3rd
October 18, 2014

The Mycobacterial Diseases of Animals (MDA) multi-state initiative (NE1201) brings together leading scientists with a shared vision of improving food security through a reduction in losses from two of the most important diseases of livestock - bovine tuberculosis (bTB) and Johne's disease (JD).
Our mission is to facilitate the development of shared research as well as the leveraging of intellectual and physical resources to address the most pressing needs and reduce health impacts and economic losses from bTB and JD.
Research Objectives
Objective 1 focuses on understanding the epidemiology and transmission of JD and TB in animals through the application of predictive modeling and assessment of recommended control practices;
Objective 2 seeks to develop and implement new generations of diagnostic tests for JD and TB;
Objective 3 focuses on improving our understanding of biology and pathogenesis of Mycobacterial diseases, as well as the host response to infection; and,
Objective 4 focuses on development of programs to create and evaluate and develop new generations of vaccines for JD and TB.
The objectives are crosscutting in nature (i.e. cut across objectives and/or address both diseases) that together help address the major animal, human, and societal issues surrounding detection and control of mycobacterial diseases in animals.
Each of our research objectives is closely linked and coordinated with our education, extension and outreach plan.
Organizational Officers
Administrative Advisor
Gary A. Thompson (Penn State) gat10@psu.edu
National Institute of Food and Agriculture Representatives
Margo Holland mholland@nifa.usda.gov
Peter J. Johnson pjohnson@nifa.usda.gov
Gary Sherman gsherman@nifa.usda.gov

Executive Committee
The NE1201 Executive Committee is responsible for all strategic, scientific, and management policy decisions for this multi-state initiative, and serve to advise the Administrative Advisor of the program. The
Chair of the Executive Committee is responsible for the implementation and facilitation of programmatic goals and will serve as the primary liaison with the USDA, Experiment Station Directors, and external stakeholders.
The current members of the Executive Committee are:
 John Bannantine (National Animal Disease Center, USDA-ARS)

Luiz Bermudez (Oregon State University, OR)

Paul Coussens (Michigan State University, MI) Chair-Elect of the Executive Committee

Yrjö Gröhn (Cornell University, NY)

Vivek Kapur (The Pennsylvania State University, PA) Chair of the Executive Committee.

Don Lein (Cornell University, NY) Chair of the External Advisory Board.

Kenneth Olson (KEO Consulting, IL) Secretary of the Executive Committee

Fred Quinn (University of Georgia)

Scott Wells (University of Minnesota, MN)
External Advisory Board
The NE 1201 External Advisory Board consists of public and private stakeholders (regulatory agencies, members of industry, and prominent scientists from related disciplines and Experiment Station Directors), to provide advice on programmatic matters, and ensure that the initiative stays true to its mission.
The Chair of the External Advisory Board is Don Lein (Cornell) who also is a member of the Executive
Committee.
To learn more about MDA, its participants, ongoing research and available resources, please visit us at our web site at http://mycobacterialdiseases.org/.

Meeting Agenda…………………………………………………………………………………………4
Program Presentation Abstracts…………………………………………………………………….5-10
Posters Abstracts……………………………………………………………………………………11-20

October 18, 2014
3 rd Annual Conference
Mycobacterial Diseases of Animals
Westin Crown Center Hotel
Kansas City, MO
Westin Crown Center Hotel, Shawnee Mission Room - Ballroom Level 3
7:00 - 8:00 AM
Opening
8:00 - 8:30 AM
Registration and Breakfast
8:30 - 9:30 AM
Opening Remarks and Introduction by Vivek Kapur, The Pennsylvania State University; Paul Coussens, Michigan
State University; Gary Thompson, The Pennsylvania State University; Peter Johnson, USDA-NIFA
Plenary Session 1: Increase understanding of the epidemiology and transmission of Mycobacterial diseases in animals, including predictive modeling.
