MYCOBACTERIAL DISEASES OF ANIMALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014673
• Multistate No.: NE-1701
• Project Start Date: Dec 26, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Project Director
Kapur, VI.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
Animal Science

Non Technical Summary
The proposed multi-state initiative will focus on two of the most important mycobacterial diseases of animals;paratuberculosis (Johne's disease; JD)and thebovine tuberculosis complex (TB). These two mycobacterial diseases represent some of the most prevalent and economically significant infections of livestock, and each has a long and rich history. The generation of new knowledge relative to the diagnosis, management, and control of mycobacterial diseases of animals is critical if we are to prevent the spread, lower the prevalence and minimize the impact of the diseases in our livestock populations. USDA NAHMS studies and other work, including the National Dairy Producer Johne's survey, have shown that while producers are increasingly aware of the diseases, they often lack knowledge relative to their management and control. Therefore, there is a critical need for developing coordinated approaches for education and outreach programs related to mycobacterial diseases of animals.Taken together, the proposed multi-state initiativewill facilitate the development of shared research as well as the leveraging of intellectual and physical resources to address some of the most important mycobacterial diseases of animals.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1090	25%
311	3410	1100	50%
311	3410	1170	25%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1100 - Bacteriology; 1170 - Epidemiology;

Keywords

mycobacterium

johne's

tuberculosis

disease

cattle

ruminant

diagnostics

vaccines

epidemiology

paratuberculosis

Goals / Objectives
Objective 1 will focus on understanding the epidemiology and transmission of JD and TB in animals through the application of predictive modeling and assessment of recommended control practices. To accomplish our overall objective of developing a better understanding of the epidemiology and transmission of JD and TB.


Objective 2 will seek to develop and implement new generations of diagnostic tests for JD and TB. Improved methods for the rapid, specific, sensitive, and cost-efficient diagnosis of JD or TB-infected remain a major priority. Objective 3 will focus on improving our understanding of biology and pathogenesis of Mycobacterial diseases, as well as the host response to infection It is well recognized that the ability to identify the route of invasion and the host-pathogen interactions at a molecular level is important for the future development of strategies to prevent infections or to limit the spread of the infection. Similarly, the elucidation of gene products specific to in vivo growth holds great promise in identifying new antigens for diagnostics or vaccine development, as well as products essential to pathogenesis.
Hence, as part of the proposed multi-state initiative, we envision studies of the basic biology of the causative organisms of JD and TB and their interaction with the host. Specifically, we anticipate studies that will employ state-of-the-art microbiological, molecular biology, genomic, proteomic, metabolomic, immunology, and or bioinformatic approaches. Objective 4 will focus on development of programs to create and evaluate and develop new generations of vaccines for JD and TB. Under the auspices of this multi-state initiative, we propose specific research projects to help achieve each of the 4 objectives and include a strong education and extension plan. We envision many of the projects to be crosscutting in nature (i.e. cut across objectives and/or address both diseases) that will together help address the major animal, human, and societal issues surrounding detection and control of mycobacterial diseases in animals. It is important to note that our research objectives are closely linked and coordinated with our education, extension and outreach plan.

Project Methods
Objective 1will focus onunderstanding the epidemiology and transmission of Mycobacterial diseases in animals.To accomplish our overall objective of developing a better understanding of the epidemiology and transmission of JD and TBObjective 2will seek todevelop and implement new generations of diagnostic tests for JD and TB. Improved methods for the rapid, specific, sensitive, and cost-efficient diagnosis of JD or TB infected remain a major priority. Hence, as part of this multi-state initiative.Objective 3will focuson improving our understanding of biology and pathogenesis of Mycobacterial diseases of animals, as well as the host response to infection.Objective 4will focus on theevaluation and development of new generations of vaccines for JD and TB.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Scientists, veterinarians, producers, regulatory agencies, educators, policy makers, and the general public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained one undergraduate student, two graduate students, and two postdoctoral fellows.Graduate students - trained in Molecular biology and Immunology experiments. Collaborated with scientists in the United Kingdom, India and Ethiopia to move the project forward. Attended domestic and international conferences to present research findings. Undergraduate students were trained by the graduate students to assist in the lab with basic lab procedures. Postdoctoral Scientists trained in developing SNP-based RT-PCR. Collaborated with scientists at USDA to analyze Whole genome data. How have the results been disseminated to communities of interest?The results have been disseminatedthrough scientific publications and through presentations at scientific and at key stakeholder meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue to refine and roll-out the new DIVA skin test for bovine tuberculosis, including working with the OIE. We will continue torefine estimates of the burden of bTB and zoonotic tuberculosis in endemic country settings. We will continue to assess the direct and indirect efficacy of BCG as a vaccine for control of bovine tuberculosis.

