Proceedings of the 10th International Colloquium on Paratuberculosis

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#176 A reference proteomic pr<strong>of</strong>ile <strong>of</strong> <strong>the</strong> cell wall <strong>of</strong> <strong>the</strong> Mycobacterium avium complex Zhiguo He, Jeroen De Buck,University <strong>of</strong> Calgary, Canada All four M. avium subspecies (avium, silvaticum, hominissuis, paratuberculosis) and M. intracellulare are collectively known as <strong>the</strong> Mycobacterium avium complex (MAC) and possess a high degree <strong>of</strong> genetic similarity but can inhabit diverse environments and infect a diverse range <strong>of</strong> host species. Despite <strong>the</strong>ir divergent phenotypes and <strong>the</strong> diseases <strong>the</strong>y cause, <strong>the</strong> genomes <strong>of</strong> M. avium subspecies share greater than 97% nucleotide identity and 91% with M. intracellulare. Therefore, a better understanding <strong>of</strong> <strong>the</strong> host specificity and differences in pathogenicity within <strong>the</strong> MAC could come from a thorough proteomic comparison. The cell wall proteins are particular interesting in this respect. The aim <strong>of</strong> this study was to construct a reference cell wall protein map for <strong>the</strong> MAC and identify differences between <strong>the</strong> MAC members. Method: A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize <strong>the</strong> cell wall associated proteins <strong>of</strong> all 5 members <strong>of</strong> <strong>the</strong> MAC. An enzymatic cell surface shaving method was used to determine <strong>the</strong> surface-exposed proteins. Results: Between 300 and 400 cell wall proteins and more than 40 surface-exposed proteins were identified for each MAC member. The overlap <strong>of</strong> <strong>the</strong> different cell wall proteomes was determined and mapped against a consensus reference MAC cell wall protein pr<strong>of</strong>ile. Detailed information about functional classification, signal peptides and number <strong>of</strong> transmembrane domains were added to <strong>the</strong> reference pr<strong>of</strong>ile. Conclusion: We have generated a comprehensive pr<strong>of</strong>ile <strong>of</strong> <strong>the</strong> MAC cell wall subproteome. This work will help us focus in on proteins in <strong>the</strong> cell wall that might be involved in Map specific virulence mechanisms and those <strong>of</strong> <strong>the</strong> o<strong>the</strong>r MAC members. 255
#179 Rhodanine agents active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis Tim J Bull, Richard Linedale, John Hermon-Taylor, Jason Hinds,St George's University <strong>of</strong> London, United Kingdom; King's College London, United Kingdom Introduction and Objective: The capacity for long term persistence inside macrophages and o<strong>the</strong>r cell lineages combined with <strong>the</strong> ability to enter a non-replicating viable (NRV), <strong>of</strong>ten non-culturable state, allows MAP to develop paucimicrobial chronic infection states leading to disease. Therapy is difficult because <strong>the</strong> lack <strong>of</strong> bacterial division, diminution <strong>of</strong> cell wall antigens and low transcriptomic turnover in NRV drastically reduces <strong>the</strong> effectiveness <strong>of</strong> conventional antibiotics. In addition, MAP can manipulate host intracellular killing mechanisms and disrupt pathogen antigen presentation leading to dysregulation <strong>of</strong> host cell mediated clearance. In this study we evaluate a novel anti-MAP agent Rhodanine(D157070) that aims to re-assert <strong>the</strong> normal surveillance and activation <strong>of</strong> host cell defense mechanisms by inhibiting MAP from releasing anti-reactive nitrogen intermediates. Materials and Methods: Culturable viability (CV) and viable persistence (VP) <strong>of</strong> MAP K10 in a variety macrophage models and culture alone as a control, with and without Rhodanine(D157070) treatment was monitored over a 7 day period. CV was measured using conventional colony counting. VP was determined as <strong>the</strong> degree <strong>of</strong> ribosomal turnover represented by <strong>the</strong> qPCR DNA:RNA ratio <strong>of</strong> a 102bp pre16SrRNA leader sequence present as single copy number (DNA) but cleaved and degraded during ribosomal assembly (RNA). Full MAP genome transcriptomic pr<strong>of</strong>iles were also obtained during treatment using <strong>the</strong> ParaTBtools MAPAC microarray. Results: Rhodanine(D157070) did not kill MAP in conventional extracellular culture but transcriptome pr<strong>of</strong>iles were altered. Rhodanine(D157070) was highly active against intracellular MAP in macrophage infection models increasing MAP killing as measured by both CV and VP assays. Rhodanine(D157070) alone showed no toxicity to cell lines. Conclusion: Rhodanine(D157070) does not kill MAP directly but can enter host cells and induce inhibition <strong>of</strong> MAP derived processes that normally decrease effectiveness <strong>of</strong> intracellular killing mechanisms. This agent thus shows good promise as a <strong>the</strong>rapy directed at non-replicating intracellular MAP infection. #206 The role <strong>of</strong> TcrXY, a two component system in Mycobacterium avium subspecies paratuberculosis Michelle Pinto, Lucy Mutharia, University <strong>of</strong> Guelph, Canada Bacteria are able to survive in environments that experience continuous fluctuation in both physical and chemical aspects by adapting to <strong>the</strong>se changes using sensory-response mechanisms such as Two Component Systems. These systems involve autophosphorylation <strong>of</strong> membrane histidine protein kinase receptors on detection <strong>of</strong> certain stimuli and phosphoryl transfer to a cytoplasmic response regulator, which generally acts as a transcription regulator. Mycobacterium avium subspecies paratuberculosis (MAP), <strong>the</strong> etiological agent involved in Johne’s disease in ruminants, has ten putative two component systems in addition to one orphan histidine protein kinase and five orphan response reglulators. The TcrXY system has been studied in M. tuberculosis and infection assays using a partial deletion construct resulted in hypervirulence in SCID mice. The tcrXY promoter in MAP_K10 was used to generate promoter: lacZ fusions and used in in vitro β-galactosidase assays in Mycobacterium avium subsp. hominisuis 104 (MAA104). The tcrXY promoter was induced in acidic conditions (pH 4.5 to 5.5). It is possible that TcrXY may be responsive to changes in pH in <strong>the</strong> environment in addition to what it faces in <strong>the</strong> gut and feces <strong>of</strong> <strong>the</strong> host as well as in phagosomes. A MAP tcrXY gene knockout will be characterized by investigating <strong>the</strong> survival <strong>of</strong> <strong>the</strong> mutants in macrophages and when exposed to acidic gradients with <strong>the</strong> goal <strong>of</strong> identifying <strong>the</strong> role that TcrXY plays in virulence and pathogenesis. The recently described conditionally replicating shuttle phasmid pHAE87 is used to generate <strong>the</strong> TcrXY mutants. 256
