Location: Infectious Bacterial Diseases Research2014 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 which is to identify virulence traits of leptospirosis, and objective 2 to characterize bacteria associated with papillomatous digital dermatitis (PDD), work has proceeded in sequencing and annotating the genomes of two Leptospira strains and a Treponema strain. Despite use of numerous molecular techniques and currently available analytic tools, final completion and annotation of the Treponema and one Leptospira strain is not complete due to large numbers of repeat elements within each genome. New tools (software, analysis packages, molecular techniques) are being utilized in attempts to complete these genomes. However, both of the genomes are substantially assembled and annotated. These genomes are available for researchers to search for specific genes, proteins, or virulence factors of interest. In support of the goal in 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. T-cell responses were characterized after vaccination and also after experimental challenge. The ability of the vaccines to prevent infection and shedding of Leptospira organisms was characterized including comparison of detection of bacteria using new molecular techniques as compared to detection using standard culture methods. To understand the factors that contribute to the pathogenesis of leptospirosis in Objective 1, a small animal model of leptospirosis (hamsters) was used to characterize chronic and acute infections using different serovars. Immune responses were characterized in early stages of infection in an effort to identify critical events which influence whether the infection is quickly resolved or becomes chronic. In support of Objective 2, work to identify bacteria which colonize digital dermatitis lesions in cattle was continued. These isolates will be used to develop experimental models which replicate the disease in cattle. New collaborations were established with scientists working on a digital dermatitis-like disease in wild elk in Washington State which resulted in new bacterial isolates and epidemiologic information on this disease under field conditions.
1. Evaluation of a novel adjuvant for an improved leptospirosis vaccine. Leptospirosis is a leading cause of reproductive failure (abortions, stillbirths, low conception, and weak calves) in beef and dairy cattle. Chronically infected cattle can be reservoirs of infection for other cattle, other animals, and humans. Although serovar Hardjo is the most common isolate from cattle in the U.S., other serovars can cause infection and current vaccines do not provide sterile immunity (animals can shed Leptospirosis after infection). In an effort to develop vaccines with greater efficacy, ARS researchers compared immunologic responses of cattle after inoculation with a new vaccine candidate or commercially available vaccines. Cattle receiving the new vaccine candidate had increased cell-mediated immune responses that were detected earlier than cattle vaccinated with commercially available vaccines. Data suggests that immune responses to the new vaccine candidate may induce immunity to multiple leptospira serovars. Development of new vaccine candidates with greater efficacy will provide economic benefits to livestock owners and public health benefits by preventing disease transmission to humans.
2. Isolation and identification of bacterial isolates from digital dermatitis lesions. Digital dermatitis is an infectious hoof disease that is the leading cause of lameness in dairy cattle, but also occurs in beef cattle. This disease causes millions of dollars in economic losses due to decreased production, increased culling, and treatment costs. The disease is thought to be polymicrobial, but specific knowledge on which bacteria or other contributing factors cause the disease remains unknown. Large numbers of Treponema bacteria are usually found in the lesions, but by themselves will not recreate the lesion. In an effort to understand this complex disease, ARS researchers isolated and identified aerobic and anaerobic bacteria from digital dermatitis lesion and from normal hoof skin of cattle with the goal of using these isolates to develop an experimental model which replicates the disease. This work will assist other researchers in understanding the complex etiology causing digital dermatitis lesions, and will be beneficial in developing strategies to prevent this disease and reduce economic losses to livestock producers.
3. Characterized infection in chronic and acute Leptospira infections. Leptospirosis includes more than 200 serovars and is the most common zoonotic infection worldwide. Disease severity is dependent upon which mammalian host is infected and the serovar causing the infection. In an effort to understand why some animals are infected for a short period of time whereas others develop chronic infections, a laboratory animal model was developed to characterize infection with different Leptospira strains. Bacterial numbers, clinical signs, duration of infection, and host immune responses were characterized. In a serovar which causes chronic infection, Leptospira were found in the brain without inflammation in a manner that mimics human infection. This is of great interest as it documents persistence of the bacteria in an immune-privileged site which may allow the organism to elude host immune responses and allow reemergence at a later time. By studying the pathogenesis of Leptospira in laboratory animals, new insights into the infection process may be made that facilitate development of intervention strategies for animal and human hosts.
Wilson-Welder, J.H., Elliott, M.K., Zuerner, R.L., Bayles, D.O., Alt, D.P., Stanton, T.B. 2013. Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion. BMC Microbiology. 13:280. Available: http://www.biomedcentral.com/content/pdf/1471-2180-13-280.pdf.