Impacts
What was accomplished under these goals? We continue to make considerable progress in the development of new generations ofdiagnostic assays for both Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis. Field trials for test validation are in progress. We have also initiated field trials forthe assessment of the efficacy of Bacilllus Calette-Guerin (BCG) vaccinationfor protection against onward transmission of M. bovis in a natural transmission model. Finally, we have initiated cross-sectional surveys and molecular epidemiologic studies using whole-genome sequence informationto better estimatethe risk of zoonotic infection from members of the M. tuberculosis complex. Overall, we continue to make considerable progress in the development of new generations of diagnostic assays for both Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis. Field trials for test validation are in progress. We have also initiated field trials for the assessment of the efficacy of BCGvaccination for protection against onward transmission of M. bovis in a natural transmission model. Finally, we have initiated cross-sectional surveys and molecular epidemiologic studies using whole-genome sequence information to better estimate the risk of zoonotic infection from members of the M. tuberculosis complex. Obj. 1. Dr. Yrjo Grohn conducts experiments on the integrated approach to understand the pathways through which pathogen transmission occurs at all levels in an ecosystem. The study focuses on identifying evolutionary factors affecting the ecology and transmission of important slow transmission mycobacteria. Obj. 2. Vivek Kapur, John Bannantine, and Yrjo Grohn are investigating identifying biomarkers for early MAP infection. The team has developed a protein microarray with individual recombinant MAP proteins as a tool for sero-diagnostic antigen discovery by studying the dynamics of the humoral immune response in a baby goat model of infection from Johne's Disease Integrated Program/Mycobacterial Diseases of Animals (JDIP/MDA) vaccine project. Obj. 3. Dr. Bannantine's lab is investigating the unknown proteins encoded by the genome of MAP, and is taking a step back to describe some fundamental aspects of two proteins that they have been studying for years. These proteins, which contain NlpC/P60 domains, are being studied primarily because they are strong antigens and thus could be used to develop a diagnostic test for JD. Dr. Paul Coussens' research group analyzed Immune phenotyping data for all cows collected through 2015. Sample subjects were 256 JD-negative cows and 91 positive cows collected from eight Michigan commercial operations. The analysis included the mean relative percent of CD4+, CD8+ or sigma T cells expressing CD25. Obj. 4. Dr. Luiz Bermudez's research group has been working on oral immunization as a strategy for affecting the course of JD's disease. MAP, the cause of JD's disease, infects the host through the gastrointestinal tract. The infectious phenotype of MAP originates either in the milk or in the intestinal tract of diseased ruminants.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Li L, Bannantine JP, Campo JJ, Randall A, Grohn YT, Schilling MA, Katani R, Radzio-Basu J, Easterling L, Kapur V. Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays. Sci Rep. 2019 Nov 26;9(1):17573. doi: 10.1038/s41598-019-53973-x. PMID: 31772281; PMCID: PMC6879513.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Buza TM, Tonui T, Stomeo F, Tiambo C, Katani R, Schilling M, Lyimo B, Gwakisa P, Cattadori IM, Buza J, Kapur V. iMAP: an integrated bioinformatics and visualization pipeline for microbiome data analysis. BMC Bioinformatics. 2019 Jul 3;20(1):374. doi: 10.1186/s12859-019-2965-4. PMID: 31269897; PMCID: PMC6610863.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Srinivasan S, Subramanian S, Shankar Balakrishnan S, Ramaiyan Selvaraju K, Manomohan V, Selladurai S, Jothivelu M, Kandasamy S, Gopal DR, Kathaperumal K, Conlan AJK, Veerasami M, Bakker D, Vordermeier M, Kapur V. A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis: Proof of Concept. Front Vet Sci. 2020 Jul 24;7:391. doi: 10.3389/fvets.2020.00391. PMID: 32793643; PMCID: PMC7393633.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Duffy SC, Srinivasan S, Schilling MA, Stuber T, Danchuk SN, Michael JS, Venkatesan M, Bansal N, Maan S, Jindal N, Chaudhary D, Dandapat P, Katani R, Chothe S, Veerasami M, Robbe-Austerman S, Juleff N, Kapur V, Behr MA. Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study. Lancet Microbe. 2020 Jun;1(2):e66-e73. doi: 10.1016/S2666-5247(20)30038-0. PMID: 32642742; PMCID: PMC7325494.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Srinivasan S, Subramanian S, Balakrishnan SS, Selvaraju RK, Manomohan V, Selladurai S, Jothivelu M, Kandasamy S, Gopal DR, Kathaperumal K, Veerasami M, Bakker D, Vordermeier M, Kapur V. A defined antigen skin test that enables implementation of BCG vaccination for control of bovine Tuberculosis. Front. Vet. Sci., 24 July 2020
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Srinivasan S, Jones G, Veerasami M, Steinbach S, Holder T, Zewude A, Fromsa A, Ameni G, Easterling L, Bakker D, Juleff N, Gifford G, Hewinson RG, Vordermeier M, Kapur V. A defined antigen skin test for the diagnosis of bovine tuberculosis. Science Advances 17 Jul 2019: Vol. 5, no. 7, DOI: 10.1126/sciadv.aax4899

