Location: Infectious Bacterial Diseases Research2015 Annual Report
These studies are focused on developing an understanding of how Leptospira and Treponema species interact with their hosts to establish tissue colonization, infection, and disease manifestation. A combination of genetic, cellular, and immunological methodologies will be used to analyze how the host responds to bacteria and how bacteria respond to the host with the long-term goal of identifying pathways that can be altered to change disease outcome or that can be exploited to induce protective immunity. Objective 1. Characterize the Leptospira host-pathogen interaction using tissue culture and animal model systems to provide information regarding events that occur during infection and enable development of improved diagnostic assays and intervention strategies including effective vaccines. Subobjective 1a - Analyze genetic differences between strains that cause acute vs. persistent infections in animal models. Identify bacterial proteins expressed during infection using in vitro and in vivo models and synthesize these proteins for further analysis. Subobjective 1b – Characterize host responses to infection in model and native hosts and develop improved vaccination strategies. Objective 2. Isolate and identify bacterial species from PDD lesions and determine their role in the formation of lesions using animal models. Subobjective 2a - Identification and isolation of diverse bacterial genera in PDD lesions. Subobjective 2b – Induction of lesion formation.
Objective 1: This objective seeks to identify serovar Hardjo virulence traits and in vivo expressed proteins (Subobjective 1a); determine what proteins induce immunological memory, and develop experimental vaccines that will be tested in hamster and cattle models (Subobjective 1b). We expect these studies will identify genes that influence the outcome of infection, result in standardized hamster models of serovar Hardjo infection, and improve vaccine effectiveness. Subobjective 1a - The genome of serovar Hardjo strain 203 will be sequenced and compared to strain JB197 with the goal of identifying genetic variations that influence the clinical outcome of infection. Serovar Hardjo proteins expressed during infection will be identified and selected proteins will be cloned and expressed to enable further characterization. Subobjective 1b - Changes in patterns of transcription by bovine leukocytes in response to serovar Hardjo will be characterized. Low passage L. borgpetersonii will be used as one of the sources of antigenic proteins in assays in an effort to characterize proteins most likely to be expressed during in vivo infection. The results of these experiments will be combined with results from Subobjective 1a to identify proteins that simulate immunological memory. Experimental vaccines including these proteins will be tested in hamster and cattle infection models. A goal of these studies is to develop effective serovar Hardjo vaccines. Objective 2: Recent attempts to induce lesion formation by PDD spirochete cultures have been unsuccessful. Although spirochetes injected into the heel bulb survive at or near the injection site and can be recovered from tissue in pure culture, the tissue lacks observable pathology. A critical first step in testing our hypothesis is to identify and isolate different bacterial genera present in PDD lesions (Subobjective 2a). Our hypothesis will be tested through the use of animal models. Bacteria, as pure strains, or mixtures of pure strains, will be injected into mice to assess their capacity to induce lesion formation. Bacteria that contribute to lesion formation in mice will be used to inoculate cattle to assess their capacity to replicate PDD lesion formation (Subobjective 2b). Subobjective 2a - Diverse bacterial genera present in PDD lesions will be identified by 16S rRNA gene (RRS)-based phylotyping. RRS sequence variation is the most common basis for differentiating bacterial genera and this typing method is well suited to high throughput sequence analysis resulting in detailed analysis of the genera present in complex bacterial mixtures. Lesions will also be used as source material for bacteriological culture, resulting in isolation and characterization of diverse bacterial strains present in PDD lesions. Subobjective 2b - Pure bacterial cultures derived from PDD lesions or mixtures of pure cultures will be injected subcutaneously or intradermally to assess bacterial survival in tissue and the capacity of these bacteria to form lesions.
In support of Objective 1 to develop and improve vaccines against leptospirosis, immunologic responses to two commercially available leptospirosis vaccines and an experimental vaccine were evaluated in cattle. Immune responses were characterized after vaccination and also after experimental challenge. Data analysis is ongoing, including characterization of cellular specific immune responses using new high-throughput assays. To understand the factors that contribute to the pathogenesis of leptospirosis in Objective 1, a laboratory model of persistent leptospirosis was developed that emulates chronic infection observed in cattle. Rats are a natural reservoir host of leptospirosis and studies are characterizing colonization, shedding, transmission and in vivo responses of Leptospira in the rat host and immune responses in chronic infection. In comparison to the hamster model, the rat model will enhance our ability to evaluate pathogen and host responses due to the increased size and feasibility of the host, increased urine output (for collection of in vivo derived leptospires) and availability of reagents to evaluate immune responses. In support of objective 2, ongoing work isolating and characterizing bacteria which colonize digital dermatitis lesions in cattle continues. Isolates will be used to develop experimental models which replicate disease in cattle. The project is collaborating with university scientists to characterize and understand an emerging a digital dermatitis-like disease in wild elk in Washington and Oregon. New collaborations were established to initiate a model of digital dermatitis model in sheep.
1. Models of lepspirosis developed to understand Host-pathogen interaction. Leptospirosis is an insidious disease of economic importance that causes reproductive losses in livestock and also causes clinical disease in over 500,000 humans globally each year. Laboratory models are valuable for studies of disease pathogenesis as infections are more reproducible and costs are lower. Two novel approaches were developed to provide acquisition of in vivo derived leptospires for proteomic and/or transcriptomic studies. One newly developed laboratory model allows for collection of leptospires directly from infected kidneys via urine. In the second model, leptospires are cultured within a dialysis membrane chamber (DMC) that is implanted in the peritoneal cavity of a rat. Both models allow isolation of leptospires which have grown in the presence of mammalian host signals, which duplicates in vivo conditions that leptospires encounter during infection in other hosts. This work will allow identification of genes or proteins that are only expressed when leptospires are under in vivo conditions and may facilitate development of new diagnostics or more protective vaccines.
Polle, F., Storey, E., Eades, S., Alt, D.P., Hornsby, R.L., Zuerner, R., Carter, R. 2014. Role of intraocular Leptospira infections in the pathogenesis of equine recurrent uveitis in the Southern United States. Journal of Equine Veterinary Science. 34(11-12):1300-1306.
Wu, Q., Prager, K.C., Goldstein, T., Alt, D.P., Galloway, R.L., Zuerner, R.L., Lloyd-Smith, J.O., Schwacke, L. 2014. Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions. Diseases of Aquatic Organisms. 110(3):165-172.
Prager, K.C., Alt, D.P., Buhnerkempe, M.G., Greig, D.J., Galloway, R.L., Wu, Q., Gulland, F.M., Lloyd-Smith, J.O. 2015. Antibiotic efficacy in eliminating leptospiruria in California sea lions (Zalophus californianus) stranding with leptospirosis. Aquatic Mammals. 41(2):203-212.
Wilson-Welder, J.H., Alt, D.P., Nally, J.E. 2015. The etiology of digital dermatitis in ruminants: recent perspectives. Veterinary Medicine: Research and Reports. 2015(6):155-164.