NE1701: Mycobacterial Diseases of Animals

(Multistate Research Project)

Status: Inactive/Terminating

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[03/01/2018] [12/22/2021]

Date of Annual Report: 03/01/2018

Report Information

Annual Meeting Dates: 12/03/2017 - 12/05/2017
Period the Report Covers: 12/01/2017 - 09/30/2018

Participants

Bannantine, John - NADC-USDA-ARS
Barletta, Raul - University of Nebraska–Lincoln
Bermudez, luiz - Oregon State
Chang, Yung Fu - Cornell University
Coussens, Paul - Michigan State University
Gerdts, Volker - VIDO-InterVac
Gibbons-Bergener, Suzanne - Wisconsin Department of Public Health
Grohn, Yrjo - Cornell University
Johnson, Peter - USDA-NIFA
Kapur, Vivek - Penn State
Katani, Robab - Penn State
Kerhli, Marcus - USDA-ARS/Iowa State University
Morrow, Alex - Centre for Agriculture and Biosciences International, UK
Olea-Popelka, Francisco - Colorado State University
Olson, Ken - AAMD
Patton, Elizabeth - Wisconsin Department of Ag
Quinn, Fred - Univ. of Georgia
Robbe-Austerman, Suelee - USDA-APHIS-NVSL
Smith, Rebecca - Cornell University
Sreevatsan, Srinand - Michigan State Univ.
Talaat, Adel - Univ. Wisconsin
Wagner, Bettina - Cornell University

Brief Summary of Minutes

Minutes Attachment:

NE1707 Meeting Minutes.pdf

Accomplishments

Investigators have made considerable progress in the following areas: Dr. Wagner’s research group has established a new multiplex assay platform for serological differentiation of Johne’s disease stages. Comparison of individual MAP antigen-based assays indicated better differentiation of samples in the low positive range, improved analytical sensitivity, earlier detection of MAP infection especially with some antigens, and improved sensitivity and specificity even at individual assay level. Dr. Sreevatsan’s group focuses initially on the evaluation of pathogen-specific biomarkers for the diagnosis of tuberculosis and the objective of his research is on developing a noninvasive biomarker-based detection system specific for Mycobacterium bovis for monitoring infection in animals and humans. He uses the pathogen peptide as a biomarker as it is a MTC-specific biomarker, can differentiate between different stages of the disease, and it has specificity and sensitivity in low/high disease prevalence areas. His research team use the iTRAQ - isobaric Tags for Relative Quantification to determine the amount of proteins from different sources in a single experiment. Dr. Grohn’s research is to focus on the increase understanding of the epidemiology and transmission of Mycobacterial diseases including predictive modeling. The approach of this research is on developing a quantitative methodology for incorporating whole genome sequence data into bacterial transmission models for infectious diseases incorporating ecology, economics, molecular biology, and epidemiology, and to apply these methods and models towards better understanding the principles and dynamics governing transmission of mycobacterial infection. Dr. Barletta’s research focuses on the MAP K-10 MycomarT7 mutant library generation. A transposon insertion “hot spot” downstream from the MAP_4116c (mmaA4) gene resulted in a biased identification of essential genes. Dr. Bermudez’s research group focused on the M.bovis interaction with mucosal cells, the bacterial surface expression, and the adaptive (Ig)Immune Response. Dr. Bermudez also presented some freeze-fracture Transmission electron microscopy of M.bovis infected bovine macrophages. Dr. SueLee Robbe-Austerman’s effort has concluded that 95% of human cases in Baja CA and Southern CA originate from cows in the Baja CA non-accredited zone. Additionally, the study suggested that there are unique events that cause most of the spill over into humans, and little evidence of human to human transmission was determined. The data also suggested that possible human to cow transmission may have occurred. Dr. Talaat’s group works on live attenuated pathogens missing virulence or metabolism genes, by design or through passaging, maintain a balance between attenuation and virulence (M. tb vs. BCG), vaccine strain selection, and rational vaccine design, based on mechanistic studies. Dr. Patton’s group has refined testing protocols to including, communication/action checklist, animal worker questionnaire to identify elevated-risk tasks, animal risk assessment tool, inclusion of occupational health programs for abattoir workers, expanded interagency zoonotic disease communication plan, and targeted TB screening and testing programs for animal workers. Extension is led by Dr. Ken Olson, as they continue to collaborate with the VBJDCP to address education, share information, and provide tools for producers, other scientists, industry partners, and USDA. Dr. Olson also attends DC meetings with USDA leadership (APHIS, NIFA, ARS,), partner organizations (AVMA, IDFA, NMPF, NASDA, AFBF), and Congressional staff. Furthermore, his efforts concentrate on the awareness of the MDA MI among stakeholders, industry and government leaders.

Publications

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. Rathnaiah G, Zinniel DK, Bannantine JP, Stabel JR, Gröhn 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. Li L, Bannantine JP, Campo JJ, Randall A, Grohn YT, Katani R, Schilling M, Radzio-Basu J, Kapur V. Identification of sero-reactive antigens for the early diagnosis of Johne's disease in cattle. PLoS One. 2017 Sep 1;12(9):e0184373. doi: 10.1371/journal.pone.0184373. eCollection 2017. PubMed PMID: 28863177; PubMed Central PMCID: PMC5581170. Grohn YT, Carson C, Lanzas C, Pullum L, Stanhope M, Volkova V. A proposed analytic framework for determining the impact of an antimicrobial resistance intervention. Anim Health Res Rev. 2017 Jun;18(1):1-25. doi: 10.1017/S1466252317000019. Epub 2017 May 16. Review. PubMed PMID: 28506325. Dong H, Lv Y, Sreevatsan S, Zhao D, Zhou X. Differences in pathogenicity of three animal isolates of Mycobacterium species in a mouse model. PLoS One. 2017 Aug 24;12(8):e0183666. doi: 10.1371/journal.pone.0183666. eCollection 2017. PubMed PMID: 28837698; PubMed Central PMCID: PMC5570376. Singhla T, Boonyayatra S, Punyapornwithaya V, VanderWaal KL, Alvarez J, Sreevatsan S, Phornwisetsirikun S, Sankwan J, Srijun M, Wells SJ. Factors Affecting Herd Status for Bovine Tuberculosis in Dairy Cattle in Northern Thailand. Vet Med Int. 2017;2017:2964389. doi: 10.1155/2017/2964389. Epub 2017 May 3. PubMed PMID: 28553557; PubMed Central PMCID: PMC5434264. Chunfa L, Xin S, Qiang L, Sreevatsan S, Yang L, Zhao D, Zhou X. The Central Role of IFI204 in IFN-β Release and Autophagy Activation during Mycobacterium bovis Infection. Front Cell Infect Microbiol. 2017 May 5;7:169. doi: 10.3389/fcimb.2017.00169. eCollection 2017. PubMed PMID: 28529930; PubMed Central PMCID: PMC5418236. Li L, Bannantine JP, Campo JJ, Randall A, Grohn YT, Katani R, Schilling M, Radzio-Basu J, Kapur V. Identification of sero-reactive antigens for the early diagnosis of Johne's disease in cattle. PLoS One. 2017 Sep 1;12(9):e0184373. doi: 10.1371/journal.pone.0184373. eCollection 2017. PubMed PMID: 28863177; PubMed Central PMCID: PMC5581170. Bannantine JP, Campo JJ, Li L, Randall A, Pablo J, Praul CA, Raygoza Garay JA, Stabel JR, Kapur V. Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array. Clin Vaccine Immunol. 2017 Jul 5;24(7). pii: e00081-17. doi: 10.1128/CVI.00081-17. Print 2017 Jul. PubMed PMID: 28515134; PubMed Central PMCID: PMC5498720. Shippy DC, Lemke JJ, Berry A, Nelson K, Hines ME 2nd, Talaat AM. Superior Protection from Live-Attenuated Vaccines Directed against Johne's Disease. Clin Vaccine Immunol. 2017 Jan 5;24(1). pii: e00478-16. doi: 10.1128/CVI.00478-16. Print 2017 Jan. PubMed PMID: 27806993; PubMed Central PMCID: PMC5216426. Grant IR, Foddai ACG, Tarrant JC, Kunkel B, Hartmann FA, McGuirk S, Hansen C,  Talaat AM, Collins MT. Viable Mycobacterium avium ssp. paratuberculosis isolated  from calf milk replacer. J Dairy Sci. 2017 Dec;100(12):9723-9735. doi: 10.3168/jds.2017-13154. Epub 2017 Oct 4. PubMed PMID: 28987590. Sandoval-Azuara SE, Muñiz-Salazar R, Perea-Jacobo R, Robbe-Austerman S, Perera-Ortiz A, López-Valencia G, Bravo DM, Sanchez-Flores A, Miranda-Guzmán D, Flores-López CA, Zenteno-Cuevas R, Laniado-Laborín R, de la Cruz FL, Stuber TP. Whole genome sequencing of Mycobacterium bovis to obtain molecular fingerprints in human and cattle isolates from Baja California, Mexico. Int J Infect Dis. 2017 Oct;63:48-56. doi: 10.1016/j.ijid.2017.07.012. Epub 2017 Jul 22. PubMed PMID: 28739421. Waters WR, Vordermeier HM, Rhodes S, Khatri B, Palmer MV, Maggioli MF, Thacker TC, Nelson JT, Thomsen BV, Robbe-Austerman S, Bravo Garcia DM, Schoenbaum MA, Camacho MS, Ray JS, Esfandiari J, Lambotte P, Greenwald R, Grandison A, Sikar-Gang A, Lyashchenko KP. Potential for rapid antibody detection to identify  tuberculous cattle with non-reactive tuberculin skin test results. BMC Vet Res. 2017 Jun 7;13(1):164. doi: 10.1186/s12917-017-1085-5. PubMed PMID: 28592322; PubMed Central PMCID: PMC5463416. Malone KM, Farrell D, Stuber TP, Schubert OT, Aebersold R, Robbe-Austerman S,  Gordon SV. Updated Reference Genome Sequence and Annotation of Mycobacterium bovis AF2122/97. Genome Announc. 2017 Apr 6;5(14). pii: e00157-17. doi: 10.1128/genomeA.00157-17. PubMed PMID: 28385856; PubMed Central PMCID: PMC5383904. Bannantine JP, Campo JJ, Li L, Randall A, Pablo J, Praul CA, Raygoza Garay JA, Stabel JR, Kapur V. Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array. Clin Vaccine Immunol. 2017 Jul 5;24(7). pii: e00081-17. doi: 10.1128/CVI.00081-17. Print 2017 Jul. PubMed PMID: 28515134; PubMed Central PMCID: PMC5498720. Danelishvili L, Chinison JJJ, Pham T, Gupta R, Bermudez LE. The Voltage-Dependent Anion Channels (VDAC) of Mycobacterium avium phagosome are associated with bacterial survival and lipid export in macrophages. Sci Rep. 2017 Aug 1;7(1):7007. doi: 10.1038/s41598-017-06700-3. PubMed PMID: 28765557; PubMed Central PMCID: PMC5539096. Jeffrey B, Rose SJ, Gilbert K, Lewis M, Bermudez LE. Comparative analysis of the genomes of clinical isolates of Mycobacterium avium subsp. hominissuis regarding virulence-related genes. J Med Microbiol. 2017 Jul;66(7):1063-1075. doi: 10.1099/jmm.0.000507. Epub 2017 Jul 3. PubMed PMID: 28671535. Danelishvili L, Shulzhenko N, Chinison JJJ, Babrak L, Hu J, Morgun A, Burrows  G, Bermudez LE. Mycobacterium tuberculosis Proteome Response to Antituberculosis  Compounds Reveals Metabolic "Escape" Pathways That Prolong Bacterial Survival. Antimicrob Agents Chemother. 2017 Jun 27;61(7). pii: e00430-17. doi: 10.1128/AAC.00430-17. Print 2017 Jul. PubMed PMID: 28416555; PubMed Central PMCID: PMC5487666.  Rathnaiah G, Zinniel DK, Bannantine JP, Stabel JR, Gröhn 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.  Bannantine JP, Etienne G, Laval F, Stabel JR, Lemassu A, Daffé M, Bayles DO, Ganneau C, Bonhomme F, Branger M, Cochard T, Bay S, Biet F. Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure  of a nonribosomal peptide. Mol Microbiol. 2017 Aug;105(4):525-539. doi: 10.1111/mmi.13717. Epub 2017 Jun 15. PubMed PMID: 28558126. Park KT, Elnaggar MM, Abdellrazeq GS, Bannantine JP, Mack V, Fry LM, Davis WC. Correction: Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages. PLoS One. 2017 Jan 25;12(1):e0171059. doi: 10.1371/journal.pone.0171059. eCollection 2017. PubMed PMID: 28122067; PubMed Central PMCID: PMC5266315. Souza CD, Bannantine JP, Brown WC, Norton MG, Davis WC, Hwang JK, Ziaei P, Abdellrazeq GS, Eren MV, Deringer JR, Laws E, Cardieri MCD. A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages. J Appl Microbiol. 2017 May 14. doi: 10.1111/jam.13491. [Epub ahead of print] PubMed PMID: 28502107. Bannantine JP, Campo JJ, Li L, Randall A, Pablo J, Praul CA, Raygoza Garay JA, Stabel JR, Kapur V. Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array. Clin Vaccine Immunol. 2017 Jul 5;24(7). pii: e00081-17. doi: 10.1128/CVI.00081-17. Print 2017 Jul. PubMed PMID: 28515134; PubMed Central PMCID: PMC5498720. Venegas-Vargas C, Manning SD, Coussens PM, Roussey JA, Bartlett P, Grooms D. Bovine Leukemia Virus and Mycobacterium avium subsp. paratuberculosis Are Not Associated with Shiga Toxin-Producing Escherichia coli Shedding in Cattle. J Food Prot. 2017 Jan;80(1):86-89. doi: 10.4315/0362-028X.JFP-16-090. PubMed PMID: 28221870. 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. Ashraf A, Imran M, Yaqub T, Tayyab M, Shehzad W, Mingala CN, Chang YF. Development and validation of a loop-mediated isothermal amplification assay for  the detection of Mycoplasma bovis in mastitic milk. Folia Microbiol (Praha). 2017 Dec 14. doi: 10.1007/s12223-017-0576-x. [Epub ahead of print] PubMed PMID: 29243178.