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists, veterinarians, producers, regulatory agencies, educators, policy makers, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained two graduate students and one post-doctoral fellow. How have the results been disseminated to communities of interest?Through scientific publications and through presentations at scientific and key stakeholder meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to refine and roll-out the new DIVA skin test for bovine tuberculosis. We will obtain better estimates of the burden of bTB and zoonotic tuberculosisin endemic country settings. We will continue to assess the direct and indirect efficacy of BCG as a vaccine for control of bovine tuberculosis.

Impacts
What was accomplished under these goals? We continue to make progress on Objectives 1, 2 and 4. For bTB - we have identified specific peptides that enable the replacement of the century old PPD test and allows for differentiation of infected from BCG vaccinated animals. This paper was published in Science Advances and has recieved considerable press as a major scientific advance in this field. We continue to field test and refine this new assay. We have also developed novel assays for the sub-sepcficdifferentiation ofM. tuberculosiscomplex organisms in animals and mand andcompleted an initial survey of the zoonotic burden of bovine tuberculosis in an endemic country setting. A high profilemanuscript describing these findings is anticiapted. For JD, we have identified a set of MAPproteins that appear to enable early serological detection of infection in animals using protein microarrays. A manuscript describing these findings is anticiapted.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Srinivasan S, Jones G, Veerasami M, Steinbach S, Holder T, Zewude A, Fromsa A, Ameni G, Easterling L, Bakker D, Juleff N, Gifford G, Hewinson RG, Vordermeier HM, Kapur V. A defined antigen skin test for the diagnosis of bovine tuberculosis. Sci Adv. 2019 Jul 17;5(7):eaax4899. doi: 10.1126/sciadv.aax4899. eCollection 2019 Jul. PubMed PMID: 31328169; PubMed Central PMCID: PMC6636981.
• Type: Books Status: Awaiting Publication Year Published: 2020 Citation: Mycobacterium paratuberculosis in man and animals. Second Edition. Edited by Marcel Behr, Karen Stevenson and Vivek Kapur. Published by CABI.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Easterling, L. An estimation of the prevalence and risk factors of bovine tuberculosis in cattle in India. MS Thesis in Animal Sciences. Penn State University, PA.

