IMMUNE RESPONSE OF JOHNES DISEASE INDUCED BY MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS-SPECIFIC LIPIDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0228403
• Grant No.: 2012-67015-19379
• Project No.: COLV-2011-02882
• Proposal No.: 2011-02882
• Program Code: A1221
• Project Start Date: Apr 1, 2012
• Project End Date: Mar 31, 2016
• Grant Year: 2012

Project Director
Eckstein, T. M.

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Microbiology, Immunology and Pathology

Non Technical Summary
Johne's disease is extremely common in dairy cattle and is the major cause of loss of million of dollars. To overcome the burden of this disease animals with the disease need to be identify before the disease affects the milk production. There aren't any good tests available that would help dairy farmers to identify these animals before they get sick. We recently identified molecules that could serve in diagnostic tests. However, it is not clear when those tests are useful. In this project we propose to follow the immune response that would allow for excellent diagnostics for our newly identified molecules. It is anticipated to define the time point at which simple diagnostic test are effective to use. Furthermore, we will define which of the two diagnostic approaches is best at what time.

Animal Health Component

100%

Research Effort Categories

Basic

90%

Applied

5%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3499	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3499 - Dairy cattle, general/other;

Field Of Science
1090 - Immunology;

Keywords

johne's disease

lipids

serology

cellular immune response

goats

experimental infection

Goals / Objectives
Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, an extremely slow-growing acid-fast, lipid-rich pathogen, is a chronic granulomatous enteritis in domestic and wild ruminants. Johne's disease posts a significant problem in animal health, and this is underscored by its extreme high prevalence in US dairy herds of 95% average for large diary herd and an average prevalence of 68.1% for all dairy operation. Most approaches on diagnostics - specifically on serological diagnostics - rely on crude antigen extracts or focus on immunogenic proteins as new diagnostic tools. Although mycobacteria consist of at least 40% lipids, more than any other bacteria, lipids are widely ignored in their ability to induce specific immune responses. Furthermore, most approaches for the identification of better diagnostic tests evaluate one time-point analyses but it is widely accepted that the immune response for chronic mycobacterial diseases varies during the course of infection. We have identified a MAP-specific cell wall lipid (Para-LP-01) that is highly immunogenic and could serve as an excellent diagnostic tool. However, most of the testing is Thus, our approach will focus on the evaluation on the immune responses of Para-LP-01 in a goat model of Johne's disease over three years. Two Specific Objectives are proposed: (1) Evaluation of the serological immune responses to Para-LP-01 in a goat model, and (2) Characterization of the cellular immune response of MAP-infected goats to Para-LP-01. To identify new diagnostic approaches a thorough analysis of the complex immune responses (both humoral and cellular) during the course of the infection and disease is essential to develop new diagnostic approaches. Altogether, we advocate to study the full course of infection/disease of JD using experimental MAP infection in goats with a sufficient mount of animals in both groups (infected, control). We expect to monitor the immune responses to Para-LP-01 antigen both in the serological and cellular immune arms during the full course of infection/disease in experimentally infected goats. We expect to determine the earliest time point at which serology could be used to detect infected animals. In addition, we will identify the time frame during infection at which serological and cellular immune assays can be used accurately as diagnostic test to identify MAP infected animals. Altogether, we will provide the fundamental knowledge with regards to serological and cellular immune development during the full course of infection with MAP and the resulting Johne's disease. Ultimately, our study will determine the capacity of Para-LP-01 antigen as essential diagnostic tool.