Impact Statements

Establishment of genomic, SNP analysis and bioinformatics related to mycobacterial diseases.

Date of Annual Report: 12/22/2021

Report Information

Annual Meeting Dates: 12/22/2021 - 12/22/2021
Period the Report Covers: 01/01/2021 - 12/31/2021

Participants

Vivek Kapur, Penn State;
Robab Katani, Penn State;
Don Lein, Cornell University;
Elizabeth Patton, University of Wisconsin;
Ken Olson, KEO consulting;
Yrjo Grohn, Cornell University;
Scott J. Wells, University of Minnesota;
John Bannantine, USDA;
Fred Quinn, Univ. of Georgia;
Luiz Bermudez, Oregon State University;
Paul Coussens, Michigan State University;
Becky Smith, Cornell University;
Matt Wilson, West Virginia University;

Brief Summary of Minutes

The 2021 annual meeting of the Multistate team (NE1701) was held on 12/22/2021. The members met for two hours via Zoom link. The goals were to update the members on their recent accomplishments. We also hoped to further enhance our objectives by expanding membership to include producers groups and recruiting new leadership by having the younger generation of scientists and researchers in the MDA field.

The Executive Committee of MDA evaluated the options of holding a smaller meeting this year to review the nominations suggest recruitment strategies, and assess opportunities to improve interdependency among participants and other projects and agencies.

In summary,

The team evaluated options in identifying new members

Each member was asked to nominate several new researchers and younger generation of scientists and share their credentials with the EC of the MDA team.

The MDA will plan another meeting in the next quarter to discuss and evaluate our options and discuss the recruitment strategies.

We also evaluated joining the Brucella group, which meets every year in conjunction with CRWAD.

MDA members also suggested other options to hold the meeting in conjunction with other conferences or virtual.

During the next meeting, some of the following steps will be finalized.

Accomplishments

Below are some of the findings of the Executive Committee of the MDA from the past three years. The list of the outputs/publications from other members of the MDA are listed following the summaries. This summary includes reporting on work related to Mycobacterium paratuberculosis, Bovine TB, and zoonotic TB. Dr. Vivek Kapur, Penn State, PA In collaboration with other institutions and with funding from the Melinda and Bill Gate’s Foundation, Dr. Kapur's team has been working on a program titled, “Accelerating control of bovine tuberculosis in low- and middle-income countries (India and Ethiopia)”. Dr. Kapur’s group has made considerable progress towards MDA overall objectives and implementation. Below are some of the findings and impacts. <ul> <li>A defined antigen skin test (DST), comprising peptide antigens covering the sequences of ESAT-6, CFP-10, and Rv3615c, was developed as part of this project, which is currently being validated in the field (DOI: 10.1126/sciadv.aax4899). Safety studies with both escalated and repeat dosing procedures were performed in India under GLP conditions in cross-bred calves. Thus far, the performance of this test has been validated in 543 buffaloes in India and 450 cattle in the Sebata / Addis area in Ethiopia. The performance of DST (at two different doses) is being benchmarked against the fusion protein formulation of the defined antigen skin test (developed in APHA, UK) and the PPDs. Collaborators at the University of Cambridge have helped analyze this data and perform latent class analyses. A publication summarizing this dataset is currently being prepared for publication. A manuscript describing the performance of DST with PPDs in buffaloes (n = 543) has been designed and is being reviewed by co-authors. The performance of DST in BCG-vaccinated cross-bred calves was assessed in a field trial conducted at TANUVAS (Chennai, India), and the data is now published in Frontiers in Veterinary Science (DOI: 10.3389/fvets.2020.00391). These results provided strong evidence that the DST is particular and enables the ability to differentiate between infected and vaccinated animals (DIVA) in skin and IGRA assay formats. This, in turn, allows for implementing BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible. A similar trial to assess DIVA capability of DST in BCG-vaccinated buffaloes is currently being planned at LUVAS, Hisar, Haryana. Natural transmission trials to estimate BCG vaccine direct efficacy and impact on onward transmission are presently being performed in Ethiopia. Another trial to assess the duration of immunity conferred by BCG and the optimal revaccination intervals is also in progress.</li> <li>Kapur’s lab also performed a systematic review of the literature and a meta-analysis to estimate bTB prevalence in cattle in India and provide a foundation for the future formulation of rational disease control strategies and the accurate assessment of economic and health impact risks. The literature search was performed in accordance with PRISMA guidelines and identified 285 cross-sectional studies on bTB in cattle in India across four electronic databases and handpicked publications. Of these, 44 articles were included, contributing 82,419 cows and buffaloes across 18 states and one union territory in India. Based on a random-effects (RE) meta-regression model, the Analysis revealed a pooled prevalence estimate of 7.3% (95% CI: 5.6, 9.5), indicating that there may be an estimated 21.8 million (95% CI: 16.6, 28.4) infected cattle in India—a population more significant than the total number of dairy cows in the United States. The analyses further suggest that production system, species, breed, study location, diagnostic technique, sample size, and study period are likely moderators of bTB prevalence in India and need to be considered when developing future disease surveillance and control programs. Together with the projected increase in intensification of dairy production and the subsequent increase in the likelihood of zoonotic transmission, the results of our study suggest that attempts to eliminate tuberculosis from humans will require simultaneous consideration of bTB control in the cattle population in countries such as India.</li> <li>Kapur’s lab also developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and differentiate infected from vaccinated animals (DIVA). In laboratory assays and experimentally or naturally infected animals, the results demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the actual standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.</li> <li>In parallel, Dr. Kapur’s lab evaluated the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0-week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is particular and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.</li> <li>Kapur’s lab also aimed to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosiscomplex (MTBC) at a large referral hospital in India. They did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or inconclusive on PCR were subject to whole-genome sequencing (WGS) and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analyzed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. Identifying M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis.</li> <li>Another on-going project at Dr. Kapur’s lab was to conduct a systematic review and meta-analysis to determine the direct efficacy of BCG against bTB challenge in cattle and performed scenario analyses with dynamic transmission models incorporating direct and indirect vaccinal effects (“herd-immunity”) to assess the potential impact on herd level disease control. The analysis shows a relative risk of infection of 0.75 (95% CI: 0.68, 0.82) in 1,902 vaccinates as compared with 1,667 controls, corresponding to a direct vaccine efficacy of 25% (95% CI: 18, 32). Importantly, scenario analyses considering both direct and indirect effects suggest that disease prevalence could be driven down close to Officially TB-Free (OTF) status (<0.1%), if BCG were introduced in the next 10-year time period in low to moderate (<15%) prevalence settings, and that 50–95% of cumulative cases may be averted over the next 50 years even in high (20–40%) disease burden settings with immediate implementation of BCG vaccination. Taken together, the analyses suggest that BCG vaccination may help accelerate control of bTB in endemic settings, particularly with early implementation in the face of dairy intensification in regions that currently lack effective bTB control programs.</li> <li>Another on-going research in Dr. Kapur’s lab is on the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovisBCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.</li> <li>Finally, Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovisand related pathogenic members of the  tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 μg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes. However, the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 μg DST cocktail correctly classified eight, 10, and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 μg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72–1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9–1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55–0.98). Overall, the results suggest that, like tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.</li> </ul> Dr. John Bannatine, USDA- ARS, IA Dr. Bannatine’s projects include several genomic and vaccine studies. <ol> <li>His lab recently completed the genome sequence of a type III ovine strain of Mycobacterium avium paratuberculosis.</li> <li>He also reported on four draft genome sequences consisting of two Bison-Type and two Sheep-Type strains of Mycobacterium avium paratuberculosis.</li> <li>With a critical mass of whole-genome sequences of Mycobacterium avium paratuberculosis available, Dr. Bannantine and collaborators analyzed every IS 900 insertion present in all these genomes. The objective of Dr. Bannatine study is to characterize the distribution of the IS900element and how it affects genomic evolution and gene function of Map. A secondary goal was to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS900. IS900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the BstEII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS900 and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The number of IS900 copies ranged from 16 in the C-type isolate to 22 in the S-type subtype I isolate. A loci-by-loci sequence alignment of all IS900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. Preferential insertion motif sequences were identified for IS900 along with genes bordering all IS900 insertions. Rarely did IS900 insert within coding sequences as only three genes were disrupted in this way.</li> <li>Bannantine and collaborators also used completed genome sequences to better understand the genetic diversity among the Mycobacterium aviumsubspecies. That study demonstrated the MAP genome is more stable than its closest members belonging to the MAC complex.</li> </ol>   <ul> <li>Among the diagnostics projects, Mycobacterium avium subsp. paratuberculosis (MAP)-derived lipopeptides (L3P and L5P), which are compounds present in the MAP cell wall, were evaluated as antigens in a milk ELISA test. Dr. Bannatine’s group used L3P and L5P as capture antigens in an in-house milk ELISA to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of the milk ELISA via L3P and L5P varied by the source of milk samples, in which the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P. To clarify whether the positivity status of milk samples in milk ELISA L3P/L5P was predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.</li> <li>Bannantine also conducted a comparative genomic study to identify diagnostic sequences that distinguish M. avium subspecies strains. Several news genes were identified as diagnostic following bioinformatic approaches which were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.</li> <li>In another study, Dr. Bannatine’s objective was to define the components present in the EtOH extract, a well-known antigen of MAP. They showed that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of Map which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits. Still, this protein is not strongly immunogenic in cattle with Johne's disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in bovis EtOH extracts. A fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle.</li> <li>On the vaccine front, Dr. Bannantine’s group at USDA tested a subunit vaccine in two independent calf trials and demonstrated reduced colonization of Map in vaccinated animals in both trials. This work has attracted commercial interest as well as numerous press releases.</li> <li>Finally, this group along with Drs. Kapur and Hines have completed an immunological evaluation of a goat vaccine trial conducted a few years ago. A manuscript describing immunological parameters that led to protection with one of the vaccines is currently being drafted.</li> </ul>     Dr. Yrjo Grohn, Cornell University, NY   <ul> <li>Grohn’s research focused on linking genomic data with demographic field data; strain-specific differences in spreading patterns could be quantified for a densely sampled dairy herd. Mixed infections of dairy cows with MAP are common, and some strains spread more successfully. Infected cows remain susceptible for co-infections with other MAP genotypes. The model suggested that cows acquired infection from 1-4 other cows and spread the infection to 0-17 individuals. Reconstructed infection chains supported the hypothesis that high shedding animals that started to shed at an early age and showed a progressive infection pattern represented a greater risk for spreading MAP. Transmission of more than one genotype between animals was recorded. In this farm with a good MAP control management program, adult-to-adult contact was proposed as the most important transmission route to explain the reconstructed networks. For each isolate, at least one more likely ancestor could be inferred. Our study results help to capture underlying transmission processes and to understand the challenges of tracing MAP spread within a herd. Only the combination of precise longitudinal field data and bacterial strain type information made it possible to trace infection in such detail.