Progress 12/26/17 to 09/30/18

Outputs
Target Audience:Scientists, veterinarians, producers, regulatory agencies, educators, policy makers, and the general public. 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, in form of publication. What do you plan to do during the next reporting period to accomplish the goals?We constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure negative high exposure; fecal positive, ELISA negative; and both fecal and ELISA positive. The analyses identified a total of 49 candidate antigens with reactivity compared with the NL group (p < 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including 28 in the NH, 26 in the F+E-, and 17 in the F+E+ groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F+E-, and F+E+, respectively at 90% specificity. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F+E- groups, have potential utility for the early sero-diagnosis of MAP infection.

Impacts
What was accomplished under these goals? Bovine tuberculosis (bTB) is a chronic inflammatory disease of both zoonotic and economic significance caused by Mycobacterium bovis. Although the tuberculin skin test has been the primary ante-mortem diagnostic test for bTB since the 1890s, it has several limitations. Importantly, cattle vaccination with Bacille Calmette-Guérin (BCG) strain interferes with the specificity of the skin test by sensitizing vaccinated animals to tuberculin. Hence, a fit-for-purpose diagnostic test to differentiate infected and vaccinated animals (DIVA) is an essential prerequisite in order to introduce BCG vaccination alongside conventional test and slaughter control strategies. Recent research has demonstrated the DIVA potential of certain M. bovis antigens such as ESAT-6, CFP-10 and Rv3615c both in vitro interferon gamma (IFN-γ) release assays as well as in the in vivo skin test. In order to formulate these antigens into a defined skin test (DST) reagent, we synthesized chemical peptides representing these antigens. We have conducted in vitro and in vivo experiments in order to assess the performance characteristics of DST, and the results obtained thus far in experimental and field conditions are encouraging and provide important proof-of-principle for the rational design of defined antigens and the development of reliable, cost-effective and BCG vaccination-compatible bTB diagnostics where conventional test and cull strategies are neither feasible nor practicable. As next steps, we plan to validate performance characteristics of DST per OIE specifications in endemic country settings. Bovine tuberculosis (bTB) is a chronic disease caused by Mycobacterium bovis (M. bovis), a member of the Mycobacteria tuberculosis complex. While cows and buffaloes are the primary hosts, this zoonotic pathogen is capable of infecting humans and represents a serious threat to public health. Furthermore, many regions hosting the greatest number of cattle are bTB endemic and simultaneously report some of the highest rates of tuberculosis in humans. However, due to a lack of bTB surveillance and control programs, few accurate estimates of bTB prevalence in cattle exist. In order to refine and contribute to our understanding of bTB prevalence of bTB and important risk factors such as production system, breed, species, and sex, we conducted a cross-sectional survey of cattle in a major dairy producing region. In addition, we sought to explore the agreement between of two of the most common bTB diagnostic tests, the single-intradermal comparative skin test (SICCT) and the interferon gamma release-assay (IGRA), as well as to determine the effects of different methods of selecting animals for testing. Our findings demonstrated an immediate need for the implementation of standardized and rigorous surveillance programs in endemic regions as well as further study into the influence of environmental mycobacterial exposure or coinfection on bTB diagnostic results. Johne's disease (JD) is another mycobacterial disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) that causes production losses and remains endemic throughout the world, including high-income countries. However, to date, there is no gold standard diagnostic test for JD and the multiple existing diagnostic tests measure different biological processes with variable sensitivities and specificities, making diagnoses equally variable and difficult. In order to assess MAP diagnostic capabilities, a rigorous matched study was conducted using eight diagnostic tests representing four major test types across multiple herds. Preliminary analysis of the data underscores the variability between both individual tests and test types and demonstrates the importance of understanding how alterations in how cases are defined affect estimates of test performance and animal diagnosis.