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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Research Project #432150

Research Project: Characterization of the Pathogenesis and Antigen Expression in Spirochete Diseases

Location: Infectious Bacterial Diseases Research

2021 Annual Report


Objectives
These studies are focused on developing an understanding of how Leptospira and Treponema species interact with their hosts to establish colonization, infection, and clinical disease. A combination of genomic, proteomic and immunological methodologies will be used to analyze how the host responds to infection and how bacteria respond to the host, with the long-term goal of identifying pathways that can be targeted to alter disease outcomes or exploited to induce protective immunity. Objective 1. Identify and characterize the Leptospira and Treponema sp. circulating in livestock. Subobjective l.1 - Determine prevalence of leptospires circulating in local bovine herds. Subobjective l.2 - Characterize clonal isolates of Treponema from bovine digital dermatitis at the phenotypic, genomic and proteomic level. Objective 2. Develop animal models that will mimic infection, facilitate characterization of interactions between host and pathogen, and allow development of assays that will accurately identify infected individuals. Subobjective 2.1 - Characterize urinary immunoglobulin from reservoir hosts of leptospirosis. Subobjective 2.2 - Characterize the cellular immune response of reservoir hosts of leptospirosis. Subobjective 2.3 - Characterize and refine an ovine model of bovine digital dermatitis. Objective 3. Characterize spirochete antigens including those that are differentially expressed during infection. Subobjective 3.1 - Identification and characterization of leptospiral proteins that are expressed in response to mammalian host signals. Subobjective 3.2 - Characterize host humoral responses to outer membrane protein antigens derived from bacteria associated with digital dermatitis.


Approach
Objective 1: This objective seeks to identify and characterize species of Leptospira and Treponema sp. circulating in livestock. Studies will be conducted to determine the prevalence of leptospires circulating in local bovine herds (Sub-objective 1.1); and to characterize clonal isolates of Treponema from bovine digital dermatitis at the phenotypic, genomic and proteomic levels (Sub-objective 1.2). We expect these studies to determine if serovars of leptospires currently circulating in bovine populations of the Mid-West have changed over the last 20 years and to demonstrate that different phylotypes of Treponema derived from bovine digital dermatitis have unique genomic, proteomic and virulence factors. Objective 2: Development of animal models that mimic infection will facilitate characterization of interactions between host and pathogen, and allow development of assays that will accurately identify infected individuals. Urinary immunoglobulins from reservoir hosts of leptospirosis will be collected and characterized (Sub-objective 2.1); the cellular immune response of reservoir hosts of leptospirosis will also be characterized (Sub-objective 2.2); and an ovine model of bovine digital dermatitis will be further characterized and refined (Sub-objective 2.3). We will also evaluate immune activation pathways in a reservoir host model of leptospirosis using the inbred Fisher 344 rat. Studies conducted will advance the use of sheep as a ruminant model to understand the pathogenic mechanisms and involvement of treponemes in digital dermatitis. Objective 3: Characterize spirochete antigens including those that are differentially expressed during infection. Studies will be conducted to identify and characterize leptospiral proteins that are expressed in response to mammalian host signals (Sub-objective 3.1) and to characterize host humoral responses to outer membrane protein antigens derived from bacteria associated with digital dermatitis (Sub-objective 3.2).


Progress Report
This will be the final report for project 5030-32000-223-00D terminating September 30, 2021. During the past 5 years, the project investigated the pathogenesis of leptospirosis and digital dermatitis disease and developed new diagnostics and vaccines to reduce disease prevalence. The project developed a new media that enhances isolation of Leptospira from samples. This media was used to demonstrate that approximately 7% of beef cattle presented to an abattoir were actively shedding Leptospira bacteria in their urine. In collaboration with other scientists, the media has been used to identify new serovars of Leptospira from natural hosts in the U.S. The media has also been used by the Center for Disease Control to isolate Leptospira bacteria from human samples and was used to develop recombinant Leptospira strains for use in pathogenesis studies. New diagnostic assays for leptospirosis were developed and made commercially available to stakeholders. To identify immunogenic proteins for development of new vaccines and/or diagnostics, outer membrane protein profiles were compared across serovars. Novel antibody responses in urine were demonstrated in a laboratory animal model after Leptospirosis infection that appear to be common in reservoir hosts and are being evaluated for potential as a new diagnostic approach. A laboratory animal model of leptospirosis was developed to characterize host immune responses during persistent renal infection. Data indicates that local immune responses at the kidney play an important role in disease pathogenesis and that immune responses differ during acute and chronic infection. Expansion of the work into cattle demonstrated that animals chronically or intermittently shedding Leptospira in urine do not demonstrate immune recall responses to Leptospira antigens. Other work demonstrated that Leptospira bacteria recovered from urine express modified outer membrane proteins. Protein modification may minimize immune responses and may be targets for development of novel vaccines and diagnostics. Project scientists also characterized the role of Treponemes in the development of digital dermatitis in cattle and sheep and expanded the work to include lesions in free-roaming elk in the Pacific Northwest. The bacterial communities (microbiome) of digital dermatitis lesions were characterized to understand which Treponema species/strains are important for lesion development. The identification of bacteria causing disease may facilitate development of novel diagnostic and intervention strategies. A sheep model was developed to characterize the pathogenesis of digital dermatitis lesion development. The model demonstrated that antibody responses to Treponema correlate with disease severity. Project scientists isolated a unique Treponema species that had previously not been identified in the United States. Genomic studies indicated this isolate contained an antibiotic resistance gene that is not present in other Treponema species associated with digital dermatitis lesions. In evaluating therapy to prevent or treat digital dermatitis, a library of novel, environmentally friendly, antimicrobial compounds were tested on bacteria associated with lesions and results indicate some of the compounds kill or inhibit bacteria found in lesions.


Accomplishments
1. Genetic manipulation of pathogenic leptospires. Leptospira bacteria are zoonotic pathogens which cause reproductive losses and clinical diseases in numerous hosts. Performing targeted gene mutations to create attenuated strains is difficult in Leptospira. Using the CRISPR/Cas 9 system, ARS scientists in Ames, Iowa, created recombinant strains of Leptospira in which immunogenic outer membrane proteins were genetically modified. Complete silencing of LipL32, LigA, and LigB were achieved, revealing for the first time that Lig proteins are crucial for disease pathogenesis. By translation to stakeholders through presentations at scientific meetings, direct communication, and peer-reviewed manuscripts, this work will be of interest to stakeholders and researchers with interest in leptospirosis vaccine development as a faster and more economical approach for development of new vaccine candidates.

2. New Leptospira species identified in United States. Leptospirosis is a zoonotic disease that causes reproductive losses and infertility in dairy cattle. Vaccines against leptospirosis tend to only be efficacious for the serovar(s) included in the vaccine. ARS scientists in Ames, Iowa, made the first isolation of L. borgpetersenii serovar Tarassovi from a cow in the U.S. This serovar has been identified as significant cause of cattle leptospirosis in other countries such as Australia. The isolation indicates the need to perform diagnostics to determine if this serovar is prevalent in the U.S. and may suggest modification of current commercial vaccines to contain this serovar. This work will be of interest to stakeholders, regulatory personnel, and researchers with interest in leptospirosis and leptospirosis vaccines.

3. Gene expression by Leptospira. Leptospirosis is a zoonotic disease that causes reproductive losses and infertility in cattle with L. borgpeterseni serovar Hardjo being the most prevalent isolate recovered from cattle. ARS scientists in Ames, Iowa, used a new media for Leptospira to characterize gene expression of serovar Hardjo while cultured at the standard 29 C as compared to bacteria grown at body temperature (37 C). Genes were identified that had increased expression at body temperature that may be upregulated by the bacteria during in vivo infection. These genes may be targets for development of new more efficacious vaccines for cattle and may also be useful for improvements in diagnostics and/or therapeutics. By translation to stakeholders through presentations at scientific meetings, direct communication, and peer-reviewed manuscripts, this work will be of interest to stakeholders, regulatory personnel, and researchers with interest in leptospirosis and leptospirosis vaccines.


Review Publications
Wunder, E.A., Adhikarla, H., Hamond, C., Owers, K., Liang, L., Rodrigues, C.B., Bisht, V., Nally, J.E., Alt, D.P., Reis, M.G., Diggle, P.J., Felgner, P.L., Ko, A.I. 2021. A live attenuated-vaccine model confers cross-protective immunity against different species of the Leptospira genus. eLife. 10:e64166. https://doi.org/10.7554/eLife.64166.
Fernandes, L.G., Hornsby, R.L., Nascimento, A.L., Nally, J.E. 2021. Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37C. Scientific Reports. 11. https://doi.org/10.1038/s41598-021-81400-7.
Putz, E.J., Sivasankaran, S.K., Fernandes, L.G., Brunelle, B.W., Lippolis, J.D., Alt, D.P., Bayles, D.O., Hornsby, R.L., Nally, J.E. 2021. Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures. PLOS Neglected Tropical Diseases. 15(4). Article e0009320. https://doi.org/10.1371/journal.pntd.0009320.
Shiel, R.E., Nolan, C.M., Nally, J.E., Refsal, K.R., Mooney, C.T. 2021. Qualitative and semiquantitative assessment of thyroid hormone binding proteins in greyhounds and other dog breeds. Domestic Animal Endocrinology. 76:1-8. https://doi.org/10.1016/j.domaniend.2021.106623.
Almeida, A.M., Ali, A., Ceciliani, F., Eckersall, P., Hernandez-Castellano, L.E., Han, R., Hodnik, J.J., Jaswal, S., Lippolis, J.D., McLaughlin, M., Miller, I., Mohanty, A.K., Mrljak, V., Nally, J.E., Nanni, P., Plowman, J.E., Poleti, M.D., Ribeiro, D.M., Rodrigues, P., Roschitzki, B.,Schlapbach,R., Staric, J., Yang, Y., Zachut, M. 2021. Domestic animal proteomics in the 21st century: a global retrospective and viewpoint analysis. Journal of Proteomics. 241. Article 104220. https://doi.org/10.1016/j.jprot.2021.104220.
Putz, E.J., Nally, J.E. 2020. Investigating the immunological and biological equilibrium of reservoir hosts and pathogenic Leptospira: balancing the solution to an acute problem. Frontiers in Immunology. 11:2005. https://doi.org/10.3389/fmicb.2020.02005.
Wafa, E.I., Wilson-Welder, J.H., Hornsby, R.L., Nally, J.E., Geary, S.M., Bowden, N.B., Salem, A.K. 2020. A poly(diaminosulfide) microparticle-based vaccine for delivery of leptospiral antigens in cattle. Biomacromolecules 2020 21(2):534-544. https://doi.org/10.1021/acs.biomac.9b01257.
Dobson, L.K., Zimin, A., Bayles, D., Fritz-Waters, E., Alt, D., Olsen, S., Blanchong, J., Reecy, J., Smith, T.P.L., Derr, J.N. 2021. De novo assembly and annotation of the North American bison (Bison bison) reference genome and subsequent variant identification. Animal Genetics. 52(3):263-274. https://doi.org/10.1111/age.13060.
Kumar, R., Register, K.B., Christopher-Hennings, J., Moroni, P., Gioia, Gloria, Garcia-Fernandez, N., Nelson, J., Jelinski, M., Lysnyansky, I., Bayles, D.O., Alt, D.P., Scaria, J. 2020. Population genomic analysis of Mycoplasma bovis elucidates geographical variations and genes associated with host types. Microorganisms. 8(10). Article 1561. https://doi.org/10.3390/microorganisms8101561.
Stuart, K.L., Bayles, D.O., Shore, S., Nicholson, T.L. 2021. Complete genome sequence of Escherichia coli Antibiotic-Resistance Isolate AR Bank #0346. Microbiology Resource Announcements. 10(23). https://doi.org/10.1128/MRA.00305-21.
Bannantine, J.P., Bayles, D.O., Biet, F. 2021. Complete genome sequence of a type III ovine strain of Mycobacterium avium subspecies paratuberculosis. Microbiology Resource Announcements. 10. https://doi.org/10.1128/MRA.01480-20.
Bannantine, J.P., Stabel, J.R., Bayles, D.O., Condes, C., Biet, F. 2021. Diagnostic sequences that distinguish M. avium subspecies strains. Frontiers in Veterinary Science. 7. https://doi.org/10.3389/fvets.2020.620094.
Nicholson, T.L., Waack, U., Anderson, T.K., Bayles, D.O., Zaia, S.R., Goertz, I., Eppinger, M., Hau, S.J., Brockmeier, S., Shore, S. 2021. Comparative virulence and genomic analysis of streptococcus suis isolates. Frontiers in Microbiology. 11. Article 620843. https://doi.org/10.3389/fmicb.2020.620843.
Wiarda, J.E., Boggiatto, P.M., Bayles, D.O., Waters, R.W., Thacker, T.C., Palmer, M.V. 2020. Severity of bovine tuberculosis is associated with innate immune-biased transcriptional signatures of whole blood in early weeks after experimental Mycobacterium bovis infection. PLoS ONE. 15(11). https://doi.org/10.1371/journal.pone.0239938.
Bannantine, J.P., Conde, C., Bayles, D.O., Branger, M., Biet, F. 2020. Genetic diversity among Mycobacterium avium subspecies revealed by analysis of complete genome sequences. Frontiers in Microbiology. 11. https://doi.org/10.3389/fmicb.2020.01701.
Wilson-Welder, J.H., Alt, D.P., Nally, J.E., Olsen, S.C. 2021. Bovine immune response to vaccination and infection with Leptospira borgpetersenii serovar Hardjo. mSphere. 6(2). https://doi.org/10.1128/mSphere.00988-20.
Masonbrink, R.E., Alt, D.P., Bayles, D.O., Boggiatto, P.M., Edwards, H., Tatum, F.M., Williams, J.E., Wilson-Welder, J.H., Wood, M., Zimin, A., Severin, A., Olsen, S.C. 2021. A pseudomolecule assembly of the Rocky Mountain elk genome. PLoS ONE. 16(4). https://doi.org/10.1371/journal.pone.0249899.
Cranford, H.M., Taylor, M., Browne, A., Alt, D.P., Anderson, T., Hammond, C., Hornsby, R.L., Lecount, K., Schlater, L., Stuber, T., DeWilde, L., Burke-France, V.J., Ellis, E.M., Nally, J.E. 2021. Exposure and carriage of pathogenic Leptospira in livestock in St. Croix, U.S. Virgin Islands. Tropical Medicine and Infectious Disease. 6(2). https://doi.org/10.3390/tropicalmed6020085.