</li> <li>In another work, Dr. Grohn’s work was on the results of a pan-GWAS analysis involving 318 MAP isolates and dairy cow Johne's disease phenotypes, taken from these three farms. Based on our highly curated accessory gene count the pan-GWAS Analysis identified several MAP genes associated with bovine Johne's disease phenotypes scored from these three farms, with some of the genes having functions suggestive of possible cause/effect relationships to these phenotypes. The work reported a pan-genomic comparative analysis between MAP and Mycobacterium tuberculosis, assessing functional Gene Ontology category enrichments between these taxa. Finally, they also provide a population genomic perspective on the effectiveness of herd isolation, involving closed dairy farms, in preventing MAP inter-farm cross infection on a micro-geographic scale.</li> <li>Grohn’s team also evaluated the benefits of MAP control programs when the herd is also affected by mastitis, a common disease causing the largest losses in dairy production. The effect of typically suggested MAP controls were estimated under the assumption that MAP infection increased the rate of clinical mastitis. We evaluated one hundred twenty three control strategies comprising various combinations of testing, culling, and hygiene, and found that the association of paratuberculosis with mastitis alters the ranking of specific MAP control programs, but only slightly alters the cost-benefit difference of particular MAP control components, as measured by the distribution of net present value of a representative U.S. dairy operation. In particular, although testing and culling for MAP resulted in a reduction in MAP incidence, that control led to lower net present value (NPV) per cow. When testing was used, ELISA was more economically beneficial than alternative testing regimes, especially if mastitis was explicitly modeled as more likely in MAP-infected animals, but ELISA testing was only significantly associated with higher NPV if mastitis was not included in the model at all. Additional hygiene was associated with a lower NPV per cow, although it lowered MAP prevalence. Overall, the addition of an increased risk of mastitis in MAP-infected animals did not change model recommendations as much as failing to consider.</li> <li>The group also analyzed the performance of the USDA's bovine tuberculosis (bTB) elimination protocol in a 1,000-cow closed dairy herd using an agent-based simulation model under different levels of initial bTB infection. We followed the bTB test sensitivity and specificity values used by the USDA in its model assessment. We estimated the net present value over a 20-yr horizon for a bTB-free milking herd and for bTB-infected herds following the USDA protocol. They estimated the expected time to identify the infection in the herd once it is introduced, its elimination time, the reproductive number (R0), and effective reproduction number (Re) under the USDA protocol. The optimal number of consecutive negative whole-herd tests (WHT) needed to declare a herd bTB-free with a 95% confidence under different bTB prevalence levels was derived. Our results support the minimum number of consecutive negative WHT required by the USDA protocol to declare a herd bTB-free; however, the number of consecutive negative WHT needed to eliminate bTB in a herd depends on the sensitivity and specificity of the tests. The robustness of the protocol was analyzed under conservative bTB test parameters from the literature. The cost of implementing the USDA protocol when 1 infected heifer is introduced in a 1,000-cow dairy herd is about $1,523,161. The average time until detection and the time required to eliminate bTB-infected animals from the herd, after 1 occult animal is introduced in the herd, were 735 and 119 d, respectively.</li> </ul>   Dr. Scott Wells, University of Minnesota, MN   <ul> <li>Wells’ group simulated six alternative control strategies consisting of testing adult cattle (>1 year) in the herd every 3 months using one test (in vivo or in vitro) or a combination in parallel of two tests (CFT, interferon-gamma release assay-IGRA- or enzyme-linked immunosorbent assay). Results showed no significant differences overall in the time needed to reach bTB eradication (median ranging between 61 and 82 months) or official bovine tuberculosis-free status (two consecutive negative herd tests) between any of the alternative strategies and the status quo (median ranging between 50 and 59 months). However, we demonstrate how alternative strategies can significantly reduce bTB prevalence when applied for restricted periods (6, 12 or 24 months), and in the case of IGRAc (IGRA using peptide-cocktail antigens), without incurring on higher unnecessary slaughter of animals (false positives) than the status quo in the first 6 months of the programme (p-value < .05). Enhanced understanding bTB-within-herd dynamics with the application of different control strategies help to identify optimal strategies to ultimately improve bTB control and bTB eradication from dairies in Uruguay and similar endemic settings.</li> <li>In another study, Dr. Well’s group was involved in a Study where they performed research on the Control of paratuberculosis: who, why and how- a review of 48 countries. They found out that most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the principal objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation, and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. A holistic approach across all ruminant livestock industries and long-term commitment are required for control of paratuberculosis.</li> <li>In the next study, Dr. wells group worked on the association between epidemiological and production factors and ELISA results for MAP in milk was quantified using four individual-level mixed multivariable logistic regression models that accounted for clustering of animals at the farm level. The four fitted models were one global model for all the animals assessed here, irrespective of age, and one for each of the categories of < 4-year-old, 4-8-year-old, and > 8 year-old cattle, respectively. A small proportion (4.9%; n = 2222) of the 45,652 tested samples were MAP-seropositive. Increasing age of the animals and higher somatic cell count (SCC) were both associated with increased odds for MAP positive test result in the model that included all animals. In contrast, milk production, milk protein and days in milk were negatively associated with MAP milk ELISA. Somatic cell count was positively associated with an increased risk in the models fitted for < 4-year-old and 4-8 year-old cattle. Variables describing higher milk production, milk protein content and days in milk were associated with significantly lower risk in the models for 4-8 year-old cattle and for all cattle. The results suggested that testing cows with high SCC (> 26 × 1000/ml), low milk production and within the first 60 days of lactation may maximize the odds of detecting seropositive animals. These results could be useful in helping to design better surveillance strategies based in testing of milk.</li> </ul>   Dr. Fred Quinn, University of Georgia, GA   <ul> <li>Quinn’s group performed research on the consequent increase in multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) cases, which requires that we increase our arsenal of effective drugs, particularly novel therapeutic approaches. Over the millennia, host and pathogen have evolved mechanisms and relationships that greatly influence the outcome of infection. Understanding these evolutionary interactions and their impact on bacterial clearance or host pathology will lead the way toward the rational development of new therapeutics that favor enhancing a host protective response. These host-directed therapies have recently demonstrated promising results against M. tuberculosis, adding to the effectiveness of currently available anti-mycobacterial drugs that directly kill the organism or slow mycobacterial replication. Here we review the host-pathogen interactions during M. tuberculosis infection, describe how M. tuberculosis bacilli modulate and evade the host immune system, and discuss the currently available host-directed therapies that target these bacterial factors. Rather than provide an exhaustive description of M. tuberculosis virulence factors, which falls outside the scope of this review, we will instead focus on the host-pathogen interactions that lead to increased bacterial growth or host immune evasion, and existing host-directed therapies can modulate that.</li> </ul>   Dr. Paul Coussens, Michigan State University, MI   <ul> <li>Currently lacking in the model systems of Johne's disease (JD) are undefined correlates of protection and the sources of inflammation due to JD. As an alternative to commonly studied immune responses, such as the Th1/Th2 paradigm, a non-classical Th17 immune response to Mycobacterium avium subspecies paratuberculosis (MAP) has been suggested. Indeed MAP antigens induce mRNAs encoding the Th17-associated cytokines IL-17A, IL-17F, IL-22, IL-23, IL-27, and IFNγ in CD3+ T cell cultures as determined by RT-qPCR. Although not as robust as when cultured with monocyte-derived macrophages (MDMs), MAP is able to stimulate the upregulation of these cytokines from sorted CD3+ T cells in the absence of antigen-presenting cells (APCs). CD4+ and CD8+ T cells are the main contributors of IL-17A and IL-22 in the absence of APCs. However, MAP-stimulated MDMs are the main contributor of IL-23. In vivo, JD+ cows have more circulating IL-23 than JD- cows, suggesting that this proinflammatory cytokine may be important in the etiology of JD. Our data in this study continue to indicate that Th17-like cells and associated cytokines may indeed play an important role in the immune responses to MAP infection and the development or control of JD.</li> <li>In another study, Dr. Coussens’s work was on the roles of non-classical immune responses, such as those associated with Th17 cells, in response to MAP infection and the development of clinical JD are less clear. In this review, we examine literature suggesting that Mycobacterial infections, including Mycobacterium tuberculosis, Mycobacterium bovis, and MAP, are all associated with expression of Th17 promoting cytokines (IL-23, IL-22, IL-17a). We discuss the possibility that Th17 associated cytokines, particularly IL-23, may act as contributing factors in the development and maintenance of inflammation characteristic of clinical JD. An as yet relatively unexplored source of chronic inflammation due to over expression of IL-1α and IL-1β is also presented. We further discuss the fact that, as with the typical Th1-like cytokines IFNγ and TNFα , IL-17a is not significantly expressed in CD4+ T cells from cows with clinical JD, possibly due to T cell exhaustion. Finally, we present the notion that the Th17 driving cytokine IL-23 expressed by infected macrophages and associated epithelial cells may contribute to chronic inflammation during later stages of JD.</li> <li>In another related project, the commonly studied immune responses, such as the Th1/Th2 paradigm, were evaluated. Th1/Th2 do not adequately explain host responses to MAP. A potential role for non-classical immune responses to MAP, such as that mediated by Th17 cells, has been suggested. Indeed, MAP antigens induce mRNAs encoding the cytokines IL-23 and IL-17a in bovine peripheral blood mononuclear cells (PBMCs). IL-23 and IL-17a production have both been associated with Th17-like immune responses. Th17 cells are also defined by surface expression of the IL-23 receptor (IL-23R). To determine the relative prevalence of potential Th17 cells in PBMCs from MAP test positive and MAP test negative cows, PBMCs were isolated and analyzed by immunostaining and flow cytometry. Fresh PBMCs from MAP test positive cows (n = 12) contained a significantly higher proportion of IL-23R positive cells in populations of CD4+, CD8+, and Yδ + T cells than in cells from MAP test negative cows (n = 12; p < 0.05). Treatment with MAP antigens increased the percentage of all T cell subsets with surface expression of IL-23R when compared to untreated (n = 12; p < 0.05) cells. ELISA results for IL-17a secretion revealed a higher concentration of IL-17a secreted from PBMCs treated with MAP antigen (n = 20) than from PBMCs not treated with MAP antigens (n = 20) (p < 0.001), regardless of the JD test status of source cows. Also, we observed a moderate negative correlation between JD diagnostic scores for JD + cows and plasma IL-17a concentration (n = 42; r = -0.437; p-value < 0.004). Plasma with low and mid JD- scores (n = 31; n = 9; 0.1 ≤ X < 0.3) had significantly more IL-17a when compared to plasma with high JD- scores (n = 10; 0.3 ≤ X < 0.46; p-values < 0.05). Similarly, plasma with low JD + score values (0.55 ≤ X < 1.0; n = 9) had significantly more IL-17a when compared to plasma with high JD + score values (X ≥ 2.0; n = 21; p < 0.05). Overall, plasma from JD + cows (0.55 < X ≤ 2.86; n = 41) had significantly less IL-17a than plasma from JD- cows (0 < X ≤ 0.46; n = 70). Our data suggests that Th17-like cells may indeed play a role in early immune responses to MAP infection and development or control of JD.