Session Chair Scott Wells (Univ. of Minnesota)
Invited speakers Rowland Kao (Univ. of Glasgow, UK)
Yrjo Grohn (Cornell University, NY) and Scott Wells (Univ. of Minnesota)
9:30 - 10:30 AM Plenary Session 2: Develop and implement new generations of diagnostic tests for JD and TB.
Session Chair John Bannantine (USDA-NADC)
Invited speakers Tim Bull (St. George, UK)
Suelee Robbe-Austerman (APHIS)
10:30 - 11:00 AM Break/poster session (Shawnee Mission)
11:00 - 12:00 Plenary Session 3: Extension/Outreach/Human Disease: Develop and deliver education and outreach material related to JD and TBc in electronic and print form for use by various stakeholders.
Session Chair Ken Olson (KEO consulting)
Invited speakers Julie Smith (Univ. of Vermont
Jeannette McDonald (Univ. of Wisconsin–Madison)
1:00 - 2:00 PM
2:00 - 3:00 PM
Plenary Session 4: Improving our understanding of the biology and pathogenesis of Mycobacterial diseases, as well as the host response to infection.
Session Chair Srinand Sreevatsan (Univ. of Minnesota)
Invited speakers Luiz Bermudez (Oregon State)
Adel Talaat (Univ. of Wisconsin-Madison)
Plenary Session 5: Identify markers for susceptibility, and understanding of host genetics.
Session Chair Paul Coussens (Michigan State Univ.)
Invited speakers Holly Neibergs (Washington State Univ.)
Frank Griffin (Univ. of Otago, New Zealand)
3:00 - 4:00 PM Plenary Session 6: Develop programs to evaluate and develop new generations of vaccines for JD and TBc.
Session Chair Murray Hines (Univ. of Georgia)
Invited speakers Fred Quinn (Univ. of Georgia)
Gregers Jungersen (Tech. Univ. Of Denmark)
4:00 - 4:15 PM Break - refreshments will be served
4:15 - 6:00 PM Chalk talk presentation and discussion by epidemiology, diagnostic tests, host genetics, and vaccines groups
6:00 PM Adjourn
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Thinking backwards and forwards - evolutionary and epidemiological approaches to modeling high density, high resolution M. bovis sequence data in cattle and badgers
Rowland Kao
University of Glasgow, Institute of Biodiversity Animal Health, Glasgow
Whole genome sequencing is an exciting new tool in the epidemiological toolbox, with the advent of large scale, high density sampling of particular interest to the forensic epidemiologist.
Here I shall discuss two different approaches to interpreting these data one based on the evolutionary paradigm and the other on epidemiological modeling. I shall show how each is a powerful tool for elucidating basic principles about M bovis transmission dynamics, but must be interpreted at the appropriate epidemiological scale.
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The Development of in vitro model systems to address important pathogenic mechanisms of
MAP associated with the survival in ruminants
Luiz E Bermudez
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University
Johne’s Disease (caused Mycobacterium avium subsp paratuberculosis , MAP) is a complex condition that affects ruminants. The study of MAP pathogenesis is quite difficult because the disease has silent periods and takes many years to manifest clinically. The establishment of model systems that can be used to obtain crucial information that would unveil key aspects of MAP pathogenesis is much needed.
We have developed a couple models that have provided knowledge that can benefit the study of the disease. In one of the models, we have established a sequential infection system from intestinal epithelial cells (first step in the infection) through macrophages and back to epithelial cells (last step on the disease). The three stages were called stages 1, 2, and 3.To determine whether this model would be reliable, we infected epithelial cells and passed the intracellular bacteria through the sequence of steps representing Johne’s disease. Then, we and measured the production of chemokines and cytokines following the infection of the first epithelial cells
(stage 1) and following the infection of the second epithelial cells (stage 3). In a reproduction of the disease, while the initial infection did not induce inflammatory response (but induced TGFbeta), the infection of the epithelial cells after passage in macrophages (stage 3) resulted in significant release of IL-18, IL-6, IL-8. CCL5, and decrease of TGF-beta. Transcriptome and lipidome of the third passage strain (inflammatory) versus the first passage strain (non-inflammatory) showed significant change in the proteins and lipids. In fact the pattern of lipids of the 3 stage strain is quite different of the first stage one. Putting it together with the transcriptome, it is clear that degradation and synthesis of new lipids is taking place. To determine if those genes are expressed in vivo in the bovine mucosa, bacterial RNA was isolated from the intestines of cows with Johne’s Disease, and PCR was performed using specific primers for lipid metabolism related genes. The results confirmed the expression of the genes in vivo. We then used cell wall from MAP to stimulate lymphocytes and macrophages. While the cell wall of bacteria from the first passage induced only mild inflammatory response, the cell wall lipids of the third stage were associated with significant increase of the inflammatory phenotype of macrophages.
