and R. M. Roop II. Department of Microbiology and Immunology,
The brucellae can withstand the oxidative stress encountered in the macrophage by producing antioxidant enzymes that detoxify superoxide and hydrogen peroxide. When Brucella cells are pretreated with a low concentration of hydrogen peroxide before exposure to 100mM hydrogen peroxide, they survive six logs better than cells that are not pretreated. This suggests that the brucellae respond to the pretreatment by inducing genes whose gene products are involved with resistance to hydrogen peroxide. One set of genes whose gene products detoxify hydrogen peroxide are ahpC and ahpD. The alkyl hydroperoxide reductase complex (AhpC/AhpD) in other bacteria detoxifies organic peroxides and peroxynitrite as well as hydrogen peroxide. Current studies are evaluating if the B. abortus 2308 AhpCD is an important antioxidant in the detoxification of these oxidative stress molecules.
17. The Brucella abortus xthA-1 and xthA-2 gene products play overlapping roles in base excision repair and resistance to oxidative stress. Michael L. Hornback and R. Martin Roop II. Department of Microbiology and Immunology,
Brucella abortus is a facultative, intracellular pathogen whose virulence is intimately linked to survival within host macrophages. Oxidative killing is one of the primary mechanisms employed by these host phagocytes to control intracellular replication of the brucellae. Consequently, cellular defenses against oxidative damage likely play a key role in the ability to of the brucellae to maintain long-term residence in the phagosomal compartment of host macrophages. Exonuclease III, encoded by the xthA gene, is an enzyme involved in base excision repair (BER) and this system play an important role in protecting Escherichia coli from oxidative damage to DNA. Interestingly, B. abortus possesses two xthA homologs, designated xthA-1 and xthA-2. An isogenic xthA-1 mutant CAM220 constructed from virulent B. abortus 2308 displays increased susceptibility to methylmethanesulfonate (MMS), H2O2, SIN-1 (a compound that produces peroxynitrite), and bleomycin compared to the parental strain in in vitro assays, a phenotype consistent with the xthA-1 gene product participating in base excision repair of oxidatively damaged DNA. When the B. abortus xthA-1 mutant was evaluated in cultured murine macrophages and experimentally infected mice, however, this strain displayed wild-type virulence. This raises a question of whether xthA2 gene product has overlapping functions and masks the importance of XthA-1 in vivo. An xthA-1 mutant containing multiple copies of plasmid-bourne B. abortus xthA-2 is able to restore wild-type resistance to MMS and H2O2 challenge, suggesting these two gene products have overlapping function. However, attempts at determining a role of xthA-2 in BER has been difficult because creating an xthA-2 mutant using traditional methods have been unsuccessful. To address this problem, an inducible antisense RNA construct was created to make a conditional xthA-2 mutant. As demonstrated with the chromosomal xthA-1 mutant, the xthA-2 mutant displays similar levels of sensitivity when exposed to MMS, hydrogen peroxide, and bleomycin. These experimental findings suggest that XthA-2 and XthA-1 may perform overlapping functions in BER and in protecting the brucellae from oxidative stress in vitro and both may play a role within the phagosomal compartment of host macrophages.
18. Hemin utilization by Brucella abortus 2308 is dependent on the ChrSA two component regulatory system. James T. Paulley, Eric S. Anderson, J. E. Baumgartner, and R. M. Roop II. East Carolina University Department of Microbiology and Immunology,