</li> </ul>   Dr. Luiz E Bermudez, Oregon State University, OR   The title of Dr. Bermudez’s research is the study of the Pathogenesis of Mycobacterium paratuberculosis, to innovate the therapy. Below is the summary of some of the findings:   <ul> <li>Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, which results in chronic and fatal diarrhea in ruminant species and hundreds of millions of dollars in losses for the agricultural industry. Natural infection usually begins with the uptake of MAP by the epithelium of the small intestine followed by translocation and ingestion by tissue macrophages and dissemination via the lymphatic or blood system, throughout the body. To gain insights into the adaptation of MAP within phagocytic cells, a cell culture passage model mimics the interaction of MAP within intestinal epithelium, followed by uptake by macrophages and the later release of the pathogen to infect the intestinal epithelium, was previously developed. Bacteria were passed through epithelial cells for both 24 hours and ten days, and then MAP was isolated from cells and used to infected RAW 264.7 macrophages (Indirect Infection, II). Uninfected cells and RAW macrophages infected with MAP from medium without passaging via epithelial cells (Direct Infection, DI) were used as controls. After 24 hours of infection macrophages were lysed and separated from bacteria. Macrophage proteomics were analyzed by electrospray ionization tandem Mass Spectrometry. Approximately 2,700 proteins were identified in uninfected, DI and II infection groups. The comparison within the subsets of proteins showed the expression of more than two-fold identified 85, 33, and 39 proteins in DI and II infections with 24h epithelial cell passaged MAP and II with 10 days epithelial cell passaged bacteria, respectively. The heat map analysis for gene enrichment revealed the predominant functional groups in the DI group were related to cytokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. These responses were absent in the II 24 hour and II 10 days groups of the macrophage infection. In those II macrophages, we observed enrichment in cellular pathways such as cell cycle, healing, cell surface integrins and SNARE trafficking signaling in these groups. It was hypothesized that macrophages infected with the passaged II MAP would initiate stronger binding to the endothelial cells and lead to bacterial spread and dissemination. Identification of specific changes in the macrophage proteome during MAP infection significantly expands our understanding on the signaling pathways of the phagocytic cell and reflects the immune response that may allow the pathogen to survive within the host for an extended time.</li> </ul>   <ul> <li>Macrophage model description : To readily understand the progression of infection in the bovine host without using a prohibitive and not practical bovine model , our lab developed an in vitro cell culture system that mimics the interactions between the bacterium and the host intestine over the course of infection (Everman et al.). Our goal in this study was to understand at a molecular level how MAP manipulates the host mechanisms after crossing the intestinal epithelial barriers. It is unknown if MAP enters the enterocytes and immediately transverse to the lamina propria and enters macrophages or if it remains within this stages of infection for an extended period of time. To represent both possibilities infected epithelial cells for both 24 hours and 10 days. After 10 days of infection, we observed that the infected cells remained visually intact and healthy. Importantly, the bacterial cells remained within the cells and did not exit to the supernatant. After ten days, we finally lysed the MDBK cells to recover the bacterium to progress to the next stage of infection.</li> <li>Passage of MAP in epithelial cells increased the uptake and survival within the macrophage: It has been previously established that the passage of bacteria through the epithelial cells resulted in changes in bacterial phenotypes (Everman et al.). We also investigated if this change in phenotype would impact in the bacteria’s ability to be phagocytized and survive within the host. MDBK cells were infected with MAP for 24 hours followed by recovery of bacteria and infection of macrophages. Bacteria were recovered from the MDBK cells and then used to infect the Raw cells (indirect infection). The timepoints of the macrophage infection were 4 hours, 72 hours, and 120 hours. The time points were chosen to demonstrate both early and late stages of infection. After being plated and allowed to grow for four weeks, we found that there was a difference in survival and uptake between the DI and II infections. The percent uptake was increased at 4 hours in the II infection. Over the course of the infection, there was a slight increase in survival of the bacteria which had been passaged when compared to non-passage bacteria. The results indicated that passage through the epithelium alters the bacteria in a way that allows for better recognition and uptake as well as survival within macrophages.</li> <li>Macrophage protein profile changes during passage model: To determine more precisely how the changes in the bacterial phenotype during infection may alter the host response, we used the cell culture passage model for quantitative proteomics. We used plate-grown bacteria (Direct Infection (DI) or non-passaged), and epithelial cell passaged bacteria for 24 hours or 10 days (Indirect Infection (II) or passaged) to infected Raw 264.7 cells for 24 hours. The uninfected control macrophages were passaged with HBSS to serves as a control. The cells were lysed, and the proteins synthesized during the time of the infection were extracted for proteomic Analysis. It was found that approximately a total of 2,900 proteins were expressed after 24 hours in the DI, II, and uninfected groups. A total of 252 proteins from the DI infected cells were upregulated; 115 of which were statically significant. A total of 227 proteins expressed in the 24-hour II infection were upregulated and 77 of those were statically significant. A total of 237 proteins were upregulated in the indirect ten days infection, but only 78 were statically significant. The heatmap of gene ontology (GO) categorizes the proteins into KEGG pathways. These pathways are a collection of pathway maps representing molecular interaction, reaction, and relation networks. We found that these systems were different between the direct and indirect 24 hours and ten-day groups.  Gene ontology for DI proteins were cytokine-mediated signaling, cytokine production, response to interferon-gamma, cellular response to lipopolysaccharide, regulation of NF-kappa B kinase signaling, regulation of inflammatory response, cytosolic DNA-sensing pathway, and positive regulation of defense response. The II 24 hours and the II 10 days pathways included NABA extracellular matrix regulation, maintenance of location, and hyaluronan metabolic process. When the proteins of the II 24 hours and II 10 days were compared, there is some overlap of significance pathways, including those associated with ECM, exocytosis, insulin-like growth factors, wound healing, and interleukin 4 13 signalings. These confirm the less inflammatory phenotype of the host immune cells. However, the II 24 hour infection profile also has pathways for nitric oxide processing and regulation of oxidative stress which could indicate that this particular infection type is more inflammatory than the II 10 days infection. This observation may suggest that extended dwelling in the enterocyte or epithelial cell has a positive impact on the ability of MAP to remain undetected by the host immune system. Proteins associated with apoptosis were found to down-regulated in all groups. As expected, cytokine signaling was downregulated in both of the indirect infection groups while proteins associated with the TCA cycle were downregulated in the direct infection groups.</li> <li>Integrin upregulation: Itga5, Fn1, CCL4 proteins were upregulated in all groups. Adam8 upregulated in DI protein group but severely downregulated in the II 24 and II 10 days. Protein comp/fibulin1, CD36, and Itgam were upregulated in the II 24 hour, II 10 days. Interestingly, Itgam was severely downregulated in the DI infection.</li> <li>Binding assay and Endothelial migration transwell assay: Table 1 shows that after 1 hour of exposure to the endothelial cell monolayer, the II macrophages attach significantly more firmly, even after three washes. The level of fluorescence was more abundant in the II exposure of macrophage to BAOEC than in the DI infection. This indicates that the upregulation of these integrins is associated with a stronger attachment to the endothelium and possible movement to distance location throughout the body. The Table below shows that the anti-integrin CD11b was associated with the blocking of the attachment of macrophages with indirect infection but not the macrophages DI of endothelial cells. Anti-Transferrin was used as antibody control, and have no detected activity in preventing bind of macrophages to endothelial cells.</li> </ul>   Exp Groups                                  # cells attached                                   # cells attached with Ab Control Macrophages                    1143 + 11                                                  1140 + 14          Directed infected Mo                    1282 + 361                                                  1251 + 331 Indirect infected Mo                     7102 + 241,2                      Anti-CD11b    3203 + 141,3                                                                                                 Anti-CD71       7316 + 291,2 <ol> <li>P< 0.05 compared to control</li> <li>P< 0.05 compared with Directed infected macrophages</li> <li>P< 0.05 compared with anti-CD71 antibody</li> </ol>     Macrophages with II or DI bacteria were incubated with anti-CD11b or anti-CD 71 antibodies at 370C for one h, stained with DAPI, and then added to a confluent monolayer of endothelial cells. After one additional hour, monolayers were washed with HBSS, and the number of adherent phagocytic cells was quantified by fluorescence. To examine if the attachment led to actual movement across a membrane, we used a polarized membrane.   Conclusions: Characterization of how innate immune cells respond to changing bacterial phenotypes is the ultimate goal of this study. These data provide a new perspective of JD pathogenesis. Proteomics studies provide a vital source for interrogating complex disease processes and gaining insights. However, massive data banks can only be helpful when navigated in light and knowledge of current literature and understanding of the subject matter. It is also essential to pair proteomic findings with biological assays to validate the proteomic results. In this study, we used both bioinformatics and branch top assays to investigate the host-pathogen interaction.   Dr. Kenneth Olson, KEO Consulting, Extension/Outreach, IL We have been active in joining a variety of coalition letters in support of research and research funding. Each of these has included contact with all steering committee members to assure agreement in joining the letter. Below are some examples of the letters the MDA steering committee agreed to support in the past three years: MDA MI joined over 350 national, regional, and local stakeholder groups in a letter to House and Senate Agriculture Committee leaders requesting at least $11.5 billion for research infrastructure at U.S. colleges of agriculture over a period of five years. The letter argues that such an “investment is necessary to advance the critical work being done at institutions across the country to support American jobs, recruit a diversity of talent for the agricultural science pipeline, address our climate challenges, and ensure on-going U.S. leadership in food and agricultural innovation.” (May 3, 2021) (Attached as Appendix 1). MDA MI joined 42 other members of the AFRI Coalition in supporting increased funding for AFRI in the FY2021 budget (March 20, 2020)  (Attached as Appendix 2). MDA MI was among the 35 organizations requesting support of $40 million for the Agricultural Genome to Phenome Initiative that was established in the 2018 Farm Bill. The initiative recognizes the critical need for increased federal investment to advance genomics in agriculturally important plant and animal species.(April 4, 2019) (Attached as Appendix 3).