In the second model system, we used Acanthamoeba castellanii as a surrogate of bovine macrophages and Alamar Blue as a measure of metabolic activity, to screen a transposon library of
MAP for attenuation. The screen takes one day and the read out is colorimetric.
We examine whether 30 of the MAP strains identified in the screen were attenuated in primary bovine macrophages. Since we could confirm that all the strains were in fact attenuated, we have began the examination of the mechanisms of pathogenesis.
Concluding, the ability to establish system models in vitro which represent in vivo stages of
Johne’s disease can help to dissect the mechanism used by MAP to survive in the host and to develop novel approaches for prevention and treatment.
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Selection for Heritable Resistance to Control Johne’s Disease in Farmed Deer!
J F T Griffin 1 , S Liggett 1 , L Brennan 1 , C Mackintosh 2 & R O’Brien 1
1 Otago University, PO Box 56, Dunedin, New Zealand; 2 AgResearch Invermay, PO Box 50034, Mosgiel,
New Zealand
Different breeds of ruminants may display heritable resistance (R) or susceptibility (S) to
Johne’s disease (JD). Nonetheless, little progress has been made to identify genetic markers associated with either R or S traits in domestic livestock.
Our current findings are derived a decade long study of 7 disparate breeds of European red deer (Cervus elaphus), in a herd that had been chronically infected with M. ptb for more than 5 years. Sires with a confirmed Resistant (R) or Susceptible (S) phenotype were used in an AI programme with crossbred females, and the phenotype of more than 50 progeny was confirmed immunologically, microbiologically and histopathologically, following experimental infection with virulent M. ptb. In a two year breeding programme, 78% of progeny from R sires displayed the R phenotype, while 84% of the S progeny expressed a S phenotype. Detailed RNA-seq and transcriptome analysis of 2 R and 2S animals was used to determine the relative expression levels of immune genes, measured by qPCR. Multiple genes involved in functional pathways of innate and adaptive immunity have been evaluated using a ‘systems biology’ approach to target overall cell function.
Animals with a S genotype expressed significantly higher levels of genes associated with proinflammatory reactions and genes related to chemotaxis and Type 1 interferons. Higher levels of adaptive immune markers and increased levels of Apoptosis was seen in R animals.
Animals with S phenotypes appear express dysfunctional innate immunity and are incapable of clearing mycobacterial pathogens. By contrast, R animals express markers of adaptive immunity, which when combined with adequate levels of innate immunity, promote protection and containment of infection.
Disparate breeds of wild deer that have evolved in geographic isolation and have undergone domestication through selective breeding, display polarized phenotypes. Our findings from the study of Johne’s disease in NZ farmed will be contrasted with recent studies in cattle and sheep.
