Registration: 7:30

59^th

Annual Brucellosis

Research Conference

Marriott Downtown Magnificent Mile

Chicago, IL

Program and Abstracts

December 2-3, 2006

59^th Annual Brucellosis Research Conference

December 2-3, 2006

Chicago Marriott Hotel - Chicago, IL

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)

59^th Annual Brucellosis Research Conference

December 2-3, 2006

Chicago Marriott Hotel - Chicago, IL

Chicago Ballroom Salon D

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 Azerbaijan, Georgia, Kazakhstan, and Uzbekistan. Participating in programs sponsored by the Defense Threat Reduction Agency (DTRA), these brucella researchers are accompanied by interpreters and administrators. Supporting agencies include CRDF, BNI, SAIC-TRSC, and TMC. They are here to share the research from their countries and to learn about the science discussed by our membership. We welcome them to our meeting!

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 Seville, Spain and completed residency training in Anatomic Pathology at Washington University School of Medicine in St. Louis. He completed post-doctoral fellowships in Immunology (University of Texas Health Science Center) and Molecular Biology (Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri) and joined the faculty of the Department of Pathology as Assistant Professor in 1991. He was promoted to the rank of Associate Professor in 1996 and to Professor in 2001. In addition, Dr. Nunez was named the first Paul H. de Kruif Endowed Professor of Pathology in 2001. Dr. Nunez is the author of more than 150 peer-reviewed publications. His laboratory is funded by grants from the National Institute of Health, Crohn’s and Colitis

Foundation and the Broad Medical Research Program.

Dr. Nunez is board certified in Anatomic Pathology.

Campus Address:
4219 CCGC 0938
1500 East Medical Center Drive
Ann Arbor, Michigan 48109-0938
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 Krishna Gopaul. Veterinary Laboratories Agency, Addlestone, United Kingdom, KT15 3NB.

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, Surrey, United Kingdom, KT15 3NB.

10:00 am

Isolation of a Brucella species from marine mammals in North America. Darla R. Ewalt,¹ Betsy Bricker,² Dyanna M. Lambourn,³ Ole Nielsen,⁴ Jennifer Maratea,⁵ Patricia Geer,¹ Lorry B. Forbes,⁶ Lena Measures,⁷ Steven J. Jeffries³. ¹USDA, APHIS, VS, NVSL, Ames, Iowa; ² USDA, ARS, NADC, Ames, IA; ³Washington Department of Fish and Wildlife, Tacoma, Washington; ⁴Department of Fisheries and Oceans, Winnipeg, Manitoba, Canada; ⁵Department of Pathobiology, University of Connecticut, Storrs, CT; ⁶Health of Animals Laboratory, Program Laboratories Directorate, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada; ⁷Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada.

10:15 am

DTRA Presentation 1 – Current Brucellosis Situation in Azerbaijan

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, University of Massachusetts, Amherst, MA.

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, University of Massachusetts, Amherst, MA.

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, University of Wisconsin-Madison, 53706, USA.

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, Texas A & M, College Station, TX. ³Dept of Biostatistics, University of Michigan, MI. ⁴USDA/Boyce Thompson Institute, Boyce Thompson Institute, Cornell University, Ithaca, NY. ⁵Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA.

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. Ficht^2,3, and Paul de Figueiredo^1,3,4. ¹Department of Plant Pathology and Microbiology. ² Department of Veterinary Pathobiology; ³ Faculty of Genetics; ⁴ Program in Biotechnology; Texas A&M University, College Station, TX 77843.

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 Nimes, France.
david.ocallaghan@univ-montp1.fr

3:00 pm

DTRA Presentation 2 - Brucellosis in Kazakhstan

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. Elzer^{1, 2}. ¹Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 and ²Department of Veterinary Science, LSU AgCenter, Baton Rouge, LA 70803.

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 Argentina.

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, East Carolina University School of Medicine, Greenville NC 27834.

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, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.

5:00 pm

DTRA Presentation 3 - Status of Brucellosis in Georgia

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, Greenville, NC 27834.

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, Texas A&M University and Texas Agricultural Experiment Station, College Station, TX 77843-4467. ²Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100094, China.

8:30 am

Targeting the virulome of the intracellular pathogen Brucella suis: Inhibition of virulence factors prevents intramacrophagic multiplication and reveals a strategy for the definition of novel antibacterial agents. Stephan Köhler¹, Pascale Joseph¹, Marie-Rose Abdo², Jean-Yves Winum², Jean-Louis Montero², Jean-Pierre Liautard¹, and Rose-Anne Boigegrain¹. ¹Institut National de la Santé et de la Recherche Médicale (INSERM) U-431, ²Laboratoire de Chimie Biomoléculaire, UMR 5032 CNRS. Université Montpellier II. Montpellier, France.

8:45 am Keynote Speaker

Nod-like Receptors: Role of in Bacterial Recognition and Host Defense. Gabriel Nuñez. Department of Pathology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA.

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-Ficht^1,2. ¹Dept. of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University; ²Dept. of Medical Biochemistry and Genetics, College of Medicine, Texas A&M University Health Science Center.

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, Uzbekistan; ²LSU AgCenter, Dept. Veterinary Science, Baton Rouge, LA.

10:15 am

BREAK

10:45 am

DTRA Presentation 4 - Brucellosis in Uzbekistan

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*, Adam Rupper¹, R. Martin Roop II², James A. Cardelli¹. ¹Louisiana State University Health Sciences Center, Shreveport, LA; ²East Carolina University School of Medicine, Greenville, NC; ³Iowa State University, College of Veterinary Medicine, Ames, IA.

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, University of California at Davis, Davis, CA 95616

P3. Effects of TLR4-directed RNA interference on cell-mediated immune response to Brucella infection. T. E. Todd¹, Y. He^1,2. ¹Unit for Laboratory Animal Medicine, ²Dept. of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI.

P4. Pharmacological studies support the use of Drosophila S2 cells as a model system for studying Brucella infection of host cells. Qingming Qin ^*1, Jianwu Pei ², Brian D. Shaw ¹, Thomas A. Ficht ^2,3, and Paul de Figueiredo ^1,3,4.¹Department of Plant Pathology and Microbiology, ²Department of Veterinary Pathobiology, ³Faculty of Genetics, ⁴Program in Biotechnology; Texas A&M, College Station, TX 77843.

