2008 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: Changes in gene and protein expression by BoAEC to infection by Leptospira were characterized. There is a temporal change in expression of selected cytokine genes during interaction of bacteria with host cells. Current studies are focused on identifying signaling mechanisms associated with this temporal change in host response to infection. Transcriptional changes in response to iron and nutrient starvation were studied in Leptospira using microarrays. Analysis of these responses is important to develop an understanding of how these bacteria survive transmission and infection cycles. Leptospirosis in marine mammals presents a potential source for zoonotic transmission. We continued development of molecular diagnostic techniques to improve detection of Leptospira DNA from clinical samples obtained from infected California sea lions. Through a collaboration with Monash University, Melbourne, Australia, the genome sequence of Leptospira biflexa, a non-pathogenic saprophyte was deduced. Comparative analysis of Leptospira genomes resulted in identification of genes unique to pathogenic Leptospira. Comparative sequence analysis of a large operon was completed from Leptospira resulting in development of reference sequences for secY, a target for diagnostic PCR. This study resulted in a new method for accurately determining Leptospira species based on a small, easily amplified genomic target. A small animal model was developed enabling analysis of chronic and acute Leptospira borgpetersenii serovar Hardjo infections. Completion of a duration of immunity to serovar Hardjo study provided new data on the development of acquired immunity. Expression of selected bacterial and host proteins during infection is being analyzed by immunofluorescence microscopy to correlate in vitro studies to experimental infection of animals. Refinement of these in vitro and in vivo models is providing new information on the progression of infection and corresponding host responses leading to identification of potential vaccine candidates. Papillomatous digital dermatitis (PDD) studies: Analysis of bacteria. By altering media composition, we have knowledge of the nutritional requirements of PDD spirochetes that will allow characterization of alterations in protein expression by these bacteria. By analyzing responses to different growth environments we gain new understanding of disease transmission. A mixed strain infection model showed strain differences in eliciting a humoral response despite equal representation in the inoculum. Lesions produced with a mixed strain inoculum were reduced in comparison to the single strain inoculum indicating that treponemes likely interact within the lesion site in a manner that affects lesion development. This project addresses NP103, component 3, problem statement 3B. New information on characterization of these bacteria, survival in transmission and infection cycles, and genetic characterization addresses Performance measure 3.2.1. Development of improved diagnostic methods and vaccines addresses Performance measure 3.2.3. 4.Accomplishments 1. Development of a small animal model to study Leptospira borgpetersenii serovar Hardjo infections. Small animal models for studying Leptospira borgpetersenii serovar Hardjo infections were needed to accelerate analysis of the infection process. Golden Syrian hamsters have been used extensively with other pathogenic Leptospira, but have not been successfully or carefully evaluated for serovar Hardjo. In FY08 we completed studies that establish hamster models for both acute and chronic infection with serovar Hardjo. This information is useful to the scientific community for characterizing serovar Hardjo infections, and biologics firms and regulatory agencies for testing and evaluation of vaccines developed for protection against serovar Hardjo infection. This accomplishment aligns with the “countermeasures to prevent and control zoonotic diseases” action plan, of NP 103, componenent 3, problem statement 3B. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 1 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Cameron, C.E., Zuerner, R.L., Raverty, S., Colegrove, K.M., Norman, S., Lambourn, D.M., Jeffries, S.J., Gulland, F.M. 2008. Detection of Pathogenic Leptospira Bacteria in Pinniped Populations via PCR and Identification of a Source of Transmission for Zoonotic Leptospirosis in the Marine Environment. Journal of Clinical Microbiology. 46(5):1728-1733. Elliott, M.K., Alt, D.P., Zuerner, R.L. 2007. Lesion Formation and Antibody Response Induced by Papillomatous Digital Dermatitis (PDD)-associated spirochetes in a Murine Abscess Model. Infection and Immunity. 75(9):4400-4408. Gay, C.G., Zuerner, R., Bannantine, J.P., Lillehoj, H.S., Zhu, J., Green, R.D., Pastoret, P.P. 2007. Genomics and vaccine development. OIE Rev. Sci Tech. 26:49-67. Picardeau, M., Bulach, D.M., Bouchier, C., Zuerner, R.L., Zidane, N., Wilson, P.J., Creno, S., Kuczek, E.S., Bommezzadri, S., Davis, J.C., Mcgrath, A., Roche, D., Johnson, M.J., Boursaux-Eude, C., Seemann, T., Rouy, Z., Coppel, R.L., Rood, J.I., Lajus, A., Davies, J.K., Medigue, C., Adler, B. 2008. Genome Sequence of the Saprophyte Leptospira Biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis. PLoS One. 3(2):e1607. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0001607. Stanton, T.B., Humphrey, S.B., Sharma, V.K., Zuerner, R.L. 2008. Collateral effects of antibiotics - carbadox and metronidazole induce VSH-1 and facilitate gene transfer among Brachyspira hyodysenteriae strains. Applied and Environmental Microbiology. 74(10):2950-2956. Zuerner, R.L., Heidari, M., Elliott, M.K., Alt, D.P., Neill, J.D. 2007. Papillomatous Digital Dermatitis Spirochetes Suppress the Bovine Macrophage Innate Immune Response. Veterinary Microbiology. 125(2007):256-264.