Location: Infectious Bacterial Diseases Research
2021 Annual Report
Objectives
Objective 1: Define the immunopathogenesis of bovine tuberculosis at the lesion and cellular level by evaluating local cytokine and biomarker expression.
Subobjective 1.1. Characterize and compare known or implied relevant cytokine and biomarker expression in granulomas of different histopathologic stages (i.e. early vs. late) in lungs and lymph nodes from cattle experimentally inoculated with M. bovis.
Subobjective 1.2. Characterize cytokine and biomarker expression at the lesion level over time.
Subobjective 1.3. Characterize and compare lesion level cytokine and cellular responses between non-vaccinated cattle and cattle with vaccine-induced protective immune responses.
Objective 2: Using antigen mining and transcriptome analysis, develop novel diagnostic tests with improved sensitivity and specificity as compared to current methods.
Subjective 2.1. Improve specificity of diagnostic tests by developing diagnostic reagents from proteins found in M. bovis but not in non-tuberculous mycobacteria.
Subobjective 2.2: Identify proteins/genes expressed by M. bovis in vivo that may be considered as potential diagnostic test targets.
Subobjective 2.3: Use genomics/transcriptomics to characterize genes/gene profiles of M. bovis-infected vs non-infected cattle.
Objective 3: Develop novel vaccines, technologies and platforms (e.g. attenuated live vaccines and vectored vaccines) that can be used to reduce TB in cattle and white-tailed deer and interrupt disease transmission.
Subobjective 3.1. Examine duration of immunity to experimental infection provided by the vaccine M. bovis BCG in white-tailed deer.
Subobjective 3.2. Examine the effects of oral BCG vaccination of white-tailed deer on deer-to-deer transmission of virulent M. bovis.
Subjective 3.3. Determine the efficacy of simultaneous administration of parenteral BCG and a mucosally delivered bacterial-vectored subunit vaccine against aerosol M. bovis infection in neonatal calves.
Approach
Characterize and compare cytokine and biomarker expression (immune responses) at the cellular level in lungs and lymph nodes of Mycobacterium bovis-infected cattle. Comparing responses between tissues, as well as over time, will aid in understanding the host response to M. bovis within the environment where host and pathogen interact (granuloma). We aim to improve the specificity of diagnostic tests by developing diagnostic reagents from proteins found in M. bovis but not in non-tuberculous mycobacteria, thus avoiding cross-reactivity elicited by environmental mycobacteria that contributes to false positive results on cattle tuberculosis diagnostic tests. Similarly, we aim to identify proteins/genes expressed by M. bovis in vivo that may be considered as potential diagnostic test targets and to use genomics/transcriptomics to characterize genes/gene profiles of M. bovis-infected vs non-infected cattle. These data will aid diagnosis and provide insight into the immunopathogenesis of bovine tuberculosis. In terms of vaccine evaluation, we aim to examine duration of immunity to experimental infection provided by the vaccine M. bovis BCG in white-tailed deer and examine the effects of oral BCG vaccination on deer-to-deer transmission of virulent M. bovis. In cattle, we aim to determine the efficacy of simultaneous administration of parenteral M. bovis BCG and a mucosally delivered bacterial-vectored subunit vaccine against aerosol M. bovis infection in neonatal calves.
Progress Report
This will be the final report for project 5030-32000-222-00D terminating September 30, 2021. Research will continue under a bridging (or new) project. Over 100 years ago, in 1917, USDA initiated a bovine tuberculosis (bTB) eradication program that is still in place today. Although significant progress has been made, eradication has proved elusive. Obstacles to eradication include, 1) lack of rapid and accurate diagnostic tests to detect animals infected with Mycobacterium bovis (M. bovis; the cause of tuberculosis in animals), and 2) wildlife acting as a source of infection for cattle. Research activities within the ARS bTB project in Ames, Iowa, provide direct support for the USDA bTB eradication program, specifically, through development of more sensitive diagnostics, efficacious vaccines, and improved animal disease models to enhance the capability to detect, prevent and control bTB.
In FY18 one SY retired, and that position was eliminated due to lack of funding. During FY19 another SY retired, and that vacancy was filled in FY20. As such, there were necessary modifications to milestones during the term of this project. Despite significant staffing changes, substantial progress was made on all three objectives. Under Objective 1, we developed and characterized new tools to further evaluate the bovine immune response to M. bovis. These new tools allowed us to investigate immune responses at the site of infection (lung and lymph nodes), where it is believed the outcome of infection (control vs progression and dissemination) is determined. Demonstrating which key immune cell types and inflammatory mediators are present at the site of infection will result in new biomarkers for enhanced early disease detection and new vaccine targets for disease prevention.