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Programs to develop and evaluate new generations of vaccines for bovine TB
Fred Quinn
College of Veterinary Medicine, University of Georgia, Athens, GA
It is widely recognized that improved understanding of the mechanisms of pathogenesis and response of the natural host to infection by Mycobacterium bovis is needed in order to develop the next generation of more efficacious vaccines. Unfortunately, the practical and logistical challenges and overall high costs associated with appropriate studies in the relevant host (the calf or cow) have forced investigators to use less than optimal model systems. Even when investigators can use the bovine infection model, these same limitations rarely allow for the incorporation of all relevant natural infection parameters into studies, including oral vaccination, repeat oral dosing with small numbers of infecting bacilli, assessments of effects on animal-toanimal transmission, and disease outcome from long-duration controlled studies. Rodent models are not useful for studying most of these parameters, and thus the utilization of ferret and larger animals may be necessary. Using the bovine long-duration transmission model and other medium and large models that more accurately mimic aspects of the natural course of disease, the next generation of vaccine candidates, including mucosal vaccines, could be more accurately examined for disease prevention, and perhaps of more relevance, prevention of disease transmission.
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Development of a recombinant multi-stage DIVA vaccine against Johne’s disease
Gregers Jungersen 1 , Aneesh Thakur 1 , Heidi Mikkelsen 1 , Claus Aagaard 2 , Peter Andersen 2
1 Technical University of Denmark, National Veterinary Institute, Frederiksberg, Denmark; 2 Statens Serum Institute, Copenhagen, Denmark
Taking advantage of results from vaccine development against Mycobacterium tuberculosis, a new (FET11) vaccine against M. avium subsp. paratuberculosis (Map) based on recombinant antigens from acute and latent stages of Map infection was developed. A hall-mark in the development of the vaccine was a requirement not to interfere with diagnostic tests for bovine TB and Johne’s disease allowing a continued diagnosis of Map infection in vaccinated animals
(DIVA vaccine).
In two post-exposure vaccination trials with 28 calves and 15 goats, respectively, animals were orally inoculated with live Map in their third week of life and post-exposure vaccinated at different times after inoculation or with different vaccine constructs. In response to vaccination animals developed vaccine-specific antibody and cell-mediated immune responses, but no measurable antibody responses by ID Screen® ELISA, PPDj-specific IFN-γ responses or positive
PPDa or PPDb skin tests. At termination 8 or 12 months of age, relative Map burden was determined in a number of gut tissues by quantitative IS900 PCR and revealed significantly reduced levels of Map and reduced histopathology. Diagnostic tests for antibody responses and cell-mediated immune responses corroborated the observed vaccine efficacy: Five of seven non-vaccinated calves seroconverted in ID Screen® ELISA indicating the progression of infection, while only four of 14 FET11 vaccinated calves seroconverted and a later time point after inoculation. Similarly, increased PPDj-induced IFN-γ responses over time in non-vaccinated calves, while FET11 vaccinated calves had significantly reduced responses in PPDj IFN-γ assay from 40 to 52 weeks compared to non-vaccinated calves. These results indicate the FET11 vaccine can be used to accelerate eradication of paratuberculosis while surveillance or test-and-manage control programs for TB and JD remain in place.
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1. The Mycobacterial Diseases of Animals (MDA) Multistate Initiative - a cooperative effort addressing animal diseases
K.E. Olson 1 , V. Kapur 2 , P. M. Coussens 3 and D. H. Lein 4
1 KEO Consulting; 2 The Pennsylvania State University; 3 Michigan State University; 4 Cornell University
Johne’s Disease Integrated Program (JDIP) efforts are well known and documented. Primary funding was through USDA grants that allowed leveraging of additional public and private resources to expand the effort. The grants have come to an end so a plan for the future was needed.
JDIP addressed many knowledge gaps, but much work remains, so a range of options for the consortium were considered. Primary objectives were to maintain the networking, collaboration and basic infrastructure developed through JDIP, allowing participants to identify, obtain and share resources needed to address Johne’s and other mycobacterial diseases. To this end, a proposal was, developed and later, approved by USDA’s National Institute for Food and Agriculture (NIFA) to begin operation as Multistate Initiative - NE1201, "Mycobacterial Diseases of An-
imals (MDA)".