P5. Pathogenesis of the experimental infection with a Brucella melitensis 16M mutant in the goat model. Maria Ceron Cucchi¹, Sandra Conde¹, Luis Samartino¹, Agustín Venzano¹,Osvaldo Rossetti², and Silvio L. Cravero². ¹Instituto de Patobiología and ²Instituto de Biotecnología-INTA Castelar, Argentina..

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 Castelar, Argentina.

P10. Purification and biochemical characterization of Brucella suis urease. Araceli Contreras-Rodriguez^1,2, 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, East Carolina University School of Medicine, Greenville, NC 27834.

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, East Carolina University School of Medicine, Greenville, North Carolina 27834.

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, Texas A&M University, College Station TX 77843-4467

P14. Co-trimoxazole plus Lactobacillus for treatment of experimental brucellosis. Grushina T.¹, Gavrilova N.², Ratnikova I.²¹M. Aikimbayev's Kazakh Scientific Center for Quarantine and Zoonotic Diseases,²Institute of Microbiology and Virology, Kazakhstan.

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, Oklahoma State University Center for Veterinary Health Sciences, Stillwater, OK 74078

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 la Universidad Nacional Autónoma de México Av. Universidad 3000, Ciudad Universitaria, Delegación Coyoacán, CP 04510, México, DF. Telephone. +52 (55) 56225854 -57. Fax. +52 (55) 5622 -5971. ²Escuela de Ciencias Biológicas del Instituto Politécnico Nacional. ³Universidad Autónoma Metropolitana Unidad Iztapalapa.

Abstracts of Oral Presentations

Keynote Presentation

1. Nod-like Receptors: Role of in Bacterial Recognition and Host Defense.

Gabriel Nuñez. Department of Pathology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA.

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 Krishna Gopaul. Veterinary Laboratories Agency, Addlestone, United Kingdom, KT15 3NB.

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, Surrey, United Kingdom, KT15 3NB.

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 North America. Darla R. Ewalt,¹ Betsy Bricker,² Dyanna M. Lambourn,³ Ole Nielsen,⁴ Jennifer Maratea,⁵ Patricia Geer,¹ Lorry B. Forbes,⁶ Lena Measures,⁷ Steven J. Jeffries³. ¹USDA, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, Iowa, ² USDA, Agricultural Research Service, National Animal Disease Center, ³Washington Department of Fish and Wildlife, Tacoma, Washington, ⁴Department of Fisheries and Oceans, Winnipeg, Manitoba, Canada, ⁵Department of Pathobiology, University of Connecticut, Storrs, Connecticut, ⁶Health of Animals Laboratory, Program Laboratories Directorate, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada, ⁷Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada.

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 (California)	Bottlenose dolphin (Connecticut)	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
Tbilisi phage	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, University of Massachusetts, Amherst, MA

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, University of Massachusetts, Amherst, MA.

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, University of Wisconsin-Madison, 53706, USA.

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, Texas A & M, College Station, TX. ³Dept of Biostatistics, University of Michigan, MI. ⁴USDA/Boyce Thompson Institute, Boyce Thompson Institute, Cornell University, Ithaca, NY. ⁵Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA.

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, Dallas, TX, ⁴ASU, Phoenix, AZ

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 (SSCA^r, n=19) and in 43.1% of those with a susceptible genotype (SSCA^s, 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 SSCA^rgenotype 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. Ficht^2,3, and Paul de Figueiredo^1,3,4. ¹Department of Plant Pathology and Microbiology. ² Department of Veterinary Pathobiology; ³ Faculty of Genetics; ⁴ Program in Biotechnology; Texas A&M University, College Station, TX 77843.

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 Nimes, France. david.ocallaghan@univ-montp1.fr.

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. Elzer^{1, 2}. ¹Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 and ²Department of Veterinary Science, LSU AgCenter, Baton Rouge, LA 70803.

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 Latin America. Human consumption of non-pasteurized milk and milk products or direct contact with infected animals or carcasses are the modes of transmission. Completion of the genomic sequences of B. melitensis 16M and B. abortus 2308 revealed no classical virulence factors, and the chromosomes were virtually identical. However, in B. melitensis, a putative hemagglutinin gene was identified which is absent in B. abortus. The possibility of the putative hemagglutinin, Region E, being a potential virulence factor is being evaluated.

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 Argentina.

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, East Carolina University School of Medicine, Greenville NC 27834.

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, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.

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, Greenville, NC 27834.

Brucella spp. have an absolute requirement for iron during infection. Due to the central role of macrophages in heme recycling in the host, heme and heme-containing proteins may serve as relevant iron sources for the brucellae during residence within these phagocytes. We have previously shown a genetic link between the bhuA (Brucella heme utilization) gene in B. abortus 2308 and the ability of this strain to use heme as an iron source in vitro. The bhuA mutant strain also undergoes a dramatic reduction in viability after entry into stationary phase in low iron conditions and is unable to maintain chronic spleen infection in experimentally infected BALB/c mice.

Transcription of the bhuA gene is greatest under low iron conditions, during entry into stationary phase. Activity from the bhuA promoter and upregulation of bhuA mRNA levels are dependent on the genes annotated as BAB1_0369 and BAB1_0370 in the B. abortus 2308 genome. These genes are predicted to encode homologs of the two component regulatory system ChrA (Corynebacterium heme responsive activator) and ChrS (Corynbacterium heme responsive sensor) in Corynebacterium diphtheriae. A derivative of B. abortus 2308 (designated JB9) in which the putative chrA homolog has been inactivated by gene replacement is unable to activate transcription from the bhuA promoter under all of the experimental conditions that have been examined to date. Promoter activity from the bhuA promoter is restored to near wild-type levels when an intact copy of the chrA gene is present in the JB9 mutant strain. The role of the ChrSA two component regulatory system in the iron dependent and growth phase dependent regulation of bhuA transcription is currently being evaluated.

19. 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, Texas A&M University and Texas Agricultural Experiment Station, College Station, TX 77843-4467. ²Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100094, China.