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Verteramo Chiu LJ, Tauer LW, Al-Mamun MA, Kaniyamattam K, Smith RL, Grohn YT. An agent-based model evaluation of economic control strategies for paratuberculosis in a dairy herd. J Dairy Sci. 2018 Jul;101(7):6443-6454. doi:10.3168/jds.2017-13175. Epub 2018 Apr 26. PubMed PMID: 29705432.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Rathnaiah G, Zinniel DK, Bannantine JP, Stabel JR, Gr�hn YT, Collins MT, Barletta RG. Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne's Disease. Front Vet Sci. 2017 Nov 6;4:187. doi: 10.3389/fvets.2017.00187. eCollection 2017. Review. PubMed PMID: 29164142; PubMed Central PMCID: PMC5681481.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Frie MC, Sporer KRB, Kirkpatrick BW, Coussens PM. T and B cell activation profiles from cows with and without Johne's disease in response to in vitro stimulation with Mycobacterium avium subspecies paratuberculosis. Vet Immunol Immunopathol. 2017 Dec;193-194:50-56. doi: 10.1016/j.vetimm.2017.10.005. Epub 2017 Nov 7. PubMed PMID: 29129227.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Danelishvili L, Rojony R, Carson KL, Palmer AL, Rose SJ, Bermudez LE. Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread. Virulence. 2018;9(1):1287-1300.doi:10.1080/21505594.2018.1504559. PubMedPMID: 30134761; PubMed Central PMCID: PMC6177253.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Blanchard JD, Elias V, Cipolla D, Gonda I, Bermudez LE. Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin. Antimicrob Agents Chemother. 2018 Sep 24;62(10). pii: e00440-18. doi:10.1128/AAC.00440-18. Print 2018 Oct. PubMed PMID: 30012773; PubMed Central PMCID: PMC6153787.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Samba-Louaka A, Robino E, Cochard T, Branger M, Delafont V, Aucher W, Wambeke W, Bannantine JP, Biet F, H�chard Y. Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebae. Front Cell Infect Microbiol. 2018 Feb 9;8:28. doi: 10.3389/fcimb.2018.00028. eCollection 2018. PubMed PMID: 29479518; PubMed Central PMCID: PMC5811464.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Abdellrazeq GS, Elnaggar MM, Bannantine JP, Park KT, Souza CD, Backer B, Hulubei V, Fry LM, Khaliel SA, Torky HA, Schneider DA, Davis WC. A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria. Vet Res. 2018 Jun 26;49(1):53. doi:1186/s13567-018-0549-3. PubMed PMID: 29941017; PubMed Central PMCID: PMC6019527.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Everman JL, Danelishvili L, Flores LG, Bermudez LE. MAP1203 Promotes Mycobacterium avium Subspecies paratuberculosis Binding and Invasion to Bovine Epithelial Cells. Front Cell Infect Microbiol. 2018 Jun 27;8:217. doi:10.3389/fcimb.2018.00217. eCollection 2018. PubMed PMID: 29998085; PubMed Central PMCID: PMC6030366.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bermudez LE, Rose SJ, Everman JL, Ziaie NR. Establishment of a Host-to-Host Transmission Model for Mycobacterium avium subsp. hominissuis Using Caenorhabditis elegans and Identification of Colonization-Associate Genes. Front Cell Infect Microbiol. 2018 Apr 24;8:123. Doi: 10.3389/fcimb.2018.00123.eCollection 2018. PubMed PMID: 29740544; PubMed Central PMCID: PMC5928147
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Li L, Wagner B, Freer H, Schilling M, Bannantine JP, Campo JJ, Katani R, Grohn YT, Radzio-Basu J, Kapur V. Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays. PLoS One. 2017 Dec 19;12(12):e0189783. doi:10.1371/journal.pone.0189783. eCollection 2017. PubMed PMID: 29261761; PubMed Central PMCID: PMC5736219.