Project Methods
Goats will be divided into two groups: (1) 10 goat kids (less than 4 months old as recommended) to be experimentally infected with MAP, and (2) 10 goat kids for the control group. SAS Proc Power was used for power analysis. We propose that 1 percent of non-JD goats (control group) will have detectable levels of a particular cytokine or serology (false positive) and 90 percent of JD goats (infection group) will have detectable levels of the same cytokine/serology (positive). We consider a conservative level of alpha = 0.005. With 10 subjects per group, the power for a one-sided Fisher's Exact Test is 0.941. All animals for this study (independent from their future infection/control status) will be subjected to blood draw and fecal sampling for initial data collection and establishment of a baseline background determination. All animals including the control group goats will be kept in-house to avoid different conditions influencing the outcome of the study. Goats will be infected twice orally as recommended by the International Committee of Johne's Disease Researchers (ICJDR). The oral route is considered to be the most likely natural way of infection with MAP and seems to be the best route to administer the pathogen. We will use the recommended daily dose of 100 microliter wet pellet equivalent to 10 to the 9th cfu in bottle-fed milk on two consecutive days. Blood draw will be performed biweekly on every goat, whereas fecal samples for MAP culturing will be performed monthly. A positive fecal culture result at 14 days after inoculation will be considered as establishing the infection. The plasma from those samples will be used to determine the antibody response. This will be determined by commercially available tests such as Parachek (Prionics) and/or IDEXX MAP Ab Test and the in-house AGID. In addition, we will perform the Para-LP-01-based Lipid-ELISA. Although the commercially available tests as well as the AGID are based on crude water-soluble antigens and would not provide information about seroreactivity towards Para-LP-01 they will give us an important general indication on the serological response. For the cellular immune response testing we will perform T cell overlay-assays onto caprine dendritic cells. Thereafter, we will monitor expression of specific cytokines (IFNγ, IL-10, IL-4, IL-17) using fluorescence intracellular staining and flow cytometry analyses. We were able to generate dendritic cells from caprine buffy coats after 7 to 10 days by differentiating monocytes with bovine GM-CSF-FLt3L and IL-4. Furthermore, as demonstrated above, we were able to purify caprine T cells. In addition, we will perform experiments with white blood cells present in the blood buffy coat. For this purpose, we will stimulate the cells with Para-LP-01 and determine the released cytokines focusing on IFNγ and IL-10. While we were able to establish an ELISpot for IFNγ, which provides a much higher sensitivity, we will measure IL-10 release through the standard cytokine ELISA.

Progress 04/01/15 to 03/31/16

Outputs
Target Audience:No changes to previous reports. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The recent results have been presented at the General Meeting of the American Society for Microbiology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Diagnostic testing as proposed was still in progress throughout the fourth year of this study. Most of the infected animals reacted with one or the other diagnostic test. See also final report.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: https://portal.nifa.usda.gov/portal/front/login?service=http%3A%2F%2Fportal.nifa.usda.gov%2Fportal%2F
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 1. Fletcher, D.M., H. E. Pirner, S. K. Meyer, A. M. Hess, and T. M. Eckstein. 2015. Environmental Factors influencing the weight development of goats experimentally infected with Mycobacterium avium subsp. paratuberculosis. 115th General Meeting of the American Society for Microbiology, May 30 to June 2 New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 2. Genis, A. B., D. M. Fletcher, A. M. Hess, and T. M. Eckstein. 2015. Cytokine Enzyme-linked Immunosorbent Assays For The Detection of Johnes disease During Pre-clinical Stages in Experimentally Infected Goats. 115th General Meeting of the American Society for Microbiology, May 30 to June 2 New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 3. Fletcher, D. M., M. Henao-Tamayo, A. M. Hess, and T. M. Eckstein. 2015. Leukocyte Subset Population Changes in Experimentally Infected Goats with Johne's disease. 115th General Meeting of the American Society for Microbiology, May 30 to June 2 New Orleans, LA.

Progress 04/01/12 to 03/31/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?All data were presented at regional, national, and international meeting as well as published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The overall goal of this study was to identify diagnostic approaches during the silent and preclinical stages of Johne's disease. We experimentally infected goat after birth and followed them throughout four years of infection. Included in this study were ten uninfected goats of same age and breed as the negative control group. Goats were housed at corrals of 2-3 acres space with individual barns for the infected and uninfected goats. None of the infected goats developed clinical Johne's disease within their first four years of life. We tested three different antigens: PPD M. bovis, PPD Johnin, and Para-LP-01. The following diagnostic tests were performed: Skin test, Elispot, cytokine ELISA, serology, fecal culture/PCR One of the ten originally infected goats died during the first six months of other reasons than Johne's disease. Of the nine remaining goats one goat was fecal culture positive once. Four goats of the remaining eight goats were skin test strongly positive and two were positive. Of the two goats that were skin test negative, one goat was positive by cytokine ELISpot. In summary, of the nine infected goats eight goats could be identified as positive at some time point with one of the five diagnostic tests during the first 2.5 years. The additional 1.5 years did not show additional useful results. At no time point we had more than 2 goats positive for one diagnostic test except for the skin test. The skin test was the only test that had six out of eight goats tested positive at the same time. Thus, skin test at the age of 2 years might be the best diagnostic test to detect Johne's disease during the preclinical stages.