Smooth Brucella inhibit macrophage apoptosis, whereas rough Brucella induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current report, a random mutagenesis approach was used to create a mutant bank consisting of 11354 mutants by mariner transposon mutagenesis using B. melitensis rough mutant 16MDmanBA as parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (10% or less of LDH release). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes from 51 mutants were identified by DNA sequence analysis and represented interruptions in virB encoding the type four secretion system (T4SS) (n=36), in vjbR, encoding a LuxR-like regulatory element previously shown to be required for virB expression (n=3), and additional mutants (n=12) among which several also have predicted roles in virB expression. These results suggest that T4SS is associated with Brucella cytotoxicity in macrophage. To verify this observation, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (∆manBA) and the T4SS (∆virB). As predicted, deletion of virB from the 16M∆manBA strain resulted in a complete loss of cytotoxicity from rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme MOI>1000. Taken together, these results demonstrate that Brucella cytotoxicity in macrophage is T4SS dependent.

20. Targeting the virulome of the intracellular pathogen Brucella suis: Inhibition of virulence factors prevents intramacrophagic multiplication and reveals a strategy for the definition of novel antibacterial agents. Stephan Köhler¹, Pascale Joseph¹, Marie-Rose Abdo², Jean-Yves Winum², Jean-Louis Montero², Jean-Pierre Liautard¹, and Rose-Anne Boigegrain¹. ¹Institut National de la Santé et de la Recherche Médicale (INSERM) U-431, ²Laboratoire de Chimie Biomoléculaire, UMR 5032 CNRS. Université Montpellier II. Montpellier, France.

Constantly increasing numbers of antibiotic-resistant pathogens and the threat of bioterrorism are major challenges for the development of alternative treatments of infectious diseases. The virulome analysis of Brucella is an original approach for the identification of pathogen-restricted targets for antibacterial agents acting on the bacteria specifically in their replicative niche. Two previously identified virulence factors, acetohydroxyacid synthase (AHAS) and histidinol dehydrogenase (HDH), are involved in amino acid biosynthesis. We have shown that they are essential for growth in minimal medium and for intracellular replication only, and that they can be targeted by sulfonylureas and by histidinol analogues, respectively. For both targets, bacterial growth in minimal medium or within human macrophage-like THP-1 cells was totally abolished at inhibitor concentrations ranging from 10 to 100 M. With crude or pure enzyme preparations, inhibitor concentrations of 5-10 nM were sufficient to obtain a 50%-inhibition of enzymatic activities. Due to the absence of any effect of the inhibitors in a nutrient-rich environment, the selection of spontaneously resistant mutants of the pathogen within the host was expected to be limited to the intravacuolar state. Furthermore, spontaneously sulfonylurea-resistant mutants, selected on minimal medium in the presence of the drug, were characterized by decreased intracellular viability and by reduced AHAS activity.

We showed that virulome analysis of B. suis allowed definition of alternative targets for antibacterials acting specifically on the intracellular pathogen, and we demonstrated the effectiveness of specific inhibitors against intramacrophagic brucellae.

Brucella Genetics and Vaccines

21. 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.

Brucellosis, one of the common bacterial zoonoses in the world, is caused by a gram negative, non-spore forming facultative intracellular bacteria belonging to the genus Brucella. According to primary host preference, six species of Brucella have been identified- Brucella melitensis (sheep and goat), B. suis (hogs), B. abortus (cattle), B. ovis (Sheep), B. canis (dogs), and B. neotomae (wood rats). In addition, Brucella spp. have also recently been isolated from several marine mammals. B. neotomae is not known to be a virulent bacterium and there is no evidence to indicate it causes disease in any host species.

Brucella appears to be well equipped to overcome the exposure to reactive oxygen intermediates during their intracellular residence in the host macrophages. Previous studies indicated that the bacterial Cu, Zn-cofactored superoxide dismutase (SOD) protects Brucella from respiratory burst of the host macrophage. SOD in B. abortus also plays a role in establishing chronic infections in mouse models. Unlike in all other Brucella strains, expression of SOD in B. neotomae and B. suis biovar2 could not be detected.

This study was undertaken to determine the reasons for the absence of SOD expression in B. neotomae. Our genetic analysis revealed a single nucleotide insertional mutation in the promoter region of SOD gene as the cause for the suppression of this protein expression in B. neotomae. However, restoration of SOD expression of B. neotomae through complementation did not alter its growth characteristics in J774 macrophage-like cells and in tissues of BALB/c mice.

22. 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, ²Walter Reed Army Institute of Research, Silver Spring, MD.

Brucella spp. are classified as a “class B” bioterrorism agent and cause undulant fever in humans and abortions in a variety of land and marine mammals. As a prophylaxis for this pathogen, the USDA approved B. abortus strain RB51 is used as a vaccine against bovine brucellosis in the USA, Chile, Mexico, and Venezuela and is also being introduced in Brazil, India, Iraq and Iran. For expression of a gene encoding a heterologous antigen (i.e. from other pathogen) in B. abortus strain RB51, a plasmid vector containing an antibiotic resistant gene has been employed. This practice has always invited criticism as the vaccine has the potential to introduce an antibiotic resistance gene into normal flora as well as pathogens in the vaccinated animals. Construction of a selectable plasmid without an antibiotic resistance gene would present a minimum of medical and environmental risk. B. abortus strain RB51 lacking the leuB gene necessary for leucine synthesis would not readily survive as the normal site of replication is in macrophages, a known nutrient limiting environment. Complementation of a leuB mutant with a plasmid carrying a functional leuB⁺gene would facilitate the survival of strain RB51 inside macrophages as well as select for plasmid maintenance. A disruption of the B. abortus strain RB51 leuB gene was performed using cre-lox technology. This technology employs a site-specific recombinase enzyme that excises the antibiotic resistance gene in the leuB to create an unmarked mutant. The resultant leuB mutant of strain RB51 did not grow in leucine deficient minimal medium unless complemented with a plasmid carrying a wild type leuB⁺ gene. In addition, a heterologous gene encoding green fluorescent protein (GFP) was also cloned in the leuB⁺ containing plasmid. The leuB deficient B. abortus strain RB51 maintains the leuB⁺plasmid and expresses GFP inside infected J774 murine macrophages. To demonstrate the utility of the cre-lox protocol, a leuBmutant of B. abortus strain 2308 was also created. The absence of the antibiotic resistance markers in the genome associated with the creation of these mutants was demonstrated by southern blot analysis. To validate the utility of this plasmid in the context of a platform vaccine, mice immunized with strain RB51 over-expressing the leuB + GFP will be assessed for the presence of: a) plasmid in strain RB51 recovered from tissues and b) specific GFP antibody in sera.