Publications

<ul> <li>Avila LN, Goncalves VSP, Perez AM. Risk of Introduction of Bovine Tuberculosis (TB) Into TB-Free Herds in Southern Bahia, Brazil, Associated With Movement of Live Cattle. Front Vet Sci. 2018;5:230; doi: 10.3389/fvets.2018.00230.</li> <li>Barandiaran S, Marfil MJ, Capobianco G, Perez Aguirreburualde MS, Zumarraga MJ, Eirin ME, et al. Epidemiology of Pig Tuberculosis in Argentina. Front Vet Sci. 2021;8:693082; doi: 10.3389/fvets.2021.693082.</li> <li>Barandiaran S, Perez Aguirreburualde MS, Marfil MJ, Martinez Vivot M, Aznar N, Zumarraga M, et al. Bayesian Assessment of the Accuracy of a PCR-Based Rapid Diagnostic Test for Bovine Tuberculosis in Swine. Front Vet Sci. 2019;6:204; doi: 10.3389/fvets.2019.00204.</li> <li>Cardenas NC, Pozo P, Lopes FPN, Grisi-Filho JHH, Alvarez J. Use of Network Analysis and Spread Models to Target Control Actions for Bovine Tuberculosis in a State from Brazil. Microorganisms. 2021;9(2); doi: 10.3390/microorganisms9020227.</li> <li>Carneiro PA, Zimpel CK, Pasquatti TN, Silva-Pereira TT, Takatani H, Silva C, et al. Genetic Diversity and Potential Paths of Transmission of Mycobacterium bovis in the Amazon: The Discovery of M. bovis Lineage Lb1 Circulating in South America. Front Vet Sci. 2021;8:630989; doi: 10.3389/fvets.2021.630989.</li> <li>Carneiro PAM, Takatani H, Pasquatti TN, Silva C, Norby B, Wilkins MJ, et al. Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil. Front Vet Sci. 2019;6:434; doi: 10.3389/fvets.2019.00434.</li> <li>de la Cruz ML, Pozo P, Grau A, Nacar J, Bezos J, Perez A, et al. assessment of the sensitivity of the bovine tuberculosis eradication program in a high prevalence region of Spain using scenario tree modeling. Prev Vet Med. 2019;173:104800; doi: 10.1016/j.prevetmed.2019.104800.</li> <li>Duffy SC, Srinivasan S, Schilling MA, Stuber T, Danchuk SN, Michael JS, et al. Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study. Lancet Microbe. 2020;1(2):e66-e73; doi: 10.1016/S2666-5247(20)30038-0.</li> <li>Hadi SA, Brenner EP, Mani R, Palmer MV, Thacker T, Sreevatsan S. Genome Sequences of Mycobacterium tuberculosis Biovar bovis Strains Ravenel and 10-7428. Microbiol Resour Announc. 2021;10(24):e0041121; doi: 10.1128/MRA.00411-21.</li> <li>Islam SKS, Rumi TB, Kabir SML, van der Zanden AGM, Kapur V, Rahman A, et al. Bovine tuberculosis prevalence and risk factors in selected districts of Bangladesh. PLoS One. 2020;15(11):e0241717; doi: 10.1371/journal.pone.0241717.</li> <li>Kakaire, R., N. Kiwanuka, S. Zalwango, J.N. Sekandi, T.H.T. Quach, M.E. Castellanos, F. Quinn, and C.C. 2020. Whalen. Excess risk of tuberculous infection among extra-household contacts of tuberculosis cases in an African city. Clin Infect Dis. Oct 16:ciaa1556.</li> <li>Kanankege KST, Alvarez J, Zhang L, Perez AM. An Introductory Framework for Choosing Spatiotemporal Analytical Tools in Population-Level Eco-Epidemiological Research. Front Vet Sci. 2020;7:339; doi: 10.3389/fvets.2020.00339.</li> <li>Kao SZ, VanderWaal K, Enns EA, Craft ME, Alvarez J, Picasso C, et al. Modeling cost-effectiveness of risk-based bovine tuberculosis surveillance in Minnesota. Prev Vet Med. 2018;159:1-11; doi: 10.1016/j.prevetmed.2018.08.011.</li> <li>Lombard JE, Patton EA, Gibbons-Burgener SN, Klos RF, Tans-Kersten JL, Carlson BW, et al. Human-to-Cattle Mycobacterium tuberculosis Complex Transmission in the United States. Front Vet Sci. 2021;8:691192; doi: 10.3389/fvets.2021.691192.</li> <li>Martinez, L., Y. Shen, A. Handel, S. Chakraburty, C.M. Stein, L.L. Malone, W.H. Boom, D. Quinn, M.L. Joloba, C.C. Whalen and S. Zalwango. Effectiveness of WHO's pragmatic screening algorithm for child contacts of tuberculosis cases in resource-constrained settings: a prospective cohort study in Uganda. 2018. Lancet Respir Med. 6(4):276-286.</li> <li>Paudel S, Brenner EP, Hadi SA, Suzuki Y, Nakajima C, Tsubota T, et al. Genome Sequences of Two Mycobacterium tuberculosis Isolates from Asian Elephants in Nepal. Microbiol Resour Announc. 2021;10(36):e0061421; doi: 10.1128/MRA.00614-21.</li> <li>Paudel S, Sreevatsan S. Tuberculosis in elephants: Origins and evidence of interspecies transmission. Tuberculosis (Edinb). 2020;123:101962; doi: 10.1016/j.tube.2020.101962.</li> <li>Picasso-Risso C, Alvarez J, VanderWaal K, Kinsley A, Gil A, Wells SJ, et al. Modelling the effect of test-and-slaughter strategies to control bovine tuberculosis in endemic high prevalence herds. Transbound Emerg Dis. 2021;68(3):1205-15; doi: 10.1111/tbed.13774.</li> <li>Picasso-Risso C, Perez A, Gil A, Nunez A, Salaberry X, Suanes A, et al. Modeling the Accuracy of Two in-vitro Bovine Tuberculosis Tests Using a Bayesian Approach. Front Vet Sci. 2019;6:261; doi: 10.3389/fvets.2019.00261.</li> <li>Pozo P, Cardenas NC, Bezos J, Romero B, Grau A, Nacar J, et al. Evaluation of the performance of slaughterhouse surveillance for bovine tuberculosis detection in Castilla y Leon, Spain. Prev Vet Med. 2021;189:105307; doi: 10.1016/j.prevetmed.2021.105307.</li> <li>Pozo P, Romero B, Bezos J, Grau A, Nacar J, Saez JL, et al. Evaluation of Risk Factors Associated With Herds With an Increased Duration of Bovine Tuberculosis Breakdowns in Castilla y Leon, Spain (2010-2017). Front Vet Sci. 2020;7:545328; doi: 10.3389/fvets.2020.545328.</li> <li>Pozo P, VanderWaal K, Grau A, de la Cruz ML, Nacar J, Bezos J, et al. Analysis of the cattle movement network and its association with the risk of bovine tuberculosis at the farm level in Castilla y Leon, Spain. Transbound Emerg Dis. 2019;66(1):327-40; doi: 10.1111/tbed.13025.</li> <li>Pullen MF, Boulware DR, Sreevatsan S, Bazira J. Tuberculosis at the animal-human interface in the Ugandan cattle corridor using a third-generation sequencing platform: a cross-sectional analysis study. BMJ Open. 2019;9(4):e024221; doi: 10.1136/bmjopen-2018-024221.</li> <li>Rufai SB, McIntosh F, Poojary I, Chothe S, Sebastian A, Albert I, et al. Complete Genome Sequence of Mycobacterium orygis Strain 51145. Microbiol Resour Announc. 2021;10(1); doi: 10.1128/MRA.01279-20.</li> <li>Salvador LCM, O'Brien DJ, Cosgrove MK, Stuber TP, Schooley AM, Crispell J, et al. Disease management at the wildlife-livestock interface: Using whole-genome sequencing to study the role of elk in Mycobacterium bovis transmission in Michigan, USA. Mol Ecol. 2019;28(9):2192-205; doi: 10.1111/mec.15061.</li> <li>Singhla T, Boonyayatra S, Chulakasian S, Lukkana M, Alvarez J, Sreevatsan S, Wells SJ. 2019.  Determination of the sensitivity and specificity of bovine tuberculosis screening tests in dairy herds in Thailand using a Bayesian approach.  BMC Vet Res. 2019, May 16;15(1):149.</li> <li>Srinivasan S, Easterling L, Rimal B, Niu XM, Conlan AJK, Dudas P, et al. Prevalence of Bovine Tuberculosis in India: A systematic review and meta-analysis. Transbound Emerg Dis. 2018;65(6):1627-40; doi: 10.1111/tbed.12915.</li> <li>Verteramo Chiu LJ, Tauer LW, Smith RL, Grohn YT. Assessment of the bovine tuberculosis elimination protocol in the United States. J Dairy Sci. 2019;102(3):2384-400; doi: 10.3168/jds.2018-14990.</li> <li>Wanzala SI, Nakavuma J, Travis D, Kia P, Ogwang S, Waters WR, et al. Retrospective Analysis of Archived Pyrazinamide Resistant Mycobacterium tuberculosis Complex Isolates from Uganda-Evidence of Interspecies Transmission. Microorganisms. 2019;7(8); doi: 10.3390/microorganisms7080221.</li> <li>Carneiro PAM, de Moura Sousa E, Viana RB, Monteiro BM, do Socorro Lima Kzam A, de Souza DC, et al. Study on supplemental test to improve the detection of bovine tuberculosis in individual animals and herds. BMC Vet Res. 2021;17(1):137; doi: 10.1186/s12917-021-02839-4.</li> <li>de la Cruz ML, Branscum AJ, Nacar J, Pages E, Pozo P, Perez A, et al. Evaluation of the Performance of the IDvet IFN-Gamma Test for Diagnosis of Bovine Tuberculosis in Spain. Front Vet Sci. 2018;5:229; doi: 10.3389/fvets.2018.00229.</li> <li>Duffy SC, Venkatesan M, Chothe S, Poojary I, Verghese VP, Kapur V, et al. Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory. Microbiol Spectr. 2021:e0109821; doi: 10.1128/Spectrum.01098-21.</li> <li>Hadi SA, Waters WR, Palmer M, Lyashchenko KP, Sreevatsan S. Development of a Multidimensional Proteomic Approach to Detect Circulating Immune Complexes in Cattle Experimentally Infected With Mycobacterium bovis. Front Vet Sci. 2018;5:141; doi: 10.3389/fvets.2018.00141.</li> <li>Kumar T, Singh M, Jangir BL, Arora D, Srinivasan S, Bidhan D, et al. A Defined Antigen Skin Test for Diagnosis of Bovine Tuberculosis in Domestic Water Buffaloes (Bubalus bubalis). Front Vet Sci. 2021;8:669898; doi: 10.3389/fvets.2021.669898.</li> <li>Ortega J, Roy A, Alvarez J, Sanchez-Cesteros J, Romero B, Infantes-Lorenzo JA, et al. Effect of the Inoculation Site of Bovine and Avian Purified Protein Derivatives (PPDs) on the Performance of the Intradermal Tuberculin Test in Goats From Tuberculosis-Free and Infected Herds. Front Vet Sci. 2021;8:722825; doi: 10.3389/fvets.2021.722825.</li> <li>Singhla T, Boonyayatra S, Chulakasian S, Lukkana M, Alvarez J, Sreevatsan S, et al. Determination of the sensitivity and specificity of bovine tuberculosis screening tests in dairy herds in Thailand using a Bayesian approach. BMC Vet Res. 2019;15(1):149; doi: 10.1186/s12917-019-1905-x.</li> <li>Srinivasan S, Jones G, Veerasami M, Steinbach S, Holder T, Zewude A, et al. A defined antigen skin test for the diagnosis of bovine tuberculosis. Sci Adv. 2019;5(7):eaax4899; doi: 10.1126/sciadv.aax4899.</li> <li>Srinivasan S, Subramanian S, Shankar Balakrishnan S, Ramaiyan Selvaraju K, Manomohan V, Selladurai S, et al. A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis: Proof of Concept. Front Vet Sci. 2020;7:391; doi: 10.3389/fvets.2020.00391.</li> </ul>   <ul> <li>Abreu R, L. Essler, A. Loy,  Quinn, and P. Giri. 2018. Heparin inhibits intracellular Mycobacterium tuberculosisbacterial replication by reducing iron levels in human macrophages. Sci Rep. 8;8(1):7296.</li> <li>Abreu, R., P. Giri, and  Quinn. 2020. Interferon-γ promotes iron export in human macrophages to limit intracellular bacterial replication. PLOS ONE. PLoS One. Dec 8;15(12):e0240949.</li> <li>Alyamani EJ, Marcus SA, Ramirez-Busby SM, Hansen C, Rashid J, El-Kholy A, et al. Publisher Correction: Genomic Analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East. Sci Rep. 2019;9(1):20268; doi: 10.1038/s41598-019-55790-8.</li> <li>Bahr NC, Halupnick R, Linder G, Kiggundu R, Nabeta HW, Williams DA, et al. Delta-like 1 protein, vitamin D binding protein and fetuin for detection of Mycobacterium tuberculosis meningitis. Biomark Med. 2018;12(7):707-16; doi: 10.2217/bmm-2017-0373.</li> <li>Baker JJ, Abramovitch RB. Genetic and metabolic regulation of Mycobacterium tuberculosis acid growth arrest. Sci Rep. 2018;8(1):4168; doi: 10.1038/s41598-018-22343-4.</li> <li>Baker JJ, Dechow SJ, Abramovitch RB. Acid Fasting: Modulation of Mycobacterium tuberculosis Metabolism at Acidic pH. Trends Microbiol. 2019;27(11):942-53; doi: 10.1016/j.tim.2019.06.005.