Publications

Progress 04/01/14 to 03/31/15

Outputs
Target Audience: The taregt audiances are the clinicians, the dairy farmers and other researcher in the field of Johne's disease developing and using diagnostics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Yes. We published our results from the first year of the study. Furthermore, we had several poster presentations at national and international meetings including publications in proceedings of the International Colloquium on Paratuberculosis. What do you plan to do during the next reporting period to accomplish the goals? We will continue evaluating the humoral and cellular immune responses.

Impacts
What was accomplished under these goals? This is an ongoing project in which the various parameters on humoral and cellular immune responses in goats exerimentally infected with Mycobacterium avium subsp. paratuberculosis, which were monthly evaluated. In addition, the weight development was also investigated throughout the year. To confirm the infection status of the goats, a skin test with Johnin PPD was performed.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fletcher, D.M., M.B. Vogt, A.B. Genis, S.K. Meyer, H.E. Pirner, M.M. Hayes, M. Henao-Tamayo, A.M. Hess, R.A. Bowen, and T.M. Eckstein. 2014. Silent Phase of Johnes Disease in Experimentally Infected Goats  A Study on New and Established Diagnostic Approaches Using Specific and Non-Specific Parameters. GSTF J. Vet. 1(2): e4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fletcher, D.M., M. Henao-Tamayo, A. Hess, and T.M. Eckstein. 2014. Leukocyte subset changes in goats with Johnes disease. Proceedings of the 12th International Colloquium on Paratuberculosis, pp. 186-188.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fletcher, D.M., H.E. Pirner, S.K. Meyer, A.M. Hess, and T.M. Eckstein. 2014. Weight Development in goats experimentally infected with Mycobacterium avium subsp. paratuberculosis  a two- year analysis. Proceedings of the 12th International Colloquium on Paratuberculosis, pp. 22-24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Inamine, J.M. and T.M. Eckstein. 2014. Lipid profiling of various strains of Mycobacterium avium subsp. paratuberculosis from stocks made in the early 1990s and 20 years later. Proceedings of the 12th International Colloquium on Paratuberculosis, pp. 238-240.

Progress 04/01/13 to 03/31/14

Outputs
Target Audience: No changes to the target audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Results have been disseminated via publications at meeting with other researcher and scientist in the field of Johne's disease. What do you plan to do during the next reporting period to accomplish the goals? Publishing at least two papers and present the three posters.

Impacts
What was accomplished under these goals? We are just finishing year two of infection. Data of year 1 resulted in two monuscripts one of which was submitted to PLoS ONE. The other will be shortly submitted. Data for year 2 still need to be analyzed. Three posters will be presented at the 12th International Colloquium on Paratiberculosis in June 2014.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 4. Fletcher, D.M., M.B. Vogt, H.E. Pirner, A.M. Hess, R.A. Bowen, M. Henao-Tamayo, and T.M. Eckstein. 2013. Flow cytometry baseline on peripheral leukocyte cell profiles for dairy goat kids. Am. J. Anim. Vet. Sci. 8:177-89
• Type: Journal Articles Status: Published Year Published: 2013 Citation: 1. Malone, A.N., D.M. Fletcher, M.B. Vogt, S.K. Meyer, A.M. Hess, and T.M. Eckstein. 2013. Early weight development of goats experimentally infected with Mycobacterium avium subsp. paratuberculosis. PLoS ONE 8(12): e84049

Progress 04/01/12 to 03/31/13

Outputs
Target Audience: The targeted audiances are researcher and scientist focusing on the development of diagnostics and vaccine/adjuvants. Furthermore, our efforts will be of interest for the clinical diagnostic laboratories that perform diagnosti tests for Johne's disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? We presented our data at two conferences: Spring Meeting of the Rocky Mountain Brach of the American Society for Microbiology and the 113th General Meeting of the American Society for Microbiology 2013. What do you plan to do during the next reporting period to accomplish the goals? We will continue collecting data on the humoral and cellular immune response of infected and uninfected goats. We also will continue to monitor changes in the T cell and antigen-presenting cell populations.

Impacts
What was accomplished under these goals? This funded research is a long-term study on the immune responses in experimentally infected goats to better define diagnostic efforts for early and/or late diagnostics. We experimentally infected one group of goats and obtained throughout the first year of infected data on the host humoral and cellular immune responses for lipid antigen Para-LP-01 and for Johnin PPD. We also performed standard diagnostic tests (serology and fecal culture) throughout the first year. In addition, we evaluated the changed of T cell subpopulations as well as antigen-presenting cell populations throughout the first year of infection.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Allison B. Genis, Megan B. Vogt, Darcy M. Fletcher, Madeline M. Hayes, Ann M. Hess, and Torsten M. Eckstein. "Influence of Corynebacterium pseudotuberculosis infections on standard diagnostic tests for Johne's disease". Spring Meeting of the Rocky Mountain Branch of the American Society for Microbiology 2013, Fort Collins, CO; April 13, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Megan B. Vogt, Allison B. Genis, Darcy M. Fletcher, Ann M. Hess, and Torsten M. Eckstein. "Influence of Corynebacterium pseudotuberculosis exposure on new diagnostic approaches for Johne's disease". Spring Meeting of the Rocky Mountain Branch of the American Society for Microbiology 2013, Fort Collins, CO; April 13, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Darcy M. Fletcher, Megan B. Vogt, Ann M. Hess, and Torsten M. Eckstein, "Antigen-presenting cells during early phase of experimental Johne's disease in goats". Spring Meeting of the Rocky Mountain Branch of the American Society for Microbiology 2013, Fort Collins, CO; April 13, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Stephen K. Meyer and Torsten M. Eckstein. Lipid-ELISA with total cell lipids of Corynebacterium pseudotuberculosis for goats experimentally infected with Mycobacterium avium subsp. paratuberculosis. Spring Meeting of the Rocky Mountain Branch of the American Society for Microbiology 2013, Fort Collins, CO; April 13, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Alyssa N. Malone, Darcy M. Fletcher, Megan B. Vogt, Stephen K. Meyer, Ann M. Hess, and Torsten M. Eckstein. "Weight development of goats experimentally infected with Mycobacterium avium subsp. paratuberculosis to induce Johne's disease". 113th General Meeting of the American Society for Microbiology, Denver, May 18-21, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Darcy M. Fletcher, Megan B. Vogt, Ann M. Hess, and Torsten M. Eckstein. "Changes in T cell subpopulations in goats experimentally infected with Mycobacterium avium subsp. paratuberculosis". 113th General Meeting of the American Society for Microbiology, Denver, May 18-21, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Allison B. Genis, Megan B. Vogt, Darcy M. Fletcher, Madeline M. Hayes, Ann M. Hess, and Torsten M. Eckstein. "Standard diagnostic cellular immune assays in goats experimentally infected with Mycobacterium avium subsp. paratuberculosis". 113th General Meeting of the American Society for Microbiology, Denver, May 18-21, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Megan B. Vogt, Allison B. Genis, Darcy M. Fletcher, Ann M. Hess, and Torsten M. Eckstein. "New cellular immune assays for the detection of Johne's disease in experimentally infected goats". 113th General Meeting of the American Society for Microbiology, Denver, May 18-21, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Stephen K. Meyer and Torsten M. Eckstein. A Para-LP-01 based Lipid-ELISA detects goats experimentally infected with Mycobacterium avium subsp. paratuberculosis during the first year of infection. 113th General Meeting of the American Society for Microbiology, Denver, May 18-21, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Alyssa N. Malone, Darcy M. Fletcher, Megan B. Vogt, Stephen K. Meyer, Ann M. Hess, and Torsten M. Eckstein. "Effect of age on early weight development in the goat model for Johne's disease". Spring Meeting of the Rocky Mountain Branch of the American Society for Microbiology 2013, Fort Collins, CO; April 13, 2013