Life Sciences Commons^™

Quantification Of Macrophages And Mycobacterium Avium Subsp. Paratuberculosis In Bovine Intestinal Tissue During Different Stages Of Johne’S Disease, Caitlin J. Jenvey, Jesse M. Hostetter, Adrienne L. Shircliff, John Bannantine, Judith R. Stabel

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Johne’s disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in …

Knowledge Gaps That Hamper Prevention And Control Of Mycobacterium Avium Subspecies Paratuberculosis Infection, Herman W. Barkema, Karin Orsel, S.S. Nielsen, A.P. Koets, V.P.M.G. Rutten, J. P. Bannantine, G.P. Keefe, D.F. Kelton, Scott J. Wells, Richard J. Whittington, Colin G. Mackintosh, E.J. Manning, M.F. Weber, C. Heuer, T.L. Forde, C. Ritter, S. Roche, C.S. Corbett, R. Wolf, P.J. Griebel, J.P. Kastelic, J. De Buck

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

In the last decades, many regional and country-wide control programmes for Johne’s disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn’s disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of …

A Mycobacterium Avium Subsp. Paratuberculosis Predicted Serine Protease Is Associated With Acid Stress And Intraphagosomal Survival, Abirami Kugadas, Elise A. Lamont, John Bannantine, Fernanda Miyagaki Shoyama, Evan Brenner, Harish K. Janagama, Srinand Sreevatsan

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed …

Controlling Johne’S Disease: Vaccination Is The Way Forward, John Bannantine, Adel M. Talaat

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

M. avium subspecies paratuberculosis, hereafter referred to as MAP, is a significant veterinary pathogen that causes Johne’s disease in ruminants, including cattle, sheep, and goats. This chronic intestinal disease is distributed worldwide and exacts a heavy eco- nomic toll on animal producers. For example, the dairy industry incurs substantial economic losses due to reduced milk production, premature culling, and reduced slaughter value (Raizman et al., 2009). It takes years for clinical signs to appear in animals after initial infection. The bacterium is shed in high numbers in the feces during this clinical phase of disease. Transmission is by ingestion of …

Screening Of Mycobacterium Avium Subsp. Paratuberculosis Mutants For Attenuation In A Bovine Monocyte-Derived Macrophage Model, Elise A. Lamont, Adel M. Talaat, Paul M. Coussens, John Bannantine, Yrjo T. Grohn, Robab Katani, Ling-Ling Li, Vivek Kapur, Srinand Sreevatsan

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Vaccination remains a major tool for prevention and progression of Johne’s disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne’s disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne’s Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA—APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase …

Enhanced Expression Of Codon Optimized Mycobacterium Avium Subsp. Paratuberculosis Antigens In Lactobacillus Salivarius, Christopher D. Johnston, John Bannantine, Rodney Govender, Lorraine Endersen, Daniel Pletzer, Helge Weingart, Aidan Coffey, Jim O’Mahony, Roy D. Sleator

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants …

How Does A Mycobacterium Change Its Spots? Applying Molecular Tools To Track Diverse Strains Of Mycobacterium Avium Subspecies Paratuberculosis, John Bannantine, L.-L. Li, Srinand Sreevatsan, Vivek Kapur

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne’s disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed-field gel …

Genome Sequencing Of Ovine Isolates Of Mycobacterium Avium Subspecies Paratuberculosis Offers Insights Into Host Association, John Bannantine, Chia-Wei Wu, Chungyi Hsu, Shiguo Zhou, David C. Schwartz, Darrell O. Bayles, Michael L. Paustian, David P. Alt, Srinand Sreevatsan, Vivek Kapur, Adel M. Talaat

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Background: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map.

Results: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed …

Immunogenicity And Protective Efficacy Of The Mycobacterium Avium Subsp. Paratuberculosis Attenuated Mutants Against Challenge In A Mouse Model, Jenn-Wei Chen, Syed M. Faisal, Subhash Chandra, Sean P. Mcdonough, Maria A.S. Moreira, Joy Scaria, Chao-Fu Chang, J. P. Bannantine, Bruce Akey, Yung-Fu Chang

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), results in serious economic losses worldwide especially in cattle, sheep and goats. To control the impact of JD on the animal industry, an effective vaccine with minimal adverse effects is urgently required. In order to develop an effective vaccine, we used allelic exchange to construct three mutant MAP strains, leuD, mpt64 and secA2. The mutants were attenuated in a murine model and induced cytokine responses in J774A.1 cell. The leuD mutant was the most obviously attenuated of the three constructed mutant strains. Our preliminary vaccine trial in mice demonstrated different …

Evaluation Of Two Mutants Of Mycobacterium Avium Subsp. Paratuberculosis As Candidates For A Live Attenuated Vaccine For Johne’S Disease, Kun Taek Park, Andrew J. Allen, John P. Bannantine, Keun Seok Seo, Mary J. Hamilton, Gaber S. Abdellrazeq, Heba M. Rihan, Amanda Grimm, William C. Davis

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Control of Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo …

Monoclonal Antibodies Bind A Snp-Sensitive Epitope That Is Present Uniquely In Mycobacterium Avium Subspecies Paratuberculosis, John Bannantine, Judith R. Stabel, Elise A. Lamont, Robert E. Briggs, Srinand Sreevatsan

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non- paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in …

Genomic And Transcriptomic Studies In Mycobacterium Avium Subspecies Paratuberculosis, J. P. Bannantine, Adel M. Talaat

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Microarray technology is an important tool in functional genomic research. It has enabled a deeper analysis of genomic diversity among bacteria belonging to the Mycobacterium avium complex (MAC). In addition, the expression of thousands of genes can be studied simultaneously in a single experiment. With the complete genome sequence of a bovine isolate of M. avium subspecies paratuberculosis, and the independent construction of DNA microarrays in our laboratories, transcriptomic studies for this veterinary pathogen are now possible. Furthermore, the bovine genome sequence project is completed and bovine arrays have been developed to examine host responses to infection with M. avium …

The Complete Genome Sequence Of Mycobacterium Avium Subspecies Paratuberculosis, Lingling Li, J. P. Bannantine, Qing Zhang, Alongkon Amonsin, Barbara J. May, David Alt, Nilanjana Banerji, Sagarika Kanjilal, Vivek Kapur

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne’s disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial …