Location: Infectious Bacterial Diseases Research2016 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.
Bacteria and bacterial RNA have been isolated under different culture conditions and growth environments including isolation of organisms naturally shed from infected hosts. Ongoing work will compare gene and protein expression of bacteria from different conditions. Data will be used to guide future studies developing new vaccines and diagnostic assays. Further characterization of rat and bovine in vivo models of chronic Leptospirosis infection was completed. Antibody and cellular immune responses were characterized during chronic (12+ week) infection. Rats remain asymptomatic but demonstrate strong cellular and moderate humoral responses after infection. In infected cattle, progress continues on analysis of samples collected from cattle experimentally infected with Leptospira, and manuscript(s) are in preparation detailing novel host responses developed during infection. Treponema species from digital dermatitis lesions were characterized using microbiologic techniques and growth conditions for these aerobic bacteria were optimized. Data demonstrates that CD8+ T cells in cattle with digital dermatitis demonstrate robust proliferative responses to Treponema antigens. Samples obtained from cases of digital dermatitis from multiple geographic locations and different stages of lesion development were sequenced to characterize bacterial populations and analysis of the data was initiated. The analysis will identify core bacteria responsible for digital dermatitis lesions. A reproducible model of digital dermatitis was developed in sheep that will allow characterization of disease pathophysiology and allow scientific evaluation of vaccination or intervention strategies. This is the final report for project 5030-32000-107-00D. Substantial results were made over the last 5 years on the two objectives. Under objective 1, studies characterizing host-pathogen interactions in rat and bovine models of chronic leptospirosis infection were completed. These studies were used to evaluate host immune responses after vaccination or infection, and characterize surface proteins of bacteria shed from infected hosts. In objective 2, treponeme bacteria isolated from digital dermatitis lesions were characterized by molecular and biochemical methods and isolates were used to develop a reproducible model in sheep that can be used for understanding lesion development or developing efficacious vaccines. The genome of Leptospira borgpetersenii serovar Hardjo strain 203, a strain associated with chronic infection, was completed and compared to the genome of a Leptospira strain that causes acute infection to identify genetic mechanisms associated with differences in in vivo infection. Collaborative work characterized treponeme bacteria in digital dermatitis-like lesions that cause lameness in wild elk. The project also sequenced and assembled the genome of Leptospira alstonii, a new pathogenic spirochete.
1. Inbred rats are an in vivo model of chronic Leptospira infection. Leptospirosis is an insidious disease that causes acute infection in some hosts, and asymptomatic infection with bacterial shedding in maintenance hosts. Understanding why the disease persists in maintenance hosts will be beneficial for developing intervention strategies to prevent disease transmission. In a series of studies, ARS scientists in Ames, Iowa demonstrated that rats can serve as a model of chronic bovine leptospirosis. Rats remain asymptomatic, but shed bacteria and develop cellular immune responses to leptospiral antigens. This model allows evaluation of immune responses in chronic infection, and also allows characterization of bacterial adaptation to long-term colonization in vivo. This data has provided scientific data on how Leptospira bacteria evade immune detection that may be useful for vaccine and/or diagnostic development.
2. First development of a reproducible digital dermatitis model. A reproducible model of digital dermatitis was needed in order to develop scientific data on disease pathogenesis, bacterial spp. involved in lesion development, and host inflammatory and/or immunologic responses to infection. In a series of experiments, ARS scientists at Ames, Iowa used material from cattle lesions to infect the hoof of sheep and induce characteristic lesions of digital dermatitis. After experimental infection, the lesion initially presents as ulceration in the heel bulb and interdigital space, and induces histological lesions in the epidermis that are similar to characteristics of lesions in cattle. Experimentally infected sheep also produce antibodies to infecting Treponemes. Sheep are preferable over developing a model in cattle due to their small size, ease of handling, and reduced cost to feed and house. This model will be used in future studies to answer specific scientific questions related to etiology, pathogenesis and intervention strategies for digital dermatitis.
3. Completed genome sequence of Leptospira alstonii serovar Room22. Whole genome sequences provide important scientific information on the bacterial strain and facilitate new research approaches. ARS scientists at Ames, Iowa sequenced and assembled the genomic sequence of Leptospira alstonii serovar Room22, a unique spirochete serovar. This L. alstonii isolate was the first to be recovered from a mammalian host, and represents a new serovar of pathogenic leptospires. This data will be used to compare to other leptospira sequences to understand molecular differences that may influence virulence and host preference that may provide targets for development of novel vaccines or diagnostics.