The multi-state initiative (MI) is focused on two mycobacterial disease complexes - paratuberculosis (Johne’s disease; JD) and the tuberculosis complex of diseases (TBc; i.e bovine tuberculosis). The initiative includes five objectives:
Objective 1: Increase understanding of the epidemiology and transmission of MDA, including predictive modeling;
Objective 2: Develop and implement new generations of diagnostic tests for JD and TBc;
Objective 3: Improving our understanding of the biology and pathogenesis of MDA, as well as the host response to infection;
Objective 4: Develop programs to evaluate and develop new generations of vaccines for JD and
TBc; and,
Extension/Outreach: Develop and deliver JD and TBc education and outreach material in electronic and print form for use by producers and other stakeholders. Use trade media, producer organizations and other outlets to aid in dissemination of information.
Projects within each objective, with cross-cutting contributions, are designed to address major animal, human, and societal issues surrounding detection and control of mycobacterial infection, including how these organisms move and spread within cattle, small ruminant and wildlife populations.
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2. Catalyzing Innovation and Scientific Discoveries in Mycobacterial Diseases of Animals.
K.E. Olson 1 , V. Kapur
1 KEO Consulting;
2 , P. M. Coussens 3 and D. H. Lein 4
2 The Pennsylvania State University; 3 Michigan State University; 4 Cornell University
Mycobacterial diseases such as Johne’s disease and bovine tuberculosis (bTB) are extremely costly to the livestock industry in North America and throughout the world. New and innovative funding and collaboration efforts are needed for the future. Public and private research has been working to address these diseases, but much remains to be done. For example, the prevalence of Johne’s disease remains high and, in North America, new cases of bTB continue to be found. In other regions of the world, bTB remains a pressing problem and can result in significant human disease. Concerns over human health links for both diseases remain, with the link between some human Crohn’s disease cases and M. paratuberculosis becoming more evident, and new research suggesting a link between M. paratuberculosis and other autoimmune disorders in humans. Unfortunately, while the impact and threat associated with these diseases remains high, the federal and state funding base for research, education, outreach and coordination of efforts has continued to shrink. Thus, there are growing concerns that grant funding shortfalls will hamper basic and translational research and education efforts to prevent and control JD and other mycobacterial diseases in animals. To help catalyze continued innovation, and to assist producer groups, researchers, regulators, and funding agencies by promoting scientific research, education and extension activities in developing and implementing science based solutions for the prevention and control of mycobacterial diseases, we have recently developed a new structure, the American Association of Mycobacterial Diseases (AAMD).
The AAMD is organized as a not- for-profit corporation focused on promoting translational research, providing resources, and enabling networking amongst key stakeholders so as to assist producers, educators and researchers efforts to address major issues relating to mycobacterial diseases of animals. The AAMD also provides a novel platform for facilitating the continued use and development of resources developed through the Johne’s Disease Integrated Program (JDIP), and helps with the dissemination of information and development of tools to address JD and other major mycobacterial diseases in the future.
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3. The Genome of a Mycobacterium avium subspecies paratuberculosis Isolate Recovered from a Crohn’s Disease Patient reveals Close Genetic Association with Isolates Recovered from Cattle
Lingling Li 1 , Michael Mwangi 1 , Rebecca Cote 1 , John P. Bannantine 2 , Juan Antonio Raygoza
Garay 1 , Srinand Sreevatsan 3 , and Vivek Kapur 1
1 Department of Veterinary and Biomedical Science, The Pennsylvania State University, University Park,
PA; 2 National Animal Disease Center USDA-ARS, Ames, IA; 3 Veterinary Population Medicine, University of Minnesota, St. Paul, MN
Several studies have identified Mycobacterium avium subspecies paratuberculosis (MAP) in human patients with Crohn's disease (CD). Preliminary investigations have also suggested that
MAP isolates from humans bear considerable genetic similarity to MAP from cattle, but not from sheep. In order to characterize genetic differences between MAP isolates recovered from humans and those associated with bovine Johne’s disease (JD), we sequenced to completion the genome of an isolate of MAP documented to have been recovered from a patient with CD.