</li> <li>Carneiro PAM, Pasquatti TN, Takatani H, Zumarraga MJ, Marfil MJ, Barnard C, et al. Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil. Vet Med Sci. 2020;6(1):133-41; doi: 10.1002/vms3.203.</li> <li>Daniel-Wayman S, Abate G, Barber DL, Bermudez LE, Coler RN, Cynamon MH, et al. Advancing Translational Science for Pulmonary Nontuberculous Mycobacterial Infections. A Road Map for Research. Am J Respir Crit Care Med. 2019;199(8):947-51; doi: 10.1164/rccm.201807-1273PP.</li> <li>Gomez-Buendia A, Romero B, Bezos J, Lozano F, de Juan L, Alvarez J. Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis. BMC Vet Res. 2021;17(1):148; doi: 10.1186/s12917-021-02848-3.</li> <li>Grooms DL, Bolin SR, Plastow JL, Lim A, Hattey J, Durst PT, et al. Survival of Mycobacterium bovis during forage ensiling. Am J Vet Res. 2019;80(1):87-94; doi: 10.2460/ajvr.80.1.87.</li> <li>Grosse-Siestrup, B.T., T. Gupta, S. Helms, S.L. Tucker, M.I. Voskuil, D. Quinn, and R.K.  Karls. 2021. A role for Mycobacterium tuberculosissigma factor C in copper nutritional immunity. Int J Mol Sci. 22(4):2118.</li> <li>Kuo CJ, Gao J, Huang JW, Ko TP, Zhai C, Ma L, et al. Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis. Biochim Biophys Acta Gen Subj. 2019;1863(9):1351-9; doi: 10.1016/j.bbagen.2019.06.003.</li> <li>Steinbach S, Jalili-Firoozinezhad S, Srinivasan S, Melo MB, Middleton S, Konold T, et al. Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles. Sci Rep. 2021;11(1):7074; doi: 10.1038/s41598-021-86398-6.</li> <li>Verteramo Chiu LJ, Tauer LW, Grohn YT, Smith RL. Mastitis risk effect on the economic consequences of paratuberculosis control in dairy cattle: A stochastic modeling study. PLoS One. 2019;14(9):e0217888; doi: 10.1371/journal.pone.0217888.</li> <li>Wanzala SI, Nakavuma J, Travis D, Kia P, Ogwang S, Waters WR, et al. Retrospective Analysis of Archived Pyrazinamide Resistant Mycobacterium tuberculosis Complex Isolates from Uganda-Evidence of Interspecies Transmission. Microorganisms. 2019;7(8); doi: 10.3390/microorganisms7080221</li> <li>Yassine, E., R. Galiwango, W. Ssengooba, F. Ashaba, M.L. Joloba, S. Zalwango, C. Whalen, and F. Quinn. 2021. Assessing transmission of Mycobacterium tuberculosis in a defined social network using single nucleotide polymorphism threshold analysis. Microbiologyopen. 2021 Jun;10(3):e1211. doi: 10.1002/mbo3.1211.</li> </ul>   <ul> <li>Abdelaal HFM, Spalink D, Amer A, Steinberg H, Hashish EA, Nasr EA, et al. Genomic Polymorphism Associated with the Emergence of Virulent Isolates of Mycobacterium bovis in the Nile Delta. Sci Rep. 2019;9(1):11657; doi: 10.1038/s41598-019-48106-3.</li> <li>Abreu R, Giri P, Quinn F. Host-Pathogen Interaction as a Novel Target for Host-Directed Therapies in Tuberculosis. Front Immunol. 2020;11:1553; doi: 10.3389/fimmu.2020.01553.</li> <li>Ali ZI, Hanafy M, Hansen C, Saudi AM, Talaat AM. Genotypic Analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin. J Dairy Sci. 2021;104(1):211-20; doi: 10.3168/jds.2020-18214.</li> <li>Alyamani EJ, Marcus SA, Ramirez-Busby SM, Hansen C, Rashid J, El-Kholy A, et al. Genomic Analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East. Sci Rep. 2019;9(1):4474; doi: 10.1038/s41598-019-41162-9.</li> <li>Chen Y, Danelishvili L, Rose SJ, Bermudez LE. Mycobacterium bovis BCG Surface Antigens Expressed under the Granuloma-Like Conditions as Potential Inducers of the Protective Immunity. Int J Microbiol. 2019;2019:9167271; doi: 10.1155/2019/9167271.</li> <li>Gupta, T, M. LaGatta, S. Helms, R.L. Pavlicek, S.O. Owino, K. Sakamoto, T. Nagy, S.B. Harvey, M. Papania, S. Ledden, K.T. Schultz, C. McCombs, D.Quinn, and RK Karls. 2018. Evaluation of a temperature-restricted, mucosal tuberculosis vaccine in guinea pigs. Tuberculosis. 113:179-188.</li> <li>Marais, B.J., B.M. Buddle, L-M. de Klerk-Lorist, P. Nguipdop-Djomo,  Quinn, and C. Greenblatt. 2019. BCG vaccination for bovine tuberculosis; conclusions from the Jerusalem One Health Workshop. Transbound Emerging Dis. 66(2):1037-1043.</li> <li>Srinivasan S, Conlan AJK, Easterling LA, Herrera C, Dandapat P, Veerasami M, et al. A Meta-Analysis of the Effect of Bacillus Calmette-Guerin Vaccination Against Bovine Tuberculosis: Is Perfect the Enemy of Good? Front Vet Sci. 2021;8:637580; doi: 10.3389/fvets.2021.637580.</li> <li>Alvarez, J., D. Bakker, and J. Bezos, Editorial: Epidemiology and Control of Notifiable Animal Diseases. Front Vet Sci, 2019. 6: p. 43.</li> <li>Barkema HW, Orsel K, Nielsen SS, Koets AP, Rutten VPMG, Bannantine JP, Keefe GP, Kelton DF, Wells SJ, Whittington RJ, Mackintosh CG, Manning EJ, Weber MF, Heuer C, Forde TL, Ritter C, Roche S, Corbett CS, Wolf R, Griebel PJ, Kastelic JP, De Buck J. 2018. Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection. Transbound Emerg Dis. 65 Suppl 1:125-148.</li> <li>Giannitti, F., M. Fraga, R.D. Caffarena, C.O. Schild, G. Banchero, A.G. Armien, G. Traveria, D. Marthaler, S.J. Wells, and F. Riet-Correa, Mycobacterium paratuberculosis sheep type strain in Uruguay: Evidence for a wider geographic distribution in South America. J Infect Dev Ctries, 2018. 12(3): p. 190-195.</li> <li>Kanankege KS, Nicholas B. Phelps, Heidi Vesterinen, Kaylee M. Errecaborde, Julio Alvarez, Jeffrey B. Bender, Scott Wells, Andres M. Perez. 2020. Lessons learned from the stakeholder engagement in research: application of spatial analytical tools in One Health problems. Frontiers, 7:254.</li> <li>Kanankege KST, Machado G, Zhang L, Dokkebakken B, Schumann V, Wells SJ, Perez AM, Alvarez J.  2019. Use of a voluntary testing program to study the spatial epidemiology of Johne’s disease affecting dairy herds in Minnesota: A cross sectional study.  BMC Vet Research, 15:429.</li> <li>Machado, G., K. Kanankege, V. Schumann, S. Wells, A. Perez, and J. Alvarez, Identifying individual animal factors associated with Mycobacterium avium subsp. paratuberculosis (MAP) milk ELISA positivity in dairy cattle in the Midwest region of the United States. BMC Vet Res, 2018. 14(1): p. 28.</li> <li>Samba-Louaka, A., E. Robino, T. Cochard, M. Branger, V. Delafont, W. Aucher, W. Wambeke, J.P. Bannantine, F. Biet, and Y. Hechard, Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebae. Front Cell Infect Microbiol, 2018. 8: p. 28.</li> <li>Smiley Evans, T., Z. Shi, M. Boots, W. Liu, K.J. Olival, X. Xiao, S. Vandewoude, H. Brown, J.L. Chen, D.J. Civitello, L. Escobar, Y. Grohn, H. Li, K. Lips, Q. Liu, J. Lu, B. Martinez-Lopez, J. Shi, X. Shi, B. Xu, L. Yuan, G. Zhu, and W.M. Getz, Synergistic China-US Ecological Research is Essential for Global Emerging Infectious Disease Preparedness. Ecohealth, 2020. 17(1): p. 160-173.</li> <li>Stabel, J.R., J.P. Bannantine, and J.M. Hostetter, Comparison of Sheep, Goats, and Calves as Infection Models for Mycobacterium avium subsp. paratuberculosis. Vet Immunol Immunopathol, 2020. 225: p. 110060.</li> <li>Whittington, R., K. Donat, M.F. Weber, D. Kelton, S.S. Nielsen, S. Eisenberg, N. Arrigoni, R. Juste, J.L. Saez, N. Dhand, A. Santi, A. Michel, H. Barkema, P. Kralik, P. Kostoulas, L. Citer, F. Griffin, R. Barwell, M.A.S. Moreira, I. Slana, H. Koehler, S.V. Singh, H.S. Yoo, G. Chavez-Gris, A. Goodridge, M. Ocepek, J. Garrido, K. Stevenson, M. Collins, B. Alonso, K. Cirone, F. Paolicchi, L. Gavey, M.T. Rahman, E. de Marchin, W. Van Praet, C. Bauman, G. Fecteau, S. McKenna, M. Salgado, J. Fernandez-Silva, R. Dziedzinska, G. Echeverria, J. Seppanen, V. Thibault, V. Fridriksdottir, A. Derakhshandeh, M. Haghkhah, L. Ruocco, S. Kawaji, E. Momotani, C. Heuer, S. Norton, S. Cadmus, A. Agdestein, A. Kampen, J. Szteyn, J. Frossling, E. Schwan, G. Caldow, S. Strain, M. Carter, S. Wells, M. Munyeme, R. Wolf, R. Gurung, C. Verdugo, C. Fourichon, T. Yamamoto, S. Thapaliya, E. Di Labio, M. Ekgatat, A. Gil, A.N. Alesandre, J. Piaggio, A. Suanes, and J.H. de Waard, Control of paratuberculosis: who, why and how. A review of 48 countries. BMC Vet Res, 2019. 15(1): p. 198.</li> <li>Richards, V.P., A. Nigsch, P. Pavinski Bitar, Q. Sun, T. Stuber, K. Ceres, R.L. Smith, S. Robbe Austerman, Y. Schukken, Y.T. Grohn, and M.J. Stanhope, Evolutionary genomics and bacteria GWAS Analysis of Mycobacterium avium subsp. paratuberculosis and dairy cattle Johne's disease phenotypes. Appl Environ Microbiol, 2021.</li> <li>Abdellrazeq, G.S., L.M. Fry, M.M. Elnaggar, J.P. Bannantine, D.A. Schneider, W.M. Chamberlin, A.H.A. Mahmoud, K.T. Park, V. Hulubei, and W.C. Davis, Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine. Vaccine, 2020. 38(8): p. 2016-2025.</li> <li>Bannantine, J.P., J.R. Stabel, D.O. Bayles, C. Conde, and F. Biet, Diagnostic Sequences That Distinguish M. avium Subspecies Strains. Front Vet Sci, 2020. 7: p. 620094.</li> <li>Bay, S., D. Begg, C. Ganneau, M. Branger, T. Cochard, J.P. Bannantine, H. Kohler, J.L. Moyen, R.J. Whittington, and F. Biet, Engineering Synthetic Lipopeptide Antigen for Specific Detection of Mycobacterium avium subsp. paratuberculosis Infection. Front Vet Sci, 2021. 8: p. 637841.</li> <li>Cinar, M.U., B. Akyuz, K. Arslan, S.N. White, H.L. Neibergs, and K.S. Gumussoy, The EDN2 rs110287192 gene polymorphism is associated with paratuberculosis susceptibility in multibreed cattle population. PLoS One, 2020. 15(9): p. e0238631.</li> <li>Conde, C., M. Price-Carter, T. Cochard, M. Branger, K. Stevenson, R. Whittington, J.P. Bannantine, and F. Biet, Whole-Genome Analysis of Mycobacterium avium subsp. paratuberculosis IS900 Insertions Reveals Strain Type-Specific Modalities. Front Microbiol, 2021. 12: p. 660002.</li> <li>Greenstein, R.J., L. Su, P.S. Fam, J.R. Stabel, and S.T. Brown, Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay. BMC Res Notes, 2018. 11(1): p. 498.</li> <li>Jenvey, C.J., J.M. Hostetter, A.L. Shircliff, and J.R. Stabel, Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease. Vet Immunol Immunopathol, 2018. 202: p. 93-101.</li> <li>Li, L., J.P. Bannantine, J.J. Campo, A. Randall, Y.T. Grohn, M.A. Schilling, R. Katani, J. Radzio-Basu, L. Easterling, and V. Kapur, Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays. Sci Rep, 2019. 9(1): p. 17573.</li> <li>Machado G, Kanankege KST, Schumann V, Wells SJ, Perez AM, Alvarez J. Identifying individual animal factors associated with Mycobacterium avium subsp. paratuberculosis (MAP) milk ELISA positivity in dairy cattle in the Midwest region of the United States. BMC Vet Res 14(1):28.</li> <li>Picasso-Risso, C., A. Grau, D. Bakker, J. Nacar, O. Minguez, A. Perez, and J. Alvarez, Association between diagnostic tests for bovine tuberculosis and Johne's disease in cattle. Vet Rec, 2019. 185(22): p. 693.</li> </ul>   <ul> <li>Al-Mamun, M.A., R.L. Smith, A. Nigsch, Y.H. Schukken, and Y.T. Grohn, A data-driven individual-based model of infectious disease in livestock operation: A validation study for paratuberculosis. PLoS One, 2018. 13(12): p. e0203177.</li> <li>Babrak, L. and L.E. Bermudez, Response of the respiratory mucosal cells to mycobacterium avium subsp. Hominissuis microaggregate. Arch Microbiol, 2018. 200(5): p. 729-742.</li> <li>Bannantine, J.P. and D.O. Bayles, Draft Genome Sequences of Two Bison-Type and Two Sheep-Type Strains of Mycobacterium avium subsp. paratuberculosis. Microbiol Resour Announc, 2021. 10(28): p. e0052621.</li> <li>Bannantine, J.P., A. Wadhwa, J.R. Stabel, and S. Eda, Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis. Vet Sci, 2019. 6(4).</li> <li>Bannantine, J.P., C. Conde, D.O. Bayles, M. Branger, and F. Biet, Genetic Diversity Among Mycobacterium avium Subspecies Revealed by Analysis of Complete Genome Sequences. Front Microbiol, 2020. 11: p. 1701.</li> <li>Bannantine, J.P., D.K. Zinniel, and R.G. Barletta, Transposon Mutagenesis in Mycobacterium avium Subspecies Paratuberculosis. Methods Mol Biol, 2019. 2016: p. 117-125.</li> <li>Bannantine, J.P., D.O. Bayles, and F. Biet, Complete Genome Sequence of a Type III Ovine Strain of Mycobacterium avium subsp. paratuberculosis. Microbiol Resour Announc, 2021. 10(10).</li> <li>Bannantine, J.P., J.R. Stabel, J.D. Lippolis, and T.A. Reinhardt, Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies. Microorganisms, 2018. 6(4).</li> <li>Bechler, J. and L.E. Bermudez, Investigating the Role of Mucin as Frontline Defense of Mucosal Surfaces against Mycobacterium avium Subsp. hominissuis. J Pathog, 2020. 2020: p. 9451591.</li> <li>Bermudez, L.E., S.J. Rose, J.L. Everman, and N.R. Ziaie, Establishment of a Host-to-Host Transmission Model for Mycobacterium avium subsp. hominissuis Using Caenorhabditis elegans and Identification of Colonization-Associated Genes. Front Cell Infect Microbiol, 2018. 8: p. 123.</li> <li>Blanchard, J.D., V. Elias, D. Cipolla, I. Gonda, and L.E. Bermudez, 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. 62(10).</li> <li>Caldeira, J.L.A., A.C.S. Faria, E.A. Diaz-Miranda, T.J. Zilch, S.L. da Costa Caliman, D.S. Okano, J.D. Guimaraes, J.L. Pena, W.F. Barbosa, A.S. Junior, Y.F. Chang, and M.A.S. Moreira, Interaction of Mycobacterium avium subsp. paratuberculosis with bovine sperm. Theriogenology, 2021. 161: p. 228-236.</li> <li>Chiplunkar, S.S., C.A. Silva, L.E. Bermudez, and L. Danelishvili, Characterization of membrane vesicles released by Mycobacterium avium in response to environment mimicking the macrophage phagosome. Future Microbiol, 2019. 14: p. 293-313.</li> <li>Danelishvili, L., E. Armstrong, E. Miyasako, B. Jeffrey, and L.E. Bermudez, Exposure of Mycobacterium avium subsp. homonissuis to Metal Concentrations of the Phagosome Environment Enhances the Selection of Persistent Subpopulation to Antibiotic Treatment. Antibiotics (Basel), 2020. 9(12).</li> <li>Danelishvili, L., R. Rojony, K.L. Carson, A.L. Palmer, S.J. Rose, and L.E. Bermudez, Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread. Virulence, 2018. 9(1): p. 1287-1300.</li> <li>DeKuiper, J.L. and P.M. Coussens, Inflammatory Th17 responses to infection with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle and their potential role in development of Johne's disease. Vet Immunol Immunopathol, 2019. 218: p. 109954.</li> <li>DeKuiper, J.L. and P.M. Coussens, Mycobacterium avium sp. paratuberculosis (MAP) induces IL-17a production in bovine peripheral blood mononuclear cells (PBMCs) and enhances IL-23R expression in-vivo and in-vitro. Vet Immunol Immunopathol, 2019. 218: p. 109952.</li> <li>DeKuiper, J.L., H.E. Cooperider, N. Lubben, C.M. Ancel, and P.M. Coussens, Mycobacterium avium Subspecies paratuberculosis Drives an Innate Th17-Like T Cell Response Regardless of the Presence of Antigen-Presenting Cells. Front Vet Sci, 2020. 7: p. 108.</li> <li>Everman, J.L., L. Danelishvili, L.G. Flores, and L.E. Bermudez, MAP1203 Promotes Mycobacterium avium Subspecies paratuberculosis Binding and Invasion to Bovine Epithelial Cells. Front Cell Infect Microbiol, 2018. 8: p. 217.</li> <li>Franceschi, V., A.H. Mahmoud, G.S. Abdellrazeq, G. Tebaldi, F. Macchi, L. Russo, L.M. Fry, M.M. Elnaggar, J.P. Bannantine, K.T. Park, V. Hulubei, S. Cavirani, W.C. Davis, and G. Donofrio, Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells. Front Immunol, 2019. 10: p. 2859.</li> <li>Hosseiniporgham, S., F. Biet, C. Ganneau, J.P. Bannantine, S. Bay, and L.A. Sechi, A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test. Vaccines (Basel), 2021. 9(9).</li> <li>Jenvey, C.J., J.M. Hostetter, A.L. Shircliff, J.P. Bannantine, and J.R. Stabel, Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne's Disease. Vet Pathol, 2019. 56(5): p. 671-680.</li> <li>Johnson, B.K., S.M. Thomas, A.J. Olive, and R.B. Abramovitch, Macrophage Infection Models for Mycobacterium tuberculosis. Methods Mol Biol, 2021. 2314: p. 167-182.</li> <li>Kiser, J.N., Z. Wang, R. Zanella, E. Scraggs, M. Neupane, B. Cantrell, C.P. Van Tassell, S.N. White, J.F. Taylor, and H.L. Neibergs, Functional Variants Surrounding Endothelin 2 Are Associated With Mycobacterium avium Subspecies paratuberculosis Infection. Front Vet Sci, 2021. 8: p. 625323.</li> <li>Lewis, M.S., L. Danelishvili, S.J. Rose, and L.E. Bermudez, MAV_4644 Interaction with the Host Cathepsin Z Protects Mycobacterium avium subsp. hominissuis from Rapid Macrophage Killing. Microorganisms, 2019. 7(5).</li> <li>Nigsch, A., S. Robbe-Austerman, T.P. Stuber, P.D. Pavinski Bitar, Y.T. Grohn, and Y.H. Schukken, Who infects whom?-Reconstructing infection chains of Mycobacterium avium ssp. paratuberculosis in an endemically infected dairy herd by use of genomic data. PLoS One, 2021. 16(5): p. e0246983.</li> <li>Nigsh, A., Robbe-Austerman, S., Stuber, T.P., Pavinski Bitar, P.D., Gr√∂hn Y.T., Schukken, Y.H.: Who infects Whom? - Reconstructing infection chains of Mycobacterium avium ssp. paratuberculosis in an endemically infected dairy herd by use of genomic data. PLOS ONE, 2021 https://doi.org/10.1371/journal.pone.0246983</li> <li>Palcekova, Z., M. Gilleron, S.K. Angala, J.M. Belardinelli, M. McNeil, L.E. Bermudez, and M. Jackson, Polysaccharide Succinylation Enhances the Intracellular Survival of Mycobacterium abscessus. ACS Infect Dis, 2020. 6(8): p. 2235-2248.</li> <li>Phillips, I.L., J.L. Everman, L.E. Bermudez, and L. Danelishvili, Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection. Microorganisms, 2020. 8(10).</li> <li>Phillips, I.L., L. Danelishvili, and L.E. Bermudez, Macrophage Proteome Analysis at Different Stages of Mycobacterium avium Subspecies paratuberculosis Infection Reveals a Mechanism of Pathogen Dissemination. Proteomes, 2021. 9(2).</li> <li>Rojony, R., L. Danelishvili, A. Campeau, J.M. Wozniak, D.J. Gonzalez, and L.E. Bermudez, Exposure of Mycobacterium abscessus to Environmental Stress and Clinically Used Antibiotics Reveals Common Proteome Response among Pathogenic Mycobacteria. Microorganisms, 2020. 8(5).</li> <li>Rojony, R., M. Martin, A. Campeau, J.M. Wozniak, D.J. Gonzalez, P. Jaiswal, L. Danelishvili, and L.E. Bermudez, Quantitative Analysis of Mycobacterium avium subsp. hominissuis proteome in response to antibiotics and during exposure to different environmental conditions. Clin Proteomics, 2019. 16: p. 39.</li> <li>Shoyama, F.M., T. Janetanakit, J.P. Bannantine, R.G. Barletta, and S. Sreevatsan, Elucidating the Regulon of a Fur-like Protein in Mycobacterium avium subsp. paratuberculosis (MAP). Front Microbiol, 2020. 11: p. 598.</li> <li>Silva, C., R. Rojony, L.E. Bermudez, and L. Danelishvili, Short-Chain Fatty Acids Promote Mycobacterium avium subsp. hominissuis Growth in Nutrient-Limited Environments and Influence Susceptibility to Antibiotics. Pathogens, 2020. 9(9).</li> <li>Stabel, J., L. Krueger, C. Jenvey, T. Wherry, J. Hostetter, and D. Beitz, Influence of Colostrum and Vitamins A, D3, and E on Early Intestinal Colonization of Neonatal Holstein Calves Infected with Mycobacterium avium subsp. paratuberculosis. Vet Sci, 2019. 6(4).</li> <li>Zinniel, D.K., W. Sittiwong, D.D. Marshall, G. Rathnaiah, I.T. Sakallioglu, R. Powers, P.H. Dussault, and R.G. Barletta, Novel Amphiphilic Cyclobutene and Cyclobutane cis-C18 Fatty Acid Derivatives Inhibit Mycobacterium avium subsp. paratuberculosis Growth. Vet Sci, 2019. 6(2).</li> <li>Berry, A., C.W. Wu, A.J. Venturino, and A.M. Talaat, Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats. Front Microbiol, 2018. 9: p. 2284.</li> <li>Phanse, Y., C.W. Wu, A.J. Venturino, C. Hansen, K. Nelson, S.R. Broderick, H. Steinberg, and A.M. Talaat, A Protective Vaccine against Johne's Disease in Cattle. Microorganisms, 2020. 8(9).</li> <li>Stabel, JR and Bannantine, JP. Reduced tissue colonization of Mycobacterium avium subsp. paratuberculosis in neonatal calves vaccinated with a cocktail of recombinant proteins. Vaccine 39(23):3131-3140.</li> <li>Thukral, A., K. Ross, C. Hansen, Y. Phanse, B. Narasimhan, H. Steinberg, and A.M. Talaat, A single dose polyanhydride-based nanovaccine against paratuberculosis infection. NPJ Vaccines, 2020. 5(1): p. 15.</li> <li>Cochard, T., M. Branger, P. Supply, S. Sreevatsan, and F. Biet, MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains. Infect Genet Evol, 2020. 77: p. 104075.</li> <li>Kanankege, K.S.T., N.B.D. Phelps, H.M. Vesterinen, K.M. Errecaborde, J. Alvarez, J.B. Bender, S.J. Wells, and A.M. Perez, Lessons Learned From the Stakeholder Engagement in Research: Application of Spatial Analytical Tools in One Health Problems. Front Vet Sci, 2020. 7: p. 254.</li> <li>Kelly, T.R., D.A. Bunn, N.P. Joshi, D. Grooms, D. Devkota, N.R. Devkota, L.N. Paudel, A. Roug, D.J. Wolking, and J.A.K. Mazet, Awareness and Practices Relating to Zoonotic Diseases Among Smallholder Farmers in Nepal. Ecohealth, 2018. 15(3): p. 656-669.</li> <li>Verteramo Chiu, L.J., L.W. Tauer, M.A. Al-Mamun, K. Kaniyamattam, R.L. Smith, and Y.T. Grohn, An agent-based model evaluation of economic control strategies for paratuberculosis in a dairy herd. J Dairy Sci, 2018. 101(7): p. 6443-6454.</li> </ul>

Impact Statements

• Although COVID significantly impacted the outreach opportunities, several on-going efforts were made to improve the outreach and extension opportunities. • A live interview with the Brownfield Radio Ag Network during the 2018 World Dairy Expo included information on the MI and the continued need for work on Johne’s and Bovine TB. • There have been several on-going activities before and during the pandemic that MDA has participated in. We have participated in signing support letters to Congressional leaders and the administration where appropriate. We have also supported advocating for funding of agricultural research and using the results in future policymaking. Each time a request is received inviting participation in a letter, all Executive Committee members are contacted to assure that there are no objections to the letter. So far, we have supported and signed 16 in 2018, 13 in 2019, seven letters in 2020, and 16 in 2021. • The MDA MI responded to an invitation to have a Symposium in the Animal Health section of the 2018 Annual Meeting of the American Dairy Science Association (ADSA). The session was held on June 27, 2018. The title was “Bovine Tuberculosis—An On-going Animal Health Challenge.” A copy of the agenda is below.