23. Enhanced Immunogenicity and Protective Efficacy using Live Microencapsulated Vaccines against Brucellosis. A. M. Arenas¹, T. A. Ficht¹, M. Kahl¹, A. C. Rice-Ficht^1,2. ¹Dept. of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University; ²Dept. of Medical Biochemistry and Genetics, College of Medicine, Texas A&M University Health Science Center.

The development of safe and efficacious immunization systems to prevent brucellosis is needed to overcome the disadvantages of the currently licensed vaccine strains. A Novel method of immunization was examined using microspheres containing a non immunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B, VpB) conjugated to a live B. melitensis attenuated mutant and subsequently encapsulated into alginate microcapsules. The immunogenicity and protective efficacy of the capsules were evaluated in mice. A single immunization dose in BALB/c mice with encapsulated B. melitensis conferred a significant enhancement of protection compared to the un-encapsulated vaccine against wild type B. melitensis 16M challenge. VpB capsules also induced a higher and sustained IgG response compared to the un-encapsulated vaccine. In addition, cytokine secretion from spleen cells of mice vaccinated with the encapsulated mutant revealed elevated secretion of gamma interferon (INFg), Interleukin 12 (IL-12) and Interleukin 2 (IL-2), but no Interleukin 4 (IL-4), suggesting an induction of a T helper 1 (Th1) response. Together, these results suggest that microencapsulation of live Brucella produces an enhanced delivery vaccine system against brucellosis increasing the efficacy of poorly-performing un-encapsulated vaccine candidates.

24. 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, Uzbekistan; ²LSU AgCenter, Dept. Veterinary Science, Baton Rouge, LA.

Brucellosis is a zoonotic disease which causes huge economic and social damage in many countries. An important part of the complex set of measures applied in the struggle against brucellosis of ruminants in Uzbekistan is the use of brucellosis vaccines as routine preventive maintenance. Until 1995, the vaccine strain Rev - 1 Brucella melitensis was used against sheep brucellosis. This strain of weakened virulence is stable and immunogenic but causes a period of seropositivity which interferes with early differential diagnostics of vaccinated animals from natural infections.

Many vaccines may have negative side effects. Vaccine strain 19 B. abortus as the full dose when given to adult cattle resulted in an immune response in that they maintained vaccinal titers for years. The vaccine strain 82 B. abortus when given to adult cattle without a previous vaccination with strain 19 possessed аbortogenic properties, and the inactivated adjuvant vaccine from strain 45/20 B. abortus appeared weakly immunogenic in comparison with agglutinating (smooth) vaccines. Now in Uzbekistan the vaccine strain 19 B. abortus is used to protect cattle and sheep against brucellosis. This strain of weakened virulence is stable and possesses high immunogenic properties and lasts years on adult cattle when applied in small doses.

In our research laboratories new brucellae vaccine strains which possess sufficient immunogenicity and at the same time are slightly agglutinogenic have been created. Using specific criteria, UzSRIV has developed the slightly agglutinogenic vaccinal strain B. melitensis “Nevsky – 13”. This candidate vaccine strain has been studied for its cultural, morphological, biochemical, and immunogenic properties. Residual virulence, stability and abortogenicity in animals have been determined. The end result was that this B. melitensis strain met the requirements of vaccinal preparations.

Long-term research has established that strain B. melitensis "Nevsky - 13" possesses fixed stable cultural, morphological and biochemical properties. Stability of strain is confirmed after 20 years of cultivation on nutrient mediums, eightfold passages of the organism in guinea pigs and five passages in cattle and sheep. Strain "Nevsky - 13” possesses all desired properties of a B. melitensis vaccine and is a RS form of brucella.

The immunogenic properties of a freeze-dried preparation of the experimental vaccine strain "Nevsky - 13” was studied in guinea pigs and compared to a similar preparation of strain 19 B. abortus. There were 29 animals in three groups: Group 1 consisted of ten animals vaccinated with “Nevsky – 13” in a dose of 1 ml with the contents 1 billion bacterial cells, parenterally; Group 2 had 11 guinea pigs vaccinated with strain 19 in the same dose, parenterally; and Group 3, with eight animals, was not vaccinated and served as the controls. Animals were challenged with virulent strain 54 B. abortus 2.5 months post immunization with five minimal infectious doses (80 bacterial cells), parenterally. Animals were sacrificed 35-45 days later, and lymph nodes and internal organs were cultured for brucellae.

Table 1. Results B. melitensis “Nevsky – 13” vs. B. abortus strain 19

Vaccine	Dose of a vaccine	Number of animals	Infected		Immune
Vaccine	Dose of a vaccine	Number of animals	Number of animals	%	Number of animals	%
Nevsky - 13	1.0	10	2	20.0	8	80.0
St. 19	1.0	11	2	18.1	9	81.9
None	-	8	8	100.0	0	0.0

On the basis of the above experiment comparing the vaccine efficacy of B. melitensis strain "Nevsky - 13" with strain 19 B. abortus, we have determined that the two vaccines exhibit equivalent protection in the guinea pig brucellosis model against a virulent B. abortus challenge.

Abstracts of Poster Presentations

Immunology and Host-Pathogen Interactions

P1. Persistence of Brucella abortus in Gamma-Interferon stimulated human monocytes. Bryan H. Bellaire³*, Adam Rupper¹, R. Martin Roop II², James A. Cardelli¹. ¹Louisiana State University Health Sciences Center, Shreveport, LA; ²East Carolina University School of Medicine, Greenville, NC; ³Iowa State University, College of Veterinary Medicine, Ames, IA.