Massively parallel sequencing approaches were used to generate a total of 88.5 million base pairs of high quality sequence from a randomly sheared MAP genomic DNA library. These sequences were subsequently assembled into contiguous fragments and an iterative primer walking approach was applied to close gaps and areas of low quality coverage re-sequenced in order to obtain the complete circular genome. Comparative analysis with the MAP K10 and other cattle isolates revealed near identity in genome organization. The analysis showed that the genome of strain K10, the only other MAP isolate whose complete genome sequence have been characterized thus far, is ~3.0kb larger as a result of several sequence insertions, including one additional copy of the insertion sequence element, IS900. The analysis revealed over 200 single nucleotide polymorphisms (SNPs), with a majority of these occurring in protein coding regions.
Genome-wide comparisons of SNPs in multiple MAP isolates recovered from humans with CD and cattle with JD strongly suggest that MAP isolates from either human or cattle are indistinguishable based on genotype.
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4. Comparison of 3 fecal culture, 2 fecal PCR, 2 serum ELISA, and milk ELISA for diagnosis of paratuberculosis in US dairy cattle.
R. W. Sweeney 1 , I. Gardner 2 , M. Hines 3 , R. Anderson 4 , T. Byrem 5 , M. Collins 6 , A. Glaser 7 , E.
Hovingh 8 , L. Jones 3 , S. Wells 9 , B. Whitlock 10 .
1 U. of Pennsylvania; 2 U. of Prince Edward Island; 3 U. of Georgia; 4 California Dept. of Food and Agriculture; 5 Antel BioSystems; 6 U. of Wisconsin; 7 Cornell University; 8 Penn State University; 9 U. of Minnesota; 10 University of Tennessee
The objective of this study was to conduct a comparison of multiple diagnostic tests for Johne’s disease in US dairy cattle and to create a repository of well-characterized fecal, serum, and milk samples as a resource for future studies. Samples were collected from 499 cows in herds known to have infected animals, and 418 cows in dairy herds in a test-negative certification program. Fecal, serum, and milk samples were collected from cows in the second lactation or older.
Diagnostic tests applied to the samples included fecal culture on solid Herrold’s Egg Yolk Medium (HEYM), 2 different liquid culture media systems (TREK Diagnostics; BACTEC MGIT, BD Diagnostic Systems), fecal PCR using two different platforms (Tetracore, Taqman), two serum ELISA
(IDEXX, Prionics), and milk ELISA (IDEXX). Bayesian models for test evaluation when a “gold standard” test is not available were employed to estimate diagnostic sensitivity of each test, as well as “traditional” methods.
For organism based tests, the specificities were 100% for MIGT, HEYM, and Tetracore PCR;
99.2% for TaqMan PCR, 99.5% for TREK. “Traditional” sensitivity estimates were 31.5% for
HEYM, 44.0% for TREK, 40.0% for MGIT, 86.0% for both TaqMan and Tetracore PCR.
For antibody detection tests, all had specificity > 99.0%. The “Traditional” sensitivity estimates were 35.5% for IDEXX serum ELISA, 23.5 % for Prionics ELISA, and 29.1% for milk ELISA.
The results of this study provide a basis for selection of optimal diagnostic test protocols according to herd characteristics and management objectives. Repository samples are available for future study.