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
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Scott Wells - Planning Committee Ch
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Monday, August 10, 2009 - All sessi
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Kari Røste Lybeck, Anne Kristine S
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1120-1140 #88: Influence of
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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Host Response and Immunology Keynot
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#33 MERKAL AWARD LECTURE No interfe
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#75 Interleukin 17 and interleukin
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#173 Local and systemic roles for b
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#23 Role of Mycoba
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Host Response and Immunology Poster
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#28 Intestinal MAP Infection via Pe
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#73 Age susceptibility of</
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#95 Role of nitric
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#134 Analysis of <
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#157 Study of <str
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#163 Immunohistochemical expression
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#193 Development of</strong
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#209 Murine macrophages carrying an
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#224 Application of</strong
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Table 1 UK and Cyprus MAP survey Re
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#228 Mycobacterium avium ssp. parat
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#145 MERKAL AWARD LECTURE Multinomi
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#50 Assessment of
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Table 1. Posterior median (CrIs) <s
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#55 Birth clusters of</stro
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from their dam, se
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#88 Influence of b
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#104 Relation between faecal shedde
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Epidemiology Poster Abstracts 109 1
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#22 The Prevalence of</stro
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CONCLUSION IS1311-based nested Ma/M
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#42 Seroprevalence survey o
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#77 Characterisation of</st
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Environmental samples were collecte
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REFERENCES 1) Chiodini RJ, Van Krui
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#113 Effect of soi
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#136 Error-adjusted, variance parti
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Latium Region Viterbo districts RES
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#144 The suitability of</st
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#178 Age structure of</stro
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A statistically significant lower f
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#183 Sero-prevalence of</st
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#218 Johne’s disease in t
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#244 A survey to estimate t
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Control Programs Keynote Lecture Re
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#202 Milk quality assurance for par
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#141 Control of pa
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#198 Use of small
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Control Programs Poster Abstracts 1
Page 206 and 207: RESULTS Loss of al
Page 208 and 209: #40 Managing Bovine Johnes Disease
Page 210 and 211: #74 Economic analysis of</s
Page 212 and 213: #87 Evaluation of
Page 214 and 215: separately for each existing herd <
Page 216 and 217: #98 An inter-laboratory ring-trial
Page 218 and 219: #120 Paratuberculosis vaccine inter
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Page 222 and 223: COMPLEMENTARY ASPECTS On-Farm Disea
Page 224 and 225: #168 Paratuberculosis in Iran: past
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Page 238 and 239: Pathogenomics and MAP Biology Conve
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Page 244 and 245: #222 Atypical structural features <
Page 246 and 247: Pathogenomics and MAP Biology Persp
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Page 250 and 251: #52 Is ‘Indian Bison Type’ Myco
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Page 260 and 261: #230 Iron concentration dependent s
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Page 266 and 267: Abstract not available at press tim
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Page 272 and 273: Perspectives Talk: Public Health My
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Page 276 and 277: A total of 206 qua
Page 278 and 279: Feller, M., K. Huwiler, R. Stephan,
Page 280 and 281: In 2008, the Ameri
Page 282 and 283: #115 Crohn’s disease and ruminant
Page 284 and 285: International John
Page 286 and 287: #76 Towards improved reporting stan
Page 288 and 289: #97 The EU ParaTBTools Project - Th
Page 290 and 291: #241 US Vaccine Initiative through
Page 292 and 293: Paratuberculosis ELISA Reliable, si
Page 294 and 295: PRIONICS AG WAGISTRASSE 27A PHONE +
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