2007 Annual Report 1a.Objectives (from AD-416) This research is designed to obtain information leading to a detailed understanding of how two groups of spirochetes interact with the host leading to tissue colonization, infection, and the manifestation of disease. The overarching goal of this project is to identify specific steps during bacterial infection that can be disrupted or otherwise altered to gain protective immunity in livestock. To achieve this goal we will use a combination of genetic, cellular, and immunological approaches to analyze how the host responds to the presence of bacteria, identify genes expressed by bacteria during interactions with host cells, and test protein products for their potential to induce protective immunity enabling animals to resist infection. This project has three specific objectives: (1) Characterize host-pathogen interactions to identify pathogenic mechanisms and detect host cell changes during experimental infection. (2) Genetic characterization of pathogenic spirochetes. (3) Evaluation of immune response to spirochete antigens. 1b.Approach (from AD-416) Objective 1 will use in vitro methods to detail various aspects of the host response to spirochete infection using a controlled environment. These studies will help develop assay techniques and identify key markers that will be used to evaluate the host response during infection of whole animals, during fulfillment of Objective 3. Objective 2 will use a genomics-based approach to identify bacterial antigens, adhesins, and potential virulence factors to detail the processes by which pathogenic spirochetes establish infections, invade tissue, and evade clearance mechanisms of the host. Objective 3 brings research under the first two objectives together to study host-parasite interactions in animals, with the goal of identifying antigens that induce protective immunity (Leptospira) or develop model systems for future whole-animal studies (PDD spirochetes). 3.Progress Report Leptospirosis studies. Host-Leptospira interactions were analyzed in vitro by measuring changes in transcription of selected bovine aortic endothelial cell genes by real-time reverse transcriptase polymerase chain reaction (RT PCR) assays. Leptospira induce transcription of a broad array of proinflammatory cytokine genes when they attach to endothelial cells. The inflammation response may promote tissue damage leading to hemorrhage and organ failure during infection. Under NFCA 3625-32000-081-01N, Leptospira interrogans transcriptional changes in response to alterations in osmolarity and temperature were measured using microarrays. The findings of these studies are consistent with L. interrogans efficiently sensing and adapting to changes in its environment. Current studies are focused on comparing how L. borgpetersenii and L. interrogans respond to stress to determine if impairment of environmental sensing functions is responsible for serovar Hardjo being limited to a transmission cycle requiring direct contact between animals. To study protein expression during leptospirosis, several fusion proteins were expressed and immune sera prepared to selected Leptospira proteins. Immune sera were used to detect proteins in tissue during experimental infection of hamsters. These data have provided new information on tissue invasion and expression of Leptospira proteins in vivo. Data from these studies are being used to help select proteins that will be tested as vaccine candidates. In addition, infection studies in cattle were conducted to analyze various immune parameters that contribute to protective immunity during serovar Hardjo infection. Papillomatous digital dermatitis (PDD) studies. We completed a study characterizing the transcriptional response of an established bovine macrophage cell line (BoMac) to PDD Treponema antigens. In this study, Serial Analysis of Gene Expression (SAGE) and real-time RT PCR were used to measure changes in host cell transcription in response to bacterial cellular components. The findings of this study suggest that Treponema may impair innate immune and wound repair functions of macrophages thereby enabling the bacteria to establish infection and promote lesion development. This study helped identify genes that may be useful to follow progress of experimental lesion formation in a cattle model. Due to the success of adapting a murine abscess model to study pathogenesis of PDD spirochetes, we tested experimental infection models in cattle. Although these studies have not resulted in lesion or abscess formation, cattle develop antibody to PDD spirochetes and demonstrate a memory response upon secondary infection. This information is being used to identify bacterial antigens recognized during experimental infection. Extensive progress was made testing PDD spirochete growth under different environmental conditions and using various media components. As a result, we have improved bacterial isolation techniques, and additional spirochete strains were isolated from PDD lesions during this FY. Data from these studies have established a basis for developing a semi-defined medium for bacterial culture. 4.Accomplishments Improved bacterial culture of PDD spirochetes. PDD is the most common cause of lameness in dairy cattle and spirochetes isolated from PDD lesions have been isolated and grown in a very complex medium. Thus, the nutritional requirements of PDD spirochetes are poorly defined and conditions for optimized growth are unknown. Individual media constituents and various environmental conditions (temperature and pH) were tested for their contribution to optimal growth of the bacteria. These studies led to a refined culture medium that is less expensive to prepare, and improved conditions for growth. Improved growth and isolation methods enable more bacterial isolates to be obtained from PDD lesions. This advance will help us determine what pathogenic agent causes PDD. This will greatly help cattle producers affected by PDD. This accomplishment aligns with National Program 103, Animal Heath, "Strategic Goal 4: Enhance protection and safety of the Nation’s Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of papillomatous digital dermatitis. Induction of VSH-1 transcription. Brachyspira hyodysenteriae is the cause of swine dysentery, a serious infection of pigs. VSH-1, a virus-like agent found in B. hyodysenteriae that may have the ability to transfer genes to other species of Brachyspira. Our findings suggest that hydrogen peroxide may be a natural inducer of VSH-1 mediated gene transfer because it induces the virus to take up genes from the bacteria and deliver them to another bacteria. VSH-1 mediated gene transfer may contribute to exchange of virulence traits or antibiotic resistance genes, thereby facilitating propagation of disease. This work completes portions of the project that included analysis of intestinal spirochetes. This knowledge will benefit other scientists. This accomplishment aligns with National Program 103, Animal Heath, “Strategic Goal 4: Enhance protection and safety of the Nation’s Agriculture and Food Supply”, “Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks”, as it relates to intestinal spirochetosis and swine dysentery.  5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 3 Number of active CRADAs and MTAs 8 Review Publications Matsunaga, J., Wernied, K., Zuerner, R.L., Frank, A.T., Haake, D. 2006. Lipl45b is a Novel, Surface-exposed Lipoprotein Expressed during Leptospiral Dissemination in the Mammalian Host. Microbiology. 152:3777-3786. Matsunaga, J., Lo, M., Bulach, D.M., Zuerner, R.L., Adler, B., Haake, D.A. 2007. Response of Leptospira interrogans to Physiologic Osmolarity: Relevance in Signaling the Environment-to-Host Transition. Infection and Immunity. 1175(6):2864-2874. Zuerner, R.L., Bulach, D.M., Seemann, T., Coppel, R.L., Adler, B. 2006. Comparative Analysis of Pathogenic Leptospira Genomes. In: Cabello, F.C., et al., editors. Molecular Biology of Spirochetes. Amsterdam, Holland: IOS Press. p. 101-115. Bulach, D., Zuerner, R.L., Wilson, P., Seemann, T., Mcgrath, A., Cullen, P., Davies, J., Kuczek, E., Alt, D.P., Peterson-Burch, B. 2006. Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proceedings of the National Academy of Sciences. 103(39):14560-14565. Matson, E.G., Zuerner, R.L., Stanton, T.B. 2007. Induction and transcription of VSH-1, a prophage-like, gene transfer agent of Brachyspira hyodysenteriae. Anaerobe. 13(2):89-97. Lo, M., Bulach, D.M., Powell, D.R., Haake, D.A., Matsunaga, J., Paustian, M., Zuerner, R.L., Adler, B. 2006. Effects of Temperature on Gene Expression Patterns in Leptospira interrogans Serovar Lai as Assessed by Whole-Genome Microarrays. Infection and Immunity. 74(10):5848-5859.