Brucella spp. are facultative intracellular pathogens of mammals that establish and maintain long term residence within host monocytes. Limiting brucellae infections within the host requires the production of the pro-inflammatory cytokine IFN-γ. Stimulation of monocytes with IFN-γ increases their antibrucella activity in mouse and human monocytes by primarily restricting the intracellular replication of the bacteria. To determine how IFN-γ activation prevents Brucella from establishing a productive infection within monocytes, human monocytes were activated with IFN-γ and the intracellular replication and phagosome maturation of Brucella were compared against results obtained for non-activated cells. Within the first 24 hours post infection, phagosomes containing B. abortus were compositionally similar among non-activated and IFN-γ activated monocytes, where these phagosomes were found to be acidic (pH < 5.5) and LAMP1+. However, by 48 hours, Brucella in non-activated cells were observed replicating within non-acidic, LAMP1+ vesicles, while, in contrast, Brucella in IFN-γ treated monocytes, although found in acidic, LAMP1+ phagosomes, did not appear to be replicating. Furthermore, these Brucella containing phagosomes were positive for the lysosomal component cathepsin D. In non-activated cells, de-acidification appears to be critical in the transition of Brucella containing phagosomes into an intracellular niche that supports bacterial replication. Thus, IFN-γ stimulation renders the intracellular environment incompatible to the intracellular growth of Brucella, where the bacteria persist in phagolysosomes.

The Type IV secretion system (T4SS) is a virulence factor essential for persistence of B. abortus. To better understand the role of the T4SS and its putative effectors in evading host defense mechanisms, we compared host gene expression profiles of splenocytes infected with wild type B. abortus, a virB1-B2::mTn5 mutant or a virB1-B12 mutant 3 days post-infection. While wild type B. abortus elicited a pro-inflammatory response, both T4SS mutants failed to induce this response. This suggested that this difference in response is likely due to the presence of a functional T4SS since bacterial loads in the spleen were similar for all strains tested. Induction of an IFN/-dependent gene expression was observed only in mice infected with wild type Brucella. We are currently testing whether induction of this response provides favorable conditions for establishing persistent infection.

The interaction between macrophages and Brucella is critical for chronic Brucella infection. The RNA interference (RNAi) technique makes it possible to knock down individual host genes and study their specific functions in Brucella-infected macrophages. To test this approach, GAPDH silencing RNA (siRNA) was transfected into RAW 264.7 macrophages, and it showed 40-80% of knockdown at 24-48 hours post infection and did not significantly stimulate nonspecific TNFa inflammatory response. We further investigated the immunological effects of knocking down TLR4, a Toll-like receptor that acts as the primary recognition molecule for the lipopolysaccharide of Gram-negative bacteria, in RAW 264.7 macrophages. The TLR4 siRNA induced approximately 80% specific knockdown and negligible TNFa and GAPDH changes at 48 hours post infection. The effects of the TLR4 knockdown in the macrophage-Brucella interaction after infections of macrophages with smooth virulent and rough attenuated Brucella strains are being analyzed and will be discussed.

Pharmacological approaches have proven exceptionally valuable for elucidating the cell biology of diverse eukaryotic systems. Here, we exploit the power of this approach to investigate the feasibility of using Drosophila S2 macrophage-like cells as a model system for studying host-Brucella interactions. We demonstrate that Brucella uptake and replication in assorted drug treated S2 cells mimic that seen in similarly treated mammalian cells. Taken together, these data support the idea that S2 cells can be used to elucidate the host-pathogen interface.

P5.   Pathogenesis of the experimental infection with a Brucella melitensis 16M mutant in the goat model.  Maria Ceron Cucchi¹, Sandra Conde¹, Luis Samartino¹, Agustín Venzano¹,Osvaldo Rossetti², and Silvio L. Cravero².  ¹Instituto de Patobiología and ²Instituto de Biotecnología-INTA Castelar, Argentina.

A Brucella melitensis 16M null mutant in the iivA gene (Bm iivA), whose product is essential to Brucella virulence, was assessed in the caprine model, in comparison to its virulent parental strain B. melitensis 16M (Bm 16M).

In order to determine the kinetics of colonization of both strains, two groups of eight animals each were conjunctively challenged with Bm iivA or Bm 16M. Inoculation with Bm iivA strain resulted in the infection of four animals, where the strain was isolated at 14 and 28 days post inoculation (p.i.) from mandibular and parotid lymph nodes. At day 42 p.i., it was only isolated from one goat’s parotid lymph nodes.

The Bm 16M strain infected all eight goats, with the bacteria being isolated at 8 and 14 days p.i. from mandibular and parotid lymph nodes, and at day 28 p.i. from iliac and supramammary lymph nodes as well as the spleen and liver.

On the histological level, the lymphatic nodules showed lymphadenitis and vasculitis, with a depletion of lymphocytes. The most severe lesions were observed in goats inoculated with Bm 16M.

Mutant strain Bm iivA was able to colonize tissues, showing a diminished virulence in comparison to its parental virulent strain Bm 16M.

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.

We have obtained B. ovis mutants for the five members of the Omp25/Omp31 family that are thought to be present in the outer membrane of this Brucella species in order to evaluate the role of each protein in the virulence of B. ovis in mice. Virulent B. ovis PA was used as parental strain to obtain the five mutant stains with the genes omp31, omp25, omp25c, omp25d or omp22 inactivated. For inactivation of omp25, the gene was cloned and a kanamycin resistance cassette inserted close to the 3'-end. The other four genes were inactivated by replacing part of the cloned gene by the kanamycin resistance cassette. Mutant B. ovis PA strains were obtained by replacing, by double homologous recombination, the wild type gene by the corresponding inactivated gene.

BALB/c mice were intraperitoneally inoculated with approximately 5x10⁶ CFU of each mutant strain or the parental B. ovis PA strain and spleen bacterial counts determined, in five mice per group, at several time points until 11 weeks PI. Parental B. ovis PA reached mean log CFU values in spleen close to 7 from week 2 to week 5 PI and, then, progressively decreased until mean log CFU values of 4.22 at week 11 PI. Mutant strains for omp25 and omp25c gave similar counts to those obtained with the parental strain through the experiment. Difference with previous works reporting attenuation of a B. ovis ∆omp25 mutant strain might be due to differences in the infection dose, the B. ovis strains used and/or the route of inoculation. The ∆omp31 strain showed lower levels of infection than the B. ovis PA parental strain (around 1 log CFU/spleen below) from week 2 to 5 PI but it behaved as the parental strain thereafter.