Under Objective 2, we sequenced the genome of several mycobacterial species that are closely related to M. bovis, and are ubiquitous in the environment, but do not cause disease. These non-disease-causing mycobacteria often interfere with bTB diagnosis, resulting in unacceptably high numbers of false positive test results. False positive findings result in unnecessary removal and slaughter of uninfected cattle, often resulting in financial losses for producers, making them less likely to cooperate with voluntary aspects of the bTB eradication program. Using genomic data, eight proteins unique to M. bovis were identified. Using these unique proteins, novel diagnostic tests or modifications of existing tests can be evaluated with the goal of producing fewer false positive results. More accurate diagnostic tests will expedite the eradication of bTB from the U.S., decrease financial losses to producers, and improve relations between producers and USDA officials.
Using information gained from Objective 1, we identified biomarkers for use in more accurate diagnostic tests. Using whole blood from experimentally infected cattle we evaluated a panel of inflammatory mediators by both gene and protein expression and identified multiple inflammatory mediators as potential biomarkers in bTB diagnostics.
Along with Animal and Plant Health Inspection Service (APHIS) and the World Organization for Animal Health (OIE), we identified an international standard for bovine tuberculin use in cattle skin testing, an important cornerstone of the bTB eradication program. Bovine tuberculosis is a global problem, an international standard tuberculin will harmonize and improve diagnosis of bTB around the world. These findings will benefit regulatory agencies, veterinarians and livestock producers involved with maintaining a bTB-free status for the U.S.
Under Objective 3, studies continued to develop bTB vaccines and vaccine delivery systems for wild deer in areas of the country where deer transmit bTB to cattle. In cooperation with USDA, APHIS, Wildlife Services (WS) we developed a bait vaccine for wild deer. The vaccine component is the human tuberculosis vaccine, known as bacilli Calmette Guerin (BCG). Partnering with a vaccine manufacturer, we will generate bait BCG vaccines to be distributed in Michigan, in early 2023. This time frame allows obtaining appropriate approvals from regulatory agencies and development of methodology to make baits and design quality control standards. Additionally, we are collaborating with APHIS, Veterinary Service colleagues in designing and implementing a BCG vaccine trial in dairy herds in Baja California, Mexico. The field study is to begin in mid to late 2021.
In addition to supporting the missions of both USDA ARS and APHIS, studies conducted by the ARS bTB project support stakeholder (beef, dairy and deer producer groups) interests as well as interests of State Departments of Agriculture and Natural Resources. Research findings continue to be used to create or modify state and federal regulations regarding TB testing of cattle and deer, including approval of novel tests for use in the USDA Uniform Methods and Rules (UM&R) for the bTB eradication program.
Accomplishments
1. Characterization of Tuberculosis granuloma development. Bovine tuberculosis (bTb) results in production losses and lost export opportunities for U.S. cattle producers. Additionally, the presence of bTB in a herd initiates quarantines, testing, and slaughter of test-positive cattle, representing a significant economic impact for producers. There is a need for improved diagnostic tests, which recognize the disease in the early stages, and vaccines to prevent infection and disease transmission. ARS scientists in Ames, Iowa, generated a model of both early (15-30 days) and late granuloma formation (9 months) and correlated to disease pathogenesis. This was the first examination of bTB at such early time points, revealing previously unrecognized aspects of bTB. Understanding the characteristics of bacterial killing, especially early in disease onset, will aid in rationale vaccine design and development of diagnostic tests that detect acute disease, inhibit within-herd spread. and development of effective vaccines that will benefit regulatory agencies, veterinarians, and livestock producers.
Review Publications
Palmer, M.V., Thacker, T.C., Kanipe, C.R., Boggiatto, P.M. 2021. Heterogeneity of pulmonary granulomas in cattle experimentally infected with Mycobacterium bovis. Frontiers in Veterinary Science. 8. https://doi.org/10.3389/fvets.2021.671460.
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.
Stabel, J.R., Waters, W.R., Bannantine, J.P., Palmer, M.V. 2021. Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis. Veterinary Immunology and Immunopathology. 237. https://doi.org/10.1016/j.vetimm.2021.110268.