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5. Genome wide association study confirms association of Mycobacterium avium paratuber-
culosis tissue infection on BTA16 and BTA22
Jennifer Kiser 1 , Holly Neibergs 1
1 Washington State University
Johne’s disease is a contagious bacterial disease affecting cattle and other ruminants that is caused by Mycobacteriuam avium subspecies paratuberculosis (Map). A previous genome wide association study (GWAS) in Holstein cattle performed by our group identified a region on bovine chromosome 3 (BTA3), and 9 (BTA9) that were highly associated (p<5x10-7) and on bovine chromosomes BTA1, BTA16, and BTA21 that were moderately associated (P<5x10-5) with susceptibility to Map tissue infection. The objective of this project was to validate the GWAS results utilizing two previously uninvestigated populations, and explore these populations for any additional associations for susceptibility to Map tissue infection. DNA from 57 Jersey (9 males,
48 females) and 205 cull Holstein cows were genotyped using the Illumina BovineSNP50 Bead-
Chip. The resulting data was then quality filtered for individual SNP call rate, genotype call rate, minor allele frequency (MAF) and failure of Hardy-Weinberg Equilibrium. Individuals were removed if the SNP call rate was <90%, and SNPs were removed if the genotype call rate was
<90% or they had a MAF of <0.01. After filtering, 53 Jersey (16 cases, 37 controls) and 184 Holstein cattle (68 cases, 116 controls) remained for analysis. A GWAS was conducted for each of the two populations using an additive, efficient mixed-model association eXpedited (EMMAX) statistical method. EMMAX was chosen due to its ability to account for population stratification within the two populations. The GWAS in the Jersey population identified 6 loci associated with
Johne’s susceptibility; four loci on BTA22 (P<9x10 -5 ), one locus on BTA4 (P<2x10 -5 ), and on
BTA16 (P<3x10 -5 ). In the Holstein population, the GWAS identified 2 loci associated with Johne’s susceptibility; one locus on BTA21 (P<5x10 -7 ), and on BTA 1 (P<5x10 -5 ). Two of the four significant loci on BTA22 are in introns of inositol 1, 4, 5-trisphosphate receptor type 1 gene (ITPR1).
ITPR1 is associated with several biological pathways including the gastric acid secretion pathway which is involved with upper gastrointestinal protection against bacterial infections. Previous studies have identified loci on both BTA16 and BTA22. However, the BTA16 locus in this study is only 1.4Mb from the previously identified locus, while the BTA22 loci are over 33Mb away.
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6. Antigenicity of Envelope Protein Complexes of Mycobacterium avium subsp. paratubercu-
losis
F. L. L. Leite 1 , T.A. Reinhardt 2 , J.P. Bannantine 2 , J. R. Stabel 2
1 Iowa State University, Ames, IA; 2 USDA-ARS, Ames, IA
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease or paratuberculosis, a chronic enteric wasting disease of ruminant animals with worldwide distribution. MAP is part of the avium complex of mycobacteria and has an envelope comprised of a cytoplasmic membrane and a cell wall consisting of peptidoglycan, arabinogalactan, mycolic acids and proteins. Proteins associated with the cell envelope of MAP are likely to be the first to interact with the host and, therefore, the first proteins for immune recognition. In the present study, blue native PAGE electrophoresis (BN) and 2D SDS-PAGE were used to characterize protein complexes in the envelope of MAP, followed by mass spectrometry to identify individual proteins within complexes. BN PAGE is a technique that solubilizes protein complexes in their native form and allows for their isolation while 2D SDS-PAGE denatures the protein complexes to visualize their subunits. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from clinical (n=13), subclinical (n=10) and noninfected control animals (n=9).
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7. ZAP-70, CTLA-4 and proximal T cell receptor signaling in cows infected with Mycobacterium
avium subsp. paratuberculosis
Fernando L. Leite a , Livia B. Eslabão b , Bruce Pesch c , John P. Bannantine c , Timothy A. Reinhardt c ,
Judith R. Stabel c a Department of Veterinary Microbiology and Preventive Medicine, Iowa State Univ., Ames, IA 50011; b Centro de Desenvolvimento Tecnológico, Núcleo Biotecnologia, UFPel, Pelotas, RS, Brazil; c USDA-ARS, National Animal Disease Center, Ames, IA 50010
Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium
avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cellmediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain associated protein of 70 kDa) and CTLA-4
(cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in clinical
(n=6) and subclinical stages of paratuberculosis (n=6) were cultured alone (medium only), with concanavalin A (ConA), and a whole cell sonicate of MAP (MPS) for 24, 72 and 144 hours to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4+ T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. It was found that the surface expression of
CTLA-4 is significantly increased in animals in the subclinical stage of infection. In contrast, levels of ZAP-70 were significantly decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered due to MAP infection. This study demonstrates changes in crucial signaling molecules in animals infected with MAP, elucidating T cell alterations that are associated with disease progression.