On the contrary, inactivation of omp25d or omp22 drastically reduced the virulence of B. ovis PA. Thus, the ∆omp25d strain showed a significant reduction of the splenic bacterial counts after 2 weeks PI, and important (0-1 CFU/spleen in 4 out 5 mice) or complete (0 CFU/spleen in all mice) clearance of the mutant from spleens at weeks 3 and 5 PI, respectively. A more dramatic reduction of virulence was observed with the ∆omp22 mutant strain since only 2 out 5 mice were infected at week 1, and complete splenic clearance of the mutant was observed at week 2 PI. Parental and mutant strains were submitted to several tests related to the bacterial outer membrane properties and the results discussed in relation to the residual virulence results obtained in mice.

We have analyzed the humoral immune response induced in rabbits by the seven Brucella spp. outer membrane proteins of the Omp25/Omp31 family (Omp31, Omp31b, Omp25, Omp25b, Omp25c, Omp25d, and Omp22). Rabbits were immunized either with whole recombinant E. coli cells bearing the Brucella spp. OMPs in the outer membrane or with the recombinant proteins purified from the cytoplasm of E. coli. Immune sera were tested in Western blot against the purified recombinant proteins and against parental B. ovis PA, B. ovis PA mutants with the genes of the omp25/omp31 family inactivated (see accompanying poster) and the corresponding complemented mutant strains.

Regarding the immunogenicity in rabbits of the seven purified OMPs, Omp31b and Omp25d were, respectively, the most and less immunogenic proteins, and some serological cross-reactivity between the members of the Omp25/Omp31 family was evidenced in Western blot by using lysates of B. ovis strains or recombinant E. coli with the OMPs located in the outer membrane.

An antibody response against Omp31, Omp31b, Omp25, Omp25b and Omp25c was also evidenced in rabbits immunized with the OMPs located in the outer membrane of recombinant E. coli (a more similar situation to that found in Brucella cells) and cross-reactivity between some of the seven members of the Omp25/Omp31 family was also observed. Omp31b, both the purified protein and when located in the E. coli outer membrane, induced a strong antibody response that intensely cross-reacted in Western blot with Omp31 in B. ovis cells. Accordingly, although taking into account that these results were obtained in a rabbit model, Omp31b might be an interesting protective antigen against B. ovis infections, an aspect currently under evaluation in our laboratory.

Synthesis of Omp31, Omp25, Omp25c and Omp22 was evidenced in B. ovis while Omp25d could not be detected, under our conditions, in this Brucella species. As expected, according to previous studies at the DNA level, absence of both Omp31b and Omp25b in B. ovis was confirmed. Detection of the members of the Omp25/Omp31 family in the other Brucella species will be attempted in the next future.

A pool of sera from rams naturally infected with B. ovis only reacted in Western blot with recombinant purified Omp31. Therefore, Omp31b, Omp25, Omp25b, Omp25c, Omp25d and Omp22 are not expected to be good diagnostic antigens for B. ovis infections and even worse results should be obtained against infections by smooth Brucella strains.

We have previously demonstrated that the gene iivA of Brucella abortus is involved in the virulence of these bacteria. This gene encodes for an 11 kDa basic protein of unknown structure and function which is highly conserved in bacteria. The primary sequence analysis predicts two coiled-coil regions encompassing the C and N terminal halves of the molecule. The C terminal coiled-coil has a higher score than the N terminal one. IivA-self associates as a trimer by its terminal region as shown by light scattering and cross-linking experiments.

In this study, we carried out the characterization of the secondary structure of IivA protein and its behavior in different conditions. Besides, we evaluated the IivA-interaction with phospholipid and its in vitro membrane- fusion activity at neutral and acidic pH. These data could contribute to understand the molecular bases of IivA function and the intimate mechanisms of Brucella virulence.

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 Castelar, Argentina.

Due to the strong cellular and humoral immune response that it elicits, the attenuated Brucella abortus strain 19 (a live vaccine) is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express antigens of other pathogens that require the same type of immune response elicited by Brucella to control the disease they elicit. Recombinant B. abortus S19 expressing RAP1a (a conserved immunogenic antigen) of Babesia bovis were generated. rap1a gene was PCR-amplified as a complete version or without the sequence that encodes for its signal peptide. The amplicons were subcloned under different promoters and signal sequences (lacz and from B. abortus bp26 and omp19 promoters gene) to study diverse subcellular localizations for the heterologous protein. The plasmid stability and the immunogenicity of the heterologous proteins were analyzed. RAP1a as a fusion with the first amino acids of b-galactosidase and as a fusion with the amino terminal sequence of OMP19 resulted in the expression of RAP1 in association to the membrane of Brucella. Even though there was a relatively lower stability of the plasmids containing rap1a, mice inoculated with B. abortus S19pBBRAP or S19pBB19RAP developed specific immune response to RAP1. As a fusion protein with BP26, RAP1a was toxic. The expression of RAP1 in B. abortus S19 is possible and is immunostimulatory in mice.

P10. Purification and biochemical characterization of Brucella suis urease. Araceli Contreras-Rodriguez^1,2, 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.

Urease was purified to homogeneity from Brucella suis 1330. The purification procedure consisted of ion exchange chromatography, and hydrophobic interaction chromatography. The purified enzyme exhibited an isoelectric point of 5. Molecular mass estimated for the native enzyme was 120,000 Da while three subunits in SDS-PAGE were identified: two small subunits, and a major subunit of 65,000 Da. The amino terminal sequence of the large subunit corresponded to the B. suis UreC1 subunit. Urease activity was optimal at pH 7 and 28°C. The enzyme was inhibited by acetohydroxamic acid, hydroxyurea, and thiourea. The UreC1 urease subunit was recognized by sera from patients with acute and chronic brucellosis.

Iron is essential to the survival of Brucella abortus, but the mammalian host represents an extremely iron-restricted environment. In response to this restriction, Brucella synthesizes two catechol-type siderophores, 2, 3-dihydroxybenzoic acid (DHBA) and the more complex siderophore, brucebactin. Both are produced through the enzymatic activities of the products of the dhb operon, and expression of this operon is tightly regulated in response to environmental iron levels.

In addition to iron-responsive regulation, a number of bacteria modulate the expression of their siderophore biosynthesis genes through the activity of AraC-like transcriptional activators. Examples of these activator proteins are YbtA (yersiniabactin A) of Yersinia pestis and AlcR (alcaligin biosynthesis regulator) of Bordetella bronchiseptica. In these organisms, the end product siderophore serves as a co-activator in conjunction with the AraC-like protein and this activation is iron-responsive. Brucella abortus 2308 possesses a homolog of the B. bronchiseptica alcR. An isogenic B. abortus alcR mutant, BEA6, shows decreased catechol siderophore production under iron-deplete conditions, when compared to the parental 2308 strain, and data indicate that the product of this gene, termed DhbR (dihydroxybenzoic acid regulator), functions as an transcriptional activator of the genes required for biosynthesis of the siderophores 2, 3-dihydroxybenzioc acid and brucebactin utilizing the end product siderophore as a co-activator for this activation. Work is currently underway to further define the regulatory role of DhbR in B. abortus.

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, East Carolina University School of Medicine, Greenville, North Carolina 27834.

Iron is essential to the survival of Brucella, but the mammalian host represents an extremely iron-restricted environment. In an effort to circumvent this restriction, Brucella synthesizes two catechol-type siderophores, 2, 3-dihydroxybenzoic acid (DHBA) and the more complex siderophore, brucebactin. In addition, the brucellae possess a number of additional genes encoding proteins used to acquire iron and iron-containing molecules such as heme.

Two genes encoding proteins required for iron acquisition have been previously identified. These genes, bhuA (Brucella heme uptake gene A) and dhbR (dhb operon regulator) are required for the uptake of heme and the regulation of siderophore biosynthesis, respectively, and are divergently transcribed with a 444 base pair intergenic region. Recently, computer analysis has identified a putative small regulatory RNA (sRNA) within this intergenic region, and the presence of this independent transcript was verified by RT PCR. In E. coli, a Fur-regulated small RNA (sRNA), RhyB, serves to control the expression of a number of iron metabolism genes. Current data suggest that this sRNA is regulated in an iron-responsive manner. Work is currently underway to determine the iron-responsive regulator of this sRNA and determine the transcripts controlled by this sRNA in B. abortus.

Vaccines and Inhibitors

Brucellosis is a zoonotic disease of worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. Development of improved vaccine strains against Brucella species for use in animals as well as in humans, must consider the possibility of challenge infection via aerosol. The differences in immune response resulting from such exposure needs to be evaluated to properly determine vaccine efficacy. In this study, we have employed the use of a Madison aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either B. abortus or B. melitensis at varying doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes at a more rapid rate and requires higher infectious doses to obtain infection rates between animals similar to B. abortus. In either case persistence of the organism is prolonged compared to other routes of exposure and it is hypothesized that the lung may serve as a source of chronic infection that would seriously exacerbate human disease due to the absence of the clearing mechanisms documented in the murine model. However, despite these concerns unmarked deletion mutants BA∆asp24 and BM∆asp24 consistently confer superior protection in mice against homologous and heterologous aerosol challenge infection, and should be considered viable candidates as vaccine strains against brucellosis.

Treatment of brucellosis is still far from ideal. Many clinicians have used co-trimoxazole for treating human brucellosis (Daikos et al., 1973; Ariza et al, 1985; Roushan et al, 2006). The aim of the work reported here was to study the application of co-trimoxazole in combination with Lactobacillus spp. for treatment of experimental brucellosis.

Fifty mice were infected intraperitoneally with Brucella melitensis in 0.1 ml of PBS. The mice were divided into 5 equal groups to serve as control and treated groups. The animals were given Lactobacillus salivarius 8g (The patent RK #17182) and/or co-trimoxazole mixed with the feed. The mice were killed at 21 days after infection. Two parameters were used for the characteristic of infectious process: the index of contamination (IC %) and measuring the numbers of Brucella colony forming units (log₁₀ CFU) in mouse spleen.

As it has been reported earlier (Gavrilova, NN, Ratnikova, IA, Grushina, TA, Antibiot Khimioter. 2003;48(2):13-5) Lactobacillus had a suppression factor in vitro not less than 1:10000 with respect to B. melitensis, abortus, suis and Lactobacillus antagonostic activity in vivo was comparable with that of gentamicin.

In this study the regimen containing Lactobacillus plus co-trimoxazole are most effective for treatment of experimental brucellosis. Differences between control (IC100%; log₁₀ CFU 5.12±0.22) and experienced groups (IC20%; log₁₀ CFU 0.7±0.22) were significant at Р<0.05.

The results demonstrate that the Lactobacillus salivarius 8g plus co-trimoxazole regimen had a high efficacy for treatment of human brucellosis.

Acknowledgment. We are indebted to our collaborators (ISTC K-318 project): Gary A. Splitter, Louisa B. Tabatabai and Thomas A. Ficht for their encouragement.

Brucellae produce chronic and often lifelong infections in natural hosts. The persistent nature of these infections is predominately due to the capacity of these bacteria to maintain long-term intracellular residence in host macrophages. Successful antibiotic treatment requires drugs to inhibit brucellae residing within these cells. We aim to develop a method to evaluate drug efficacy for treatment of human brucellosis as part of an NIH drug-screening contract. The Mono Mac 6 cell line is the only human cell line to constitutively express phenotypic and functional features of mature monocytes. We determined that these macrophages maintain the ability to mount an oxidative burst with repeated passaging, and that infection by B. abortus 2308 proceeds without opsonization or centrifugation. MIC values were evaluated using a colorimetric broth microdilution assay based on CLSI standards. Prior to infection, the toxicity of the highest drug concentration to be used was evaluated in uninfected macrophages using the MTT assay. Neither Rifampin, Ciprofloxacin, nor Tetracycline displayed adverse affects on the cells. After an optimal multiplicity of infection was determined (50:1), Mono Mac 6 cells were incubated with B. abortus 2308 for 2 hr at 37°C and 5% CO₂,washed, and treated with Gentamicin (50 µg/ml) to remove extracellular bacteria. One ml portions (1.5 x 10⁵ cfu) were transferred to sterile 24 well plates. Rifampin, Ciprofloxacin, Tetracycline, and DMSO (the solvent control for Rifampin) were added to 8, 4, 1, 0.25, and 0.125 times the MIC. Cells were incubated as before, washed and lysed after 24 hours drug treatment. Lysates were serially diluted and plated for viable cells. MTT assays were performed on infected cells to determine if infection potentiated toxic drug interaction. This protocol will provide a useful method to examine intracellular efficacy of novel antimicrobics against the Brucella species.

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 la Universidad Nacional Autónoma de México Av. Universidad 3000, Ciudad Universitaria, Delegación Coyoacán, CP 04510, México, DF. Telephone. +52 (55) 56225854 -57. Fax. +52 (55) 5622 -5971. ²Escuela de Ciencias Biológicas del Instituto Politécnico Nacional. ³Universidad Autónoma Metropolitana Unidad Iztapalapa.

Cheese made out of raw goat milk could be a potential transmission vehicle of Brucella and other pathogens. Many factors affect the presence and survival of these bacteria in cheese such as heat tolerance, acidity, salt concentration, water activity (Aw) and microorganisms load. At the same time, the manufacture process stages such as the elaboration and storage temperature, the culture initiators, acid production, the brine, other inhibitors, as well as the cheese ripening process can affect the bacterial survival. The aim of this study was to evaluate the survival and viability of B. melitensis when it was inoculated on ripe and fresh (“Panela” style) goat cheeses during different stages of the ripening process. The stages established for fresh cheese were: at the milk reception, milk at 24º C, curd cheese, cutting, serum elimination and cheese as final product. The ripe cheese process was divided in the following stages: milk reception, pasteurization, milk at 24º C, incubation, cutting, immersion in brine (30 minutes), ripening I (5 days), ripening II (15 days) and ripening III (30 days). In every stage, fungi and yeast presence were evaluated as well as Aw and pH. In fresh cheese, as final product 1 x 10⁶CFU/g of B. melitensis was registered. In contrast, 3 x 10⁴ CFU of B. melitensis/g were isolated in the cutting stage of ripe cheese. No bacterium growth was registered in the other stages. Fresh cheese showed a 0.969_Aw and pH 6, while ripe cheese (ripening III) showed 0.527_Aw and pH 5. We conclude that the cheese ripening process is critical for B. melitensis inhibition. In addition, the elaboration process of fresh goat cheese does not ensure the product innocuousness. Therefore, we propose further studies in relation of water activity effect on Brucella melitensis development and survival in goat products.

DTRA Country Presentations

DTRA Presentation 1 – Current Brucellosis Situation in Azerbaijan

Ismail Gasanov¹ and Maqsud Gasimov². ¹Chief Veterinary Officer, Veterinary Services, Baku, Azerbaijan; ²Director, Repubican Anti-Plague Station, Baku, Azerbaijan.

Brucellosis was first registered in Azerbaijan in the late 1930s and continues to be a major public health concern for both humans and animals. From 2000 to 2006, over one million cattle, sheep, and goats were examined each year for brucellosis. There was a higher frequency of infection in sheep and goats. In an effort to control the infection, Azerbaijan vaccinates sheep and goats with the vaccine strain REV-1.

The distribution of human cases is non-uniform with the majority of registered cases in the Big and Small Caucasus and the Khura-Araks areas. Most human infections are farm-related; however, there is an increase in non-farm infections due to an increase in dairy product consumption such as cheese. The seasonal variation in human cases correlates with the goat/sheep birthing and lactation times in the early spring and summer, respectively.

Azerbaijan is introducing a strategic control program that includes:

Improved diagnostic and prophylaxis measures
Introduction of an animal identification system including passport for each animal
Implementation of a regular immunization protocol for cattle, sheep, and goats
Development of a computerized information system to track animal vaccinations and disease patterns
Implementation of a monitoring program that includes a compensation-based animal neutralization plan

DTRA Presentation 2 - Brucellosis in Kazakhstan

DTRA Presentation 3 - Status of Brucellosis in Georgia

Human Brucellosis in Georgia

N. Tsertsvadze, L. Bakanidze, E. Tsertsvadze, Sh. Tsanava, P. Imnadze

National Center for Disease Control and Medical Statistics of Georgia.

Brucellosis is wide spread in Georgia, in animals as well as in humans. However, the distribution of disease varies by region and by species. Infection with B. melitensis is most common in the eastern, northern and southern regions of the country, due to sheep farming traditions in those areas. In western Georgia, cases of B. abortus bovis infection are most common.

For the last several years, registration and counts of domestic animals in Georgia have been incomplete. During Soviet period all cattle belonged to Collective Farms” and were state property. Vaccination of cattle was provided according to the state plan. Upon collapse of the Soviet Union, milk and meat production reverted to the private citizens. The population in rural areas is mainly poor, and cannot afford vaccination of their livestock. Consequently, there has been an increase in the incidence of brucellosis in animals and humans.

According to the official registration, ~200 human brucellosis cases are reported in Georgia annually, almost the same number of cases as reported in the United States. The distribution of human brucellosis cases demonstrates seasonality with increasing transmission in spring, peaking in the summer months and decreasing in fall. The majority of cases are observed in adults (>86%).

Disease diagnosis is largely based on clinical symptoms (e.g.; undulant fever, pain in joints, gastro-intestinal symptoms) and epidemiological data/risk assessment (e.g.; affiliation with dairy/cattle farming, consumption of unpasteurized dairy products). Laboratory diagnosis relies on serological tests. The most commonly performed assay is the Wright and Hedlson agglutination test (cut-off titer was ≥1:160).

Morbidity with Brucellosis in Georgia, 1997-2006

Years	Number of cases in abs. figures	Laboratory confirmed	Children < 14	Prevalence per 100 000 population
1997	63	26	1	1.17
1998	62	2	3	1.15
1999	64	4	6	1.39
2000	70	2	10	1.52
2001	189	28	10	3.3
2002	156	9	19	3.49
2003	106	5	3	2.38
2004	152	-	14	3.47
2005	129	1	14	2.97
2006(9months)	50	3	4	1.16

The official data probably fails to reflect the actual burden of disease in Georgia as disease cases may not be identified and reported to respective agencies. For example, due to financial difficulties infected persons may not present to physicians for medical treatment instead seeking self treatment. Failure to adhere to appropriate treatment protocols frequently results in chronic illness which may lead to long term morbidity. Improved surveillance and access to medical care as well as laboratory based diagnostics will improve clinical outcomes and provide better estimates of the burden of disease due to brucellosis in Georgia.

DTRA Presentation 4 - Brucellosis in Uzbekistan