Location: Infectious Bacterial Diseases Research
2019 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
In support of Subobjective 1, ARS scientists in Ames, Iowa continue to develop a new growth media for growth of highly fastidious leptospires from animal samples. Scientists are also collaborating with scientists with the Animal and Plant Health Inspection Service (APHIS) on development of a fluorescent antibody test for diagnosis of leptospirosis in cattle. Scientists in the project also recovered and characterized the second reported isolate of Treponema brennaborense a novel spirochete previously isolated from ulcers of a cow with digital dermatitis, which highlights the fastidious growth requirements of these bacteria. Work in the past year has also included characterization of the outer membrane components of treponemes from digital dermatitis lesions to identify antigens that may be useful for development of vaccines and diagnostics.
In support of Subobjective 2, ongoing studies are further characterizing the bacterial community, or microbiome, of digital dermatitis in both experimental models and natural infections. In the past year, this work has included characterizing the pathology and immunological responses of free-roaming wild elk with a digital dermatitis like disease in the Pacific Northwest. The overall goal is to develop sufficient knowledge of bacteria playing a role in the disease such that diagnostics and intervention strategies can be developed.
Accomplishments
1. Hoof disease in free-ranging elk. ARS scientists from Ames, Iowa collaborated with state officials and university scientists in characterization of hoof disease in free-ranging elk (Cervus elaphus) in southwestern Washington State which continues to spread throughout the region. In adult elk, lesions of hoof overgrowth, sole ulcers, and sloughed hoof capsules are observed in addition to higher mortality. To characterize the pathogenesis, groups of elk at 9, 7, and 3 months of age were examined in a field study. Lesions were most severe in elk at 9 months of age and mimicked lesions in adult elk. Treponemes were identified in the lesions by immunohistochemistry and polymerase chain reaction (PCR) assays. Elk examined at 3 months of age had the mildest lesions but evidence of Treponemes was determined. Serum samples obtained from elk at 7 and 9 months of age demonstrated antibody responses to Treponemes when compared to non-infected elk. Data suggests that Treponeme-associated hoof disease (TAHD) in free-ranging elk is a debilitating and progressive condition that is similar to digital dermatitis in cattle and sheep. The work provides critical insight into the pathogenesis of this disease that may lead to intervention strategies that reduce its prevalence in the northwest U.S.
2. Treponemes in bovine digital dermatitis. In the past year, ARS scientists in Ames, Iowa isolated and characterized new strains of T. brennaborense and T. phagedenis from digital dermatitis lesions of cattle and compared them to T. phagedenis and T. pedis type strains. The isolate of T. brennaborense demonstrated very fastidious culture requirements in vitro, rapid motility under dark-field microscopy, and has an enzymatic profile identical to the only other isolate of T. brennaborense. Mass spectrometry was used to characterize outer membrane protein profiles of each strain and data indicated that each strain has a unique profile of outer membrane proteins. This work provides a basis for development of assays to identify Treponemes in digital dermatitis lesions based on outer membrane proteins and may also be useful in development of vaccines and diagnostics for the disease.
Review Publications
Hernandex-Castellano, L.E., Nally, J.E., Lindahl, J., Wanapat, M., Alhidary, I.A., Fangueiro, D., Grace, D., Ratto, M., Bambou, J.C., de Almeida, A.M. 2019. Dairy science and health in the tropics: challenges and opportunities for the next decades. Tropical Animal Health and Production. 51(5):1009-1017. https://doi.org/10.1007/s11250-019-01866-6.
Wilson-Welder, J.H., Nally, J.E., Alt, D.P., Humphrey, S.B., Olsen, S.C. 2018. Short communication: Lymphocyte proliferative responses in cattle naturally infected with digital dermatitis consists of CD8plus and gamma delta T cells but lack CD4plus T cells. Journal of Dairy Science. 101(9):8301-8307. https://doi.org/10.3168/jds.2017-13913.
Han, S., Mansfield, K., Haldorson, G., Bradway, D., Besser, T., Reed, D., Alt, D.P., Wilson-Welder, J.H. 2018. Treponeme-associated hoof disease of wild elk (Cervus elaphus spp.) in southwestern Washington State, USA. Veterinary Pathology. https://doi.org/10.1177/0300985818798108.
Nally, J.E., Hornsby, R.L., Alt, D.P., Whitelegge, J.P. 2019. Phenotypic and proteomic characterization of treponemes associated with bovine digital dermatitis. Veterinary Microbiology. 235:35-42. https://doi.org/10.1016/j.vetmic.2019.05.023.
Ferrer, M.F., Scharrig, E., Charo, N., Ripodas, A.L., Drut, R., Carrera Silva, E.A., Nagel, A., Nally, J.E., Schattner, M., Gomez, R.M., Montes de Oca, D.P. 2018. Macrophages and galectin 3 control bacterial burden in acute and subacute murine leptospirosis that determines chronic kidney fibrosis. Frontiers in Cellular and Infection Microbiology. 8:384. https://doi.org/10.3389/fcimb.2018.00384.
