2012 Annual Report
1a.Objectives (from AD-416):
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.
1b.Approach (from AD-416):
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.
Domestic livestock are ubiquitously infected with Spirochetes which can cause reproductive losses, renal disease, and other clinical syndromes. Specific spirochete organisms have been found to localize in lesions of bovine digital dermatitis (BDD); a clinical syndrome being increasingly reported particularly in dairies. Spirochetes have a unique pathophysiology in that they can remain endemic within affected herds and significantly impact production. The current focus of the research group is on Leptospira borgpetersenii serovar Hardjo and characterization of the treponema-bacteria complex associated with BDD. In order to advance knowledge of these diseases, current pursuits include studies to evaluate host-pathogen interactions and characterization of the polymicrobial cause of BDD. Work has continued with the small animal model of leptospirosis to evaluate alternate routes of inoculation and key steps in development of chronic versus acute disease, specifically host responses to different serovars were evaluated. Both innate and adaptive immune responses were evaluated as current literature indicates a link between these two arms of the immune system in effective bacterial clearance. This information will be beneficial in helping to develop vaccines targeting specific immune pathways which elicit greater protection. Work on sequencing of several spirochetes is progressing and will allow discovery and characterization of key bacterial proteins involved in host interaction and development of disease. Understanding of these factors will enable development of better diagnostic tools and more efficacious therapeutics. The project has continued to support work from Louisiana State University School of Veterinary Medicine; the Marine Mammal Center; University of California, Los Angeles; the National Park Service and various other academic, private and public organizations. Sample submissions have resulted in additional isolates from horses exhibiting uveitis, a common sequela of leptospirosis in horses, which can lead to blindness, and also from protected wildlife species (California Sea Lions and Channel Island foxes and spotted skunks). Continued monitoring of leptospirosis in these and other non-cattle populations provide information on leptospiral prevalence and allow better responses to potential outbreaks by non-typical serovars. Two specific cooperative agreements were established to evaluate prevalence, seroreactivity, and bacterial species present in BDD in two small dairy herds (University of Minnesota and South Dakota State University). These collaborations will provide samples and materials key to research activities for evaluation of the treponeme-bacterial complex involved in production of BDD lesions over the next several years.
Selection of targets for vaccine evaluation. Analysis of sequence data from available leptospiral genomes was used to select proteins with characteristics indicating potential as vaccine targets. Several of these proteins were cloned for use in analysis of expression in the host and for use in recombinant vaccine evaluation. In collaboration with Iowa State University, ARS scientists at Ames, Iowa, selected a particular protein with potential for enzymatic function. A recombinant protein was produced and its enzymatic activity and expression during natural infection were characterized. This protein may also be a target for development of more protective vaccines. Improved vaccines will decrease impacts on animal production due to disease and help reduce the risk to workers in the animal industry.
Evaluation of early immune response. One early response in innate immunity is the development of neutrophil extracellular traps (NETs) which can trap and kill bacterial invaders. In a series of experiments, the ability of Leptospira organisms to induce development of NETs was characterized, and the ability of NETs to kill the bacteria was determined. Leptospira do induce formation of NETs but surprisingly, the NETs do not appear to have an effect on the leptospira. Basic knowledge of the early pathogenesis of leptospiral infection will allow us to more effectively direct host responses to resist infection.
Characterization of Treponeme isolated from Bovine Digital Dermatitis. Different treponemes have been isolated from lesions of bovine digital dermatitis (BDD), including most commonly Treponema (T) phagedenis-like spirochetes. To understand the possible role of T phagedenis-like spirochetes in BDD, ARS scientists in Ames, Iowa, characterized T phagedenis-like strain 4A by genomic, bacteriologic, and biochemical procedures. The resulting information on structure, growth and enzymatic activities will provide a baseline for comparison with other isolates from cases of BDD. Characterization of this isolate will produce information which may explain the role of this organism in development of BDD.
Evaluation of protein expression in vivo. Leptospirosis in cattle caused by Leptospira borgpetersenii serovar Hardjo can have a significant impact on production and serves as a potential health threat to workers in the animal industry. Monoclonal antibodies against leptospiral outer membrane proteins were used to probe tissues of infected animals at different stages of infection to determine if the proteins were expressed during infection. Several of the outer membrane proteins were expressed during all stages of infection, both early in development of disease and later after development of chronic infection, whereas others were not detected during infection. This work will identify Leptospira proteins expressed in the host as potential targets for vaccine development. Effective vaccines will help diminish negative impacts on animal production and decrease the threat to the health of workers in the animal industry.
Eshghi, A., Pinne, M., Haake, D.A., Zuerner, R.L., Frank, A.T., Cameron, C.E. 2012. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32. Microbiology. 158(Pt. 3):622-635.