Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat- treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated

The Kinematic and Microphysical Control of Storm Integrated Lightning Flash Extent

Objective: To investigate the kinematic and microphysical control of lightning properties, particularly those that may govern the production of nitrogen oxides (NOx) in thunderstorms, such as flash rate, type (intracloud [IC] vs. cloud-to-ground [CG] ) and extent. Data and Methodology: a) NASA MSFC Lightning Nitrogen Oxides Model (LNOM) is applied to North Alabama Lightning Mapping Array (NALMA) and Vaisala National Lightning Detection Network(TradeMark) (NLDN) observations following ordinary convective cells through their lifecycle. b) LNOM provides estimates of flash type, channel length distributions, lightning segment altitude distributions (SADs) and lightning NOx production profiles (Koshak et al. 2012). c) LNOM lightning characteristics are compared to the evolution of updraft and precipitation properties inferred from dual-Doppler (DD) and polarimetric radar analyses of UAHuntsville Advanced Radar for Meteorological and Operational Research (ARMOR, Cband, polarimetric) and KHTX (S-band, Doppler)

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model

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 I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne’s disease vaccine candidate screening and evaluation

Primary transcriptomes of Mycobacterium avium subsp. paratuberculosis reveal proprietary pathways in tissue and macrophages

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) persistently infects intestines and mesenteric lymph nodes leading to a prolonged subclinical disease. The <it>MAP </it>genome sequence was published in 2005, yet its transcriptional organization in natural infection is unknown. While prior research analyzed regulated gene sets utilizing defined, in vitro stress related or advanced surgical methods with various animal species, we investigated the intracellular lifestyle of MAP in the intestines and lymph nodes to understand the MAP pathways that function to govern this persistence.</p> <p>Results</p> <p>Our transcriptional analysis shows that 21%, 8% and 3% of the entire MAP genome was represented either inside tissues, macrophages or both, respectively. Transcripts belonging to latency and cell envelope biogenesis were upregulated in the intestinal tissues whereas those belonging to intracellular trafficking and secretion were upregulated inside the macrophages. Transcriptomes of natural infection and in vitro macrophage infection shared genes involved in transcription and inorganic ion transport and metabolism. MAP specific genes within large sequence polymorphisms of ancestral <it>M. avium </it>complex were downregulated exclusively in natural infection.</p> <p>Conclusions</p> <p>We have unveiled common and unique MAP pathways associated with persistence, cell wall biogenesis and virulence in naturally infected cow intestines, lymph nodes and in vitro infected macrophages. This dichotomy also suggests that in vitro macrophage models may be insufficient in providing accurate information on the events that transpire during natural infection. This is the first report to examine the primary transcriptome of MAP at the local infection site (i.e. intestinal tissue). Regulatory pathways that govern the lifecycle of MAP appear to be specified by tissue and cell type. While tissues show a "shut-down" of major MAP metabolic genes, infected macrophages upregulate several MAP specific genes along with a putative pathogenicity island responsible for iron acquisition. Many of these regulatory pathways rely on the advanced interplay of host and pathogen and in order to decipher their message, an interactome must be established using a systems biology approach. Identified MAP pathways place current research into direct alignment in meeting the future challenge of creating a MAP-host interactome.</p

Generation and screening of a comprehensive \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e transposon mutant bank

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and world wide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay developent. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P\u3e95% )was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped up stream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated

Circulating Mycobacterium bovis Peptides and Host Response Proteins as Biomarkers for Unambiguous Detection of Subclinical Infection

Bovine tuberculosis remains one of the most damaging diseases to agriculture, and there is also a concern for human spillover. A critical need exists for rapid, thorough, and inexpensive diagnostic methods capable of detecting and differentiating Mycobacterium bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. In a previous study, Seth et al. (PLoS One 4:e5478, 2009, doi:10.1371/journal.pone.0005478) identified 32 host peptides that specifically increased in the blood serum of M. bovis-infected animals). In the current study, 16 M. bovis proteins were discovered in the blood serum proteomics data sets. A large-scale validation analysis was undertaken for selected host and M. bovis proteins using a cattle serum repository containing M. bovis (n = 128), Mycobacterium kansasii (n = 10), and Mycobacterium avium subsp. paratuberculosis (n = 10), cases exposed to M. bovis (n = 424), and negative controls (n = 38). Of the host biomarkers, vitamin D binding protein (VDBP) showed the greatest sensitivity and specificity for M. bovis detection. Circulating M. bovis proteins, specifically polyketide synthetase 5, detected M. bovis-infected cattle with little to no seroreactivity against M. kansasii- and M. avium subsp. paratuberculosis-infected animals. These data indicate that host and pathogen serum proteins can serve as reliable biomarkers for tracking M. bovis infection in animal populations

Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle

BACKGROUND: Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P < or = 0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. CONCLUSIONS/SIGNIFICANCE: The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Survival strategies of Mycobacterium avium subsp. paratuberculosis in a variety of microenvironments.

University of Minnesota Ph.D. dissertation. June 2012. Major: Comparative and Molecular Biosciences. Advisor: Srinand Sreevatsan, M.VSc., M.P.H., Ph.D., 1 computer file (PDF); iv, 179 pages.Mycobacteria, specifically Mycobacterium avium subsp. paratuberculosis (MAP), are extreme strategists and as a rule live by deception. Mycobacteria represent a group of closely related acid-fast bacilli that encompass a wide-range of host tropisms and diseases. Mycobacteria can be divided into two complexes: the Mycobacterium tuberculosis complex and the Mycobacterium avium complex (MAC). The MAC is comprised of M. avium subsp. avium (M. avium), MAP, M. intracellulare and M. avium subsp. hominissuis (M. hominissuis), all of which share an over 90 percent nucleotide similarity. Despite its genetic similarity, MAC elicits different diseases in both animals and humans including infections of the lung, lymph nodes, bones, skin and gastrointestinal tract. MAP is a unique member of MAC as it infects and establishes itself within the intestine of ruminants and other wildlife. Furthermore, MAP lives in a quiescent state in soil and aquatic environments. Since MAP encounters numerous environments, including those with unfavorable conditions, it has developed several strategies to survive. However, the mechanisms by which MAP survival is achieved remains incompletely understood. The goal of these studies was to determine how MAP may survive and disseminate under unfavorable conditions, which included nutrient starvation and host pressures. We have identified the development of a new MAP morphotype under prolonged nutrient starved conditions. This novel MAP morphotype resembles a spore-like structure and contains dipicolinic acid, which is used to protect DNA located within the core. These novel structures are heat resistant at 70oC and can be enriched for in multiple MAP strains. Furthermore, we describe an unrecognized mechanism by which MAP takes advantage of host responses at the epithelium interface to recruit macrophages to the site of initial infection. MAP is able to safely enter into macrophages and consequently ensures its establishment, survival and dissemination throughout the host. Lastly, we demonstrate the importance of host physiological relevant temperature on successful disease progression. Infection utilizing the temperature of MAP’s natural host, the cow, enhances the speed of infection as well as host and pathogen transcriptomic profiles. Taken together, data generated from these studies will provide the basis for understanding MAP persistence and survival in diverse conditions. The mechanisms by which MAP establishes, disseminates and/or survives difficult conditions may impact new programs to control JD as well as rational vaccine/therapeutic design and the way in which we view other mycobacterioses

Monoclonal antibodies bind a SNP-sensitive epitope that is present uniquely in Mycobacterium avium subspecies paratuberculosis

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 MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mis- match) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the speci- ficity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen