Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- Wilson-Welder, J.H., Alt, D.P., Nally, J.E. 2015. Digital dermatitis in cattle: current bacterial and immunological findings. Animals. 5:114-1135. doi: 10.3390/ani5040400.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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.
Impacts
(N/A)
Publications
- 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.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- 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:
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. The two objectives of this research project are to characterize the pathogenesis of Leptospira borgpetersenii serovar Hardjo and understand the role of the treponema-bacteria complex associated with bovine digital dermatitis (DD). To advance knowledge of these diseases, current studies are evaluating leptospiral host-pathogen interactions and characterizing the polymicrobial agents causing digital dermatitis. A small animal model of leptospirosis (hamsters) has been used to characterize differences between chronic and acute infections and host responses to different serovars. Both innate and adaptive immune responses were evaluated as current literature indicates a link with effective bacterial clearance and vaccine development. Studies in the hamster model of chronic and acute leptospirosis were conducted and gene expression was characterized including genes encompassing innate and adaptive immunity, signaling, and immune activation. This information will assist in developing new vaccines that target specific immune pathways and elicit greater protection. Additionally we have evaluated the use of a quantitative PCR for leptospira in blood/tissues that will eventually be useful for evaluating vaccine efficacy in cattle after challenge. The role of bovine neutrophils in the response to leptospira infection was characterized to evaluate bacterial survival in the presence of neutrophils and neutrophil gene expression after incubation. The project has continued collaborations to sequence and annotate the genomes of several spirochetal pathogens including two Leptospira strains and a Treponema strain isolated from bovine digital dermatitis. Ongoing work is focused on completing the assembly of these genomes. This work will allow for greater characterization of bacterial gene or protein expression as it relates to host colonization and disease. The project continues efforts to clone and express immunogenic proteins as previously identified by genomic analysis but is hampered by the observation that most of the recombinant proteins are insoluble. This creates difficulty in downstream purification (removal of endotoxin to acceptable levels for use in a vaccine) and makes the proteins unsuitable for use as vaccine candidates in vivo. Collaborations were established and samples obtained from herds with high incidences of digital dermatitis. Bacterial culture methods were developed to enhance growth and facilitate isolation of anaerobes and treponema strains from samples for further characterization. A PCR based method for differentiating Treponema species has also been developed. Accomplishments 01 DNA sequencing of bacterial genomes provides useful information for agricultural research. Infection with the bacteria, leptospira, can lead to abortions, stillbirth and decreased animal productivity. ARS scientists in Ames, Iowa, used genomic techniques to sequence and assemble the genome of Leptospira borgpetersenii serovar Hardjo strain 203. Completion of this genome increases understanding of genes that contribute to the infection and pathogenesis of leptospirosis in natural hosts, and facilitates development of new and more effective vaccines. This work will be of benefit to researchers working with leptospira or related bacteria, and should also lead to improvements in controlling leptospirosis for cattle producers. 02 Neutrophils, a type of immune cell, do not control Leptospira infection in cattle. Infection with Leptospira can result in abortions, stillbirth and decreased productivity in cattle. Work in humans has suggested that neutrophils can trap leptospira as an important part of innate immunity and help in controlling leptospira infections. To determine if this also occurs in natural hosts of leptospira, ARS researchers in Ames, Iowa, characterized the role of bovine neutrophils in creating neutrophil nets for trapping and killing leptospira. Although bovine neutrophils did form nets in which leptospira bacteria could be found, there appeared to be no bactericidal effects in reducing bacteria viability or survival. This work suggests that neutrophil nets do not play a significant role in control of Leptospira infection in cattle and may contribute to chronic colonization. This work will be of interest to spirochete researchers by providing basic knowledge on immunologic responses to leptospirosis and to cattle producers in understanding how the bacteria evades the host�s innate response; thereby, leading to design of more effective vaccines. 03 Improved isolation of bacteria from cattle with observable sores near the hoof, digital dermatitis lesions. The bacteria, Treponema, are believed to contribute to the development of sores located near the hoof in cattle. These sores can be associated with lameness, resulting in decreased productivity and potentially removal from the herd. However, these bacteria are very difficult to grow and isolate. In an effort to elucidate the mechanisms leading to development of digital dermatitis lesions, ARS researchers at Ames, Iowa, developed enhanced culture techniques and PCR assays to facilitate isolation and identification of Treponema from lesion materials. These enhanced techniques have increased the numbers of isolates made from lesion material. More efficient isolation of bacteria contributing to lesion development will be beneficial in understanding the pathogenesis of digital dermatitis lesions. This work will benefit researchers working on Treponema and should also benefit cattle producers by contributing to development of control measures to prevent digital dermatitis and associated production losses.
Impacts
(N/A)
Publications
- Huntimer, L., Wilson-Welder, J.H., Ross, K., Carrillo-Conde, B., Pruisner, L., Wang, C., Narasimhan, B., Wannemuehler, M.J., Ramer-Tait, A.E. 2013. Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses. Biomedical Materials Research. 101(1):91-98.
- Prager, K.C., Greig, D.J., Alt, D.P., Galloway, R.L., Hornsby, R.L., Palmer, L.J., Soper, J., Wu, Q., Zuerner, R.L., Gulland, F.M., Lloyd-Smith, J.O. 2013. Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus). Veterinary Microbiology. 164(1-2):177-183.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. Accomplishments 01 Evaluation of protein expression in vivo. Leptospirosis in cattle cause by Leptospira borgpetersenii serovar Hardjo can have a significant impac on production and serves as a potential health threat to workers in the animal industry. Monoclonal antibodies against leptospiral outer membran 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 the host as potential targets for vaccine development. Effective vaccine will help diminish negative impacts on animal production and decrease th threat to the health of workers in the animal industry. 02 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 thes proteins were cloned for use in analysis of expression in the host and f 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 produce 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. 03 Evaluation of early immune response. One early response in innate immunity is the development of neutrophil extracellular traps (NETs) whi 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 determin Leptospira do induce formation of NETs but surprisingly, the NETs do no 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. 04 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 fr cases of BDD. Characterization of this isolate will produce information which may explain the role of this organism in development of BDD.
Impacts
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Publications
- 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.
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