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8. An Outbreak of Bovine Tuberculosis in Beef Cattle and White Tailed Deer in Minnesota
Glaser L.
1 , Carstensen M.
2 , Shaw S.
3 , Wuenschmann A.
4 , Robbe-Austerman S.
3 , and Wells SJ.
5
1 Minnesota Board of Animal Health; 2 Minnesota Department of Natural Resources; 3 USDA Animal and
Plant Health Inspection Service; 4 University of Minnesota, Veterinary Population Medicine; 5 Center for
Animal Health and Food Safety, University of Minnesota
Introduction: A five year old Minnesota cow was identified with bovine tuberculosis at a slaughter plant in 2005. The subsequent investigation traced animal movements in and out of the herd and identified livestock in contact with infected herds. Live animals were tested for bovine tuberculosis in trace and contact herds and others in the geographic region, and 41 cattle from 12 herds were eventually found infected with M. bovis in a small area of northwestern
Minnesota. In addition, 6,600 free-ranging white-tailed deer in this geographic region were tested from 2006 through 2012; 27 infected deer were identified. All cattle confirmed as M. bovis-positive were euthanized and disposed of through rendering, alkali digestion or burial.
The remaining cattle in the 12 M. bovis-positive herds were depopulated by slaughter with enhanced inspection and federal indemnity paid to the owner.
Materials and methods: An epidemiologic evaluation of the disease outbreak and control program was conducted through collaboration with involved agencies. Whole genome sequencing was performed using available M. bovis isolates.
Results: Whole genome sequencing indicated the origin of the infection was likely Mexico. After an aggressive disease eradication program and coordination between animal health and wildlife agencies, no cases have been identified since 2009.
Conclusion: This investigation highlights the identification, surveillance, epidemiology, and successful disease control of an outbreak of bovine TB in cattle and free-ranging deer. Movements of cattle among infected herds, proximity of infected deer, and genetic sequencing of isolates provide evidence of transmission pathways involved in this outbreak.
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9. Landscape context and deer-cattle interactions: A targeted approach to bovine tuberculosis surveillance
Ribeiro Lima J.
1 , Forester JD.
2 , Carstensen M.
3 , Cornicelli L.
3 , Wells SJ.
1
1 Department of Veterinary Population Medicine, University of Minnesota; 2 Department of Fisheries,
Wildlife, and Conservation Biology, University of Minnesota; 3 Minnesota Department of Natural Resources
Introduction: Wildlife that spatially overlap with livestock operations represent an additional source of infection and a susceptible population for infectious disease transmission. The objective of this analysis was to evaluate the impact posed by the landscape surrounding any given cattle farm on the risk for cattle-deer interactions.
Materials and methods: White-tailed deer (Odocoileus virginianus) were captured (n=19) and fitted with satellite-linked radio collars. The study area contained 25 private cattle farms and included a mix of state forest and wildlife management areas in addition to private recreational lands. A step selection function (SSF) was estimated using a standard conditional logistic regression (50 controls per deer location). Model selection for data from individual deer, was based on AIC, where models with ∆AIC ≤ 3 were considered.
Results: The best fitting model showed that for five of the six cattle farms with deer visits, farm locations overlap with higher resource selection areas. On average, there was a tendency for stronger selection for open areas outside forest in early Spring and Fall. In the Summer months, the selection was stronger for within forest areas, while in the Winter deer preferentially selected for the edge of forest. Broad-scale patterns in the distribution of RSF values show that the eastern portion of the study area had higher values, corresponding to higher densities of forest land.
Conclusion: In this study, we show that the type of landscape surrounding a cattle farm can predict risk of deer-cattle interactions. Understanding factors that drive habitat use by deer will aid in the implementation of targeted approaches for disease surveillance.
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