Proceedings of the 10th International Colloquium on Paratuberculosis

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#230 Iron concentration dependent stressome <strong>of</strong> Mycobacterium avium subsp. paratuberculosis Harish K Janagama 1 , Srinand Sreevatsan 1 , TMA SenthilKumar 1 , Michael Paustian 2 , John Bannantine 2 1 University <strong>of</strong> Minnesota, USA; 2 National Animal Disease Center, USDA-ARS, Ames, Iowa, USA Mycobacterium avium subsp. paratuberculosis (MAP) residing inside host macrophages regulates expression <strong>of</strong> iron acquisition, iron transport and oxidative stress response genes. Since MAP is mycobactin dependent for its growth in laboratory media functional analysis <strong>of</strong> MAP in response to iron limitation in-vitro would increase our understanding <strong>of</strong> its iron physiology in <strong>the</strong> host as well as aid in <strong>the</strong> development <strong>of</strong> improved culture methods. Therefore, we undertook a high-resolution proteomic and transcriptomic analysis <strong>of</strong> MAP using iTRAQ (isobaric tag for relative and absolute quantification) and microarrays. MAP was grown to mid-logarithmic phase in MB7H9 medium containing mycobactin J (MJ) and washed several times in PBS and inoculated separately into minimal essential medium (MEM) (nutrient starvation) (20%glycerol, K 2 HPO 4 and MgSO 4 ) containing (i) no iron or (ii) iron at 50μM. Three hours following inoculations, total cellular protein extracted from bacterial pellets was analyzed using iTRAQ labeling for protein identification and total RNA extracted was used in MAP K-10 microarrays for genome wide transcriptome analysis. Results show that under iron deplete conditions, MAP downregulated expression <strong>of</strong> iron utilizing proteins and upregulated stress response proteins similar to nutrient deprived conditions (estimated iron concentration 1µM). Fur<strong>the</strong>r, we identified two novel proteins belonging to <strong>the</strong> ESAT-6 family and a conserved hypo<strong>the</strong>tical membrane protein, which were not previously identified by genome annotation. Identification <strong>of</strong> <strong>the</strong>se proteins is significant as it opens new areas <strong>of</strong> functional characterization to understand MAP physiology during infection. The data also show that MAP may be unsuccessful in accumulating sufficient quantities iron deplete conditions and allows us to interrogate this stress induced metabalome to device better culture methods. #232 PrrA is important in Mycobacterium avium subspecies paratuberculosis virulence Chia-wei Wu, Chung-Yi Hsu, Adel M. Talaat,University <strong>of</strong> Wisconsin-Madison, USA Two-component systems are widely found in bacteria and involved in sensing environmental changes and subsequently altering gene expression patterns to adapt to various survival challenges. One <strong>of</strong> such systems, PrrA-PrrB, was shown to be required for early intracellular replication <strong>of</strong> Mycobacterium tuberculosis (M. tb) in a macrophage infection model. The response regulator, PrrA, was also shown to be auto-regulated through promoter binding. BLAST analysis indicated that <strong>the</strong> prrAB operon (MAP0833c and MAP0834c) <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (M. ap) is homologous to that <strong>of</strong> M. tb (82% identical), and those in o<strong>the</strong>r mycobacteria. Additional sequence analysis showed that M. ap prrA has a signal receiver domain and a DNA binding domain, suggesting <strong>the</strong> signal transduction and gene regulation roles <strong>of</strong> PrrA. We have screened a prrA::Tn5367 mutant from our transposon mutant library with <strong>the</strong> genetic background <strong>of</strong> M. ap ATCC 19698. In a mouse infection model <strong>of</strong> paratuberculosis, <strong>the</strong> prrA::Tn5367 mutant showed significant reduced levels <strong>of</strong> colonization in <strong>the</strong> liver and intestine compared to <strong>the</strong> wild type. Fur<strong>the</strong>rmore, <strong>the</strong> mutant also showed lessdefined granulomatous structure in pathological examinations, suggesting a reduced virulence phenotype <strong>of</strong> <strong>the</strong> mutant. In addition, microarray analysis <strong>of</strong> M. ap culture in an acidic condition (pH 5.5) showed a 3.5fold induction <strong>of</strong> <strong>the</strong> prrA gene. Currently we are testing <strong>the</strong> intracellular survival <strong>of</strong> <strong>the</strong> mutant in a mouse macrophage infection model and identifying genes under <strong>the</strong> control <strong>of</strong> prrA with microarray analysis. In summary, we showed that disruption <strong>of</strong> <strong>the</strong> prrA gene results in reduced virulence <strong>of</strong> M. ap and we believe <strong>the</strong> PrrAB two-component system is important in M. ap pathogenesis. 259
#239 Mycobacterium avium subsp. paratuberculosis (MAP) infection in cull dairy cows Robert H Whitlock, T Fyock, Y Schukken, J Van Kessel, J Karns, E Hovingh, J Smith University <strong>of</strong> Pennsylvania, USA; Cornell University, USA; USDA-ARS, Beltsville, MD, USA; Penn State University, PA, USA; University <strong>of</strong> Vermont, Burlington, VT, USA Objective: To correlate fecal shedding to MAP tissue bio-burden and to determine what proportion <strong>of</strong> fecal culture negative cows would have culture positive fecals and tissues at slaughter. Materials and Methods: Tissues and fecal samples were harvested from 230 cows culled from 3 dairy herds over four years. Five samples were collected from each animal; 2 intestinal lnn, ileum, IC valve and a fecal sample. Samples were cultured on HEYM. Results: Of <strong>the</strong> 230 cattle with harvested tissues, 17 (9.8%) were previously fecal culture positive for MAP. Fourteen <strong>of</strong> 17 animals were positive at slaughter (82%) and 3/17 (18%) cattle had all 5 samples culture negative. Of <strong>the</strong> 20 fecal culture positive cattle at <strong>the</strong> time <strong>of</strong> slaughter, 9 were heavy shedders each massively infected in <strong>the</strong> four tissues examined (>300 cfu MAP/tube). No o<strong>the</strong>r fecal culture positive or negative cows had such massive tissue infection with MAP. Interestingly 5/20 (25%) fecal culture positive cows at slaughter had negative cultures on all four tissues. Three positive fecal culture cows (very low shedders) had only one colony on <strong>the</strong> four tissues cultured, suggesting infections as adults. Of <strong>the</strong> 156 cattle with all negative fecal cultures prior to culling, 58/156 (37%) had at least one positive sample at slaughter. The lnn was positive most frequently, followed closely by ileum and IC valve. 26/58 (45%) cattle had less than 10 total cfu <strong>of</strong> MAP on 20 tubes <strong>of</strong> HEYM for <strong>the</strong> five samples suggests a more recent infection. Conclusions: 35% <strong>of</strong> fecal culture negative cattle had positive tissues at slaughter. Cattle shedding more than a few colonies <strong>of</strong> MAP or with multiple positive fecal cultures had multiple tissues positive at high MAP concentrations. 260
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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
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RESULTS Loss of al
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#40 Managing Bovine Johnes Disease
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Page 214 and 215: separately for each existing herd <
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Page 288 and 289: #97 The EU ParaTBTools Project - Th
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Page 292 and 293: Paratuberculosis ELISA Reliable, si
Page 294 and 295: PRIONICS AG WAGISTRASSE 27A PHONE +
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