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United States Department of Agriculture

Agricultural Research Service

Research Project: Control, Immunology and Genomics of Spirochete Diseases
2013 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.


3.Progress Report:
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.


4.Accomplishments
1. 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.

2. 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.

3. 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.


Review 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.

Last Modified: 10/31/2014
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