Proceedings of the 10th International Colloquium on Paratuberculosis

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#45 Identification <strong>of</strong> genes expressed by Mycobacterium avium subsp. paratuberculosis in subclinical Johne’s disease and in vitro infected macrophages Sajan George, Wayne Xu, Zeng Jin Tu, Jeremy Schefers, Srinand Sreevatsan Veterinary Population Medicine, University <strong>of</strong> Minnesota, USA; Minnesota Supercomputing Institute, University <strong>of</strong> Minnesota, USA; Veterinary Diagnostic Laboratory, University <strong>of</strong> Minnesota, USA Mycobacterium avium subsp. paratuberculosis (MAP) uses a diverse set <strong>of</strong> genes in <strong>the</strong> cellular environment for its survival and pathogenesis. We hypo<strong>the</strong>sized that genes expressed exclusively in <strong>the</strong> in tissues or in macrophages would contribute to MAP virulence. We studied <strong>the</strong> MAP gene regulation in ileum and mesenteric lymph nodes <strong>of</strong> two subclinical Johne’s disease (JD) animals or in an in vitro macrophage infection model. Total RNA was extracted from tissues or in vitro infected monocyte derived macrophages. Mycobacterial mRNA was enriched and amplified, reverse transcribed to cDNA and competitively hybridized to a MAP K10 microarrays. We evaluated only those genes differentially expressed in tissues distinct from those expressed in a nutrient rich broth environment. Genes related to cell envelope biogenesis, carbohydrate metabolism (glucose-6phosphates, glucokinases), fatty acid metabolism (acyl-coA dehydrogenases, glycosyl transferases), virulence (MCE family proteins, PPE family proteins, serine/threonine protein kinases), transcriptional regulators, ABC transporters and oxidoreductases were differentially expressed by MAP in tissues or macrophages. Gene expression patterns were similar between <strong>the</strong> infected tissues and macrophage infection at three time points and shared common functions as determined by pathway analysis. Our analysis establishes a repertoire <strong>of</strong> genes used by MAP in <strong>the</strong> host, which is expected to aid in <strong>the</strong> development <strong>of</strong> successful vaccination strategies for <strong>the</strong> effective control <strong>of</strong> JD in herd level. 239
#89 Mycobacterium avium subspecies paratuberculosis isolates recovered from infected goats in <strong>the</strong> central Spain contain deletions <strong>of</strong> mce genes and an increased capacity for persistence in macrophages Elena Castellanos, Alicia Aranaz, Lucas Dominguez, Lucia de Juan, Ana Mateos, Richard Linedale, Tim John Bull Animal Health Surveillance Center (VISAVET). Complutense University <strong>of</strong> Madrid (UCM). Animal Health Department. Veterinary School.UCM, Spain; Division <strong>of</strong> Cellular and Molecular Medicine. St. George’s University <strong>of</strong> London, United Kingdom This study used <strong>the</strong> ParaTBtools MAPAC microarray to perform comparative genomic hybridization (CGH) on MAP isolates recovered from a goat herd in Spain. These animals presented unusual features on histopathological investigation including moderate to severe diffuse lymphocytic infiltration with a heavy macrophage load but a very low cytoplasmic bacterial load as determined by Ziehl-Neelsen staining. CGH analysis revealed a novel deletion, consisting <strong>of</strong> 19 consecutive ORFs which including a complete mce gene operon, PE/PPE family genes, a transcriptional regulator and several o<strong>the</strong>r genes with ei<strong>the</strong>r putative or unknown function. Findings were confirmed by a PCR-based approach and sequencing. A panel <strong>of</strong> 97 Spanish MAP isolates was also screened using this PCR-based approach revealing 53 isolates containing <strong>the</strong> same deleted region. Epidemiological analysis correlated mce negative MAP isolates with herds having a history <strong>of</strong> animal transfer. Parallel infections <strong>of</strong> a representative mce negative isolate (MAP 464) and a reference isolate (MAP K-10) into a macrophage cell line showed a significant increase in intracellular persistence associated with MAP 464. Fur<strong>the</strong>r studies <strong>of</strong> differential mce expression between MAP 464 and MAP K-10 will also be presented. 240
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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
Page 190 and 191: #183 Sero-prevalence of</st
Page 192 and 193: #218 Johne’s disease in t
Page 194 and 195: #244 A survey to estimate t
Page 196 and 197: Control Programs Keynote Lecture Re
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Page 200 and 201: #141 Control of pa
Page 202 and 203: #198 Use of small
Page 204 and 205: 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
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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 252 and 253: #69 In vitro susceptibility <strong
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Page 266 and 267: Abstract not available at press tim
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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
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#241 US Vaccine Initiative through
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Paratuberculosis ELISA Reliable, si
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PRIONICS AG WAGISTRASSE 27A PHONE +
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