Location: Infectious Bacterial Diseases Research
2018 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
Leptospirosis is an insidious and persistent disease of cattle that causes reproductive losses, and also causes clinical disease in humans. Leptospires are excreted in urine leading to disease transmission to other co-housed animals. The gold standard for detecting shedding is culture, but this is difficult and not offered by diagnostic laboratories. During the past year, in support of Subobjective 1.1, the project used culture methods and fluorescent antibody testing to characterize Leptospira serovars currently circulating in cattle in the U.S., and demonstrated that vaccinated dairy cows shed bacteria in urine. The project continues to refine and improve culture methods for isolation of spirochete bacteria.
In support of Subobjective 2.1, antibodies in urine from infected cattle were used to identify Leptospiral antigens expressed during renal colonization and are being evaluated for use in improving vaccines and diagnostics.
In support of Subobjective 2.2, a novel rat model was developed to characterize host cellular immune responses during persistent renal colonization. Data suggests the renal lymph node contains CD4+ T cells reactive to Leptospira and highlights the role of local immune responses during renal infection. Identification of immune responses associated with persistent infection in the murine model may provide insight into disease pathogenesis in cattle. In addition, the animal model provides a source of in vivo derived leptospires for characterization of gene and protein expression during infection.
In support of Subobjectives 2.3 and 3.2, the developed ovine model was used to understand pathogenic mechanisms of bovine digital dermatitis. Samples were collected at various times after infection and used to characterize microbial profile and histologic changes during disease progression. Immune response during disease pathogenesis were also evaluated. The project also characterized outer membrane components of treponeme isolates in an effort to understand their role in disease pathogenesis.
Accomplishments
1. Isolation and characterization of pathogenic leptospires. Leptospirosis is a bacterial disease transmitted from animals to humans worldwide. As Leptospires infect the kidney of natural hosts and are excreted in the urine, contact with infected urine or contaminated water can result in disease. Bovine leptospirosis is endemic in the United States and infection in cattle causes reproductive losses. Current commercial vaccines are based on strains isolated from cattle over 25 years ago. ARS scientists in Ames, Iowa, characterized leptospires currently circulating in cattle. The study found that 7.2 percent of sampled cattle were actively excreting leptospires and all isolates were found to be serovar Hardjo, historically the predominant serovar infecting cattle in the United States and worldwide.
2. An inbred rat model to study renal infection. Pathogenic species of leptospira cause leptospirosis, a zoonotic disease with a global distribution. In natural hosts, leptospires localize in the kidney and are excreted in urine. Reservoir hosts of leptospirosis are typically asymptomatic and excrete leptospires for months and years. Surprisingly, little is known about host cellular immune response that facilitate persistent renal infection. ARS scientists at Ames, Iowa, developed a laboratory animal model of persistent renal Leptospira colonization. Data demonstrated the inbred rats developed persistent renal infections with excretion of leptospires in urine. Results implicated the local renal lymph node as an important site for immunologic responses to Leptospira rather than systemic immune responses. These data allow understanding of immunologic responses that contribute to persistent renal infection and provides critical information on disease pathogenesis.
3. Leptospires modulate protein expression during infection. Pathogenic leptospires are inherently difficult to grow under laboratory conditions in part because in vitro conditions differ from the in vivo environment. Growth under conditions that replicate their natural environment allows characterization of how bacteria adapt to colonization of a natural host. ARS researchers at Ames, Iowa, developed a novel method for culturing leptospires that more closely mimics natural infection. This unique culture method resulted in expression of Leptospira proteins normally found during infection that are usually not expressed under laboratory conditions. Data demonstrate that leptospires modify their protein content under in vivo conditions. In addition to differences in quantity of expressed protein under natural conditions, some proteins are modified in a manner that influences their function. These data provide novel insights on colonization and persistence infection in natural hosts.
4. Development of an ovine digital dermatitis model. Digital dermatitis is a leading cause of lameness in cattle and causes economic losses. Although the etiology of the disease is not fully understood, numerous studies have implicated bacteria from the genus Treponema and other anaerobic bacteria as contributing to lesion development. ARS scientists in Ames, Iowa, developed an ovine model to allow characterization of bacteria involved in lesion pathogenesis. Sheep, having similar immune responses as cattle, are naturally susceptible to digital dermatitis, and are easy to handle. After inoculation of sheep hooves with material from bovine digital dermatitis infection, lesions develop within 4 weeks and mimic the disease in cattle. Treponemes were confirmed within lesions in sheep and the disease could be horizontally transmitted to other sheep. Development of a digital dermatitis model in sheep, allows investigation into host-pathogen interactions, the role of treponemes in disease pathogenesis, and evaluation of the efficacy of novel vaccines.
Review Publications
Wilson-Welder, J.H., Nally, J.E., Alt, D.P., Palmer, M.V., Cotaney, J., Plummer, P. 2017. Experimental transmission of bovine digital dermatitis to sheep: development of an infection model. Veterinary Pathology. 55(2):245-257. https://doi.org/10.1177/0300985817736572.
Nally, J.E., Schuller, S. 2017. Proteomic analysis of lung tissue by DIGE. Methods in Molecular Biology. 1664:167-183. https://doi:10.1007/978-1-4939-7268-5_14.
Furuya, Y., Kirimanjeswara, G., Roberts, S., Racine, R., Wilson-Welder, J.H., Sanfilippo, A., Salmon, S., Metzger, D.W. 2017. Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice. Vaccine. 35(37):4997-5005. https://doi.org/10.1016/j.vaccine.2017.07.071.
Lippolis, J.D., Nally, J.E. 2018. Considerations for farm animal proteomics experiments: An introductory view gel based versus non-gel based approaches. In: de Almeida A., Eckersall D., Miller I., editors. Proteomics in Domestic Animals: from Farm to System Biology. Basel, Switzerland: Springer International Publishing AG. p. 7-16. https://doi.org/10.1007/978-3-319-69682-9_2.
Nally, J.E., Wilson-Welder, J.H., Hornsby, R.L., Palmer, M.V., Alt, D.P. 2018. Inbred rats as a model to study persistent renal colonization and associated cellular immune responsiveness. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2018.00066.
Nally, J.E., Hornsby, R.L., Alt, D.P., Bayles, D.O., Wilson-Welder, J.H., Bauer, N.E. 2018. Isolation and characterization of pathogenic leptospires associated with cattle. Veterinary Microbiology. 218:25-30. https://doi.org/10.1016/j.vetmic.2018.03.023.
Nally, J.E., Grassmann, A.A., Planchon, S., Sergeant, K., Renaut, J., Sesku, J., McBride, A., Caimano, M.J. 2017. Pathogenic leptospires modulate protein expression and post-translational modifications in response to mammalian host signals. Frontiers in Cellular and Infection Microbiology. 7:362. https://doi.org/10.3389/fcimb.2017.00362.
Isani, G., Ferlizza, E., Nally, J.E. 2018. Proteomic research in urine and other fluids. Book Chapter. 121-147. https://doi.org/10.1007/978-3-319-69682-9_7.
