Proceedings of the 10th International Colloquium on Paratuberculosis

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endoscopic criteria and histopathology findings. Most patients had a history <strong>of</strong> long-standing CD. Patients underwent surgery for different reasons, usually stenosis. The disease was localized in <strong>the</strong> ileum in three patients (9%), in terminal ileum in 15 (45%), in cecum in two (6%), in ascending colon in five (15%), in transverse colon in two (6%), in descending colon in two (6%) and in rectosigmoid in four (12%) patients. In <strong>the</strong> control group, tumour was localized in cecum in three patients (8%), in ascending colon in one (3%), in transverse colon in 10 (26%), in descending colon in 2 (5%), in sigmoid colon in seven (18%) and in rectum in 16 (41%). Specimens <strong>of</strong> <strong>the</strong> individual patients which showed <strong>the</strong> most pronounced lesions (transmural infiltration, cryptitis, crypt abscesses, epi<strong>the</strong>loid granulomas) were selected for fur<strong>the</strong>r analysis. All specimens were stained according to Kreyberg trichrome method and histologically examined. DNA extraction. Formalin-fixed, paraffin-embedded specimens were one to 19 years old. Five 6μm sections were cut from each tissue block. The microtome was cleaned with xylene (Merck) and a new knife was used for each tissue block. All five sections were transferred into a 1.5-ml tube. Tubes were coded and processed blind. A total <strong>of</strong> 1200 μl <strong>of</strong> xylene was added to each tube and vortexed vigorously to remove <strong>the</strong> paraffin. After centrifugation at full speed for 5 min at room temperature, <strong>the</strong> supernatant was removed by pipetting. 1200 μl <strong>of</strong> absolute ethanol was added to <strong>the</strong> pellet to remove <strong>the</strong> residual xylene, mixed gently by vortexing and removed by careful pipetting after centrifugation at full speed for 5 min. Ethanol washing <strong>of</strong> <strong>the</strong> pellet was repeated once again. The tissue was <strong>the</strong>n air-dried. DNA was extracted from all samples using <strong>the</strong> commercially available High Pure PCR Template Preparation Kit according to <strong>the</strong> manufacturer’s instructions (Roche Diagnostics). Detection <strong>of</strong> Map-specific IS900. For <strong>the</strong> PCR assay, IS900-specific primers described previously, namely Av1 (5’-ATG TGG TTG CTG TGT TGG ATG G-3’) and Av2 (5’-CCG CCG CAA TCA ACT CCA G-3’), were used to amplify a 298 bp fragment <strong>of</strong> <strong>the</strong> IS900 gene (3). A 50-μl PCR reaction mixture contained 1� Expand High Fidelity reaction buffer containing 1.5 mM MgCl2, 10% dimethyl sulfoxide, 0.2 mM <strong>of</strong> each dNTP, 200 μM <strong>of</strong> each primer and 3.5 U <strong>of</strong> Expand High Fidelity Taq polymerase (Expand High Fidelity PCR System, Roche Diagnostics). The amplification conditions were as follows: 94°C for 5 min followed by 40 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 3 min, with a final extension at 72°C for 7 min. Amplification products were detected by electrophoresis in 1.5% agarose gels stained by ethidium bromide and visualized by UV transilluminator and camera (Bio Imaging System, Gene Genious, Syngene). Each batch was run with a process control, i.e. archival formalinfixed, paraffin-embedded sections from cattle with Johne’s disease, and with a positive PCR control (DNA from Map strain ATCC 43015). Negative PCR samples were tested for inhibition by spiking 200 ng <strong>of</strong> DNA positive control to one <strong>of</strong> <strong>the</strong> specimens <strong>of</strong> each CD patient and control patient after getting negative PCR Map results. Sterile PCR-grade distilled water was used as negative PCR control. Statistical analysis. The Statistical Package for <strong>the</strong> Social Sciences (SPSS), v.17.0.0. for Windows, was used for <strong>the</strong> statistical analysis (t-test). Statistical significance was accepted at p
All controls were negative to Map by IS900 PCR (Fig. 1). Map was detected in six <strong>of</strong> 164 (4%) specimens obtained from four patients between 26 and 41 years old. Three specimens belonged to a single patient and <strong>the</strong> o<strong>the</strong>r three to <strong>the</strong> remaining three patients. In total, Map DNA was detected in <strong>the</strong> formalin-fixed, paraffin-embedded tissue from four <strong>of</strong> 33 (12%) CD patients. Four positive samples were obtained from terminal ileum, one from rectum and one from mesenteric lymph node taken from <strong>the</strong> ileal resection specimen. Statistical significance was observed when comparing IS900-specific DNA detection rate between CD and non-IBD group (p=0.022). % <strong>of</strong> CD patients or control patients 100% 50% 0% Comparison <strong>of</strong> Map-positive specimens from CD patients vs. control patients 88.0 100 12.0 0 CD patients control patients Fig.1: Comparison <strong>of</strong> Map-positive CD patients vs. control patients Map-negative Map-positive DISCUSSION Extraction <strong>of</strong> nucleic acids from formalin-fixed, paraffin-embedded tissues enables different retrospective studies. The technique has been used to detect Map as a possible infectious agent in <strong>the</strong> pathogenesis <strong>of</strong> CD (Baksh et al., 2004; Cheng et al., 2005; Ryan et al., 2002). In this study we were able to detect Map DNA in <strong>the</strong> formalin-fixed, paraffin-embedded intestine samples from Slovenian CD patients using IS900 PCR. Not all CD patients tested positive to Map, but no Map DNA was detected in control specimens. The detection <strong>of</strong> Map in archival formalin-fixed and paraffin-embedded tissue sections is especially challenging. The target DNA may be fragmented or cross-linked to proteins, which leads to poor or no PCR amplification. This may be <strong>the</strong> reason for some potentially false negative results in our study. Moreover, extraction <strong>of</strong> Map DNA may also fail due to very low Map concentrations in CD patients and due to strong, and even for mycobacteria unconventional cell wall (Hermon- Taylor, 2001). To overcome this limitation, more specimens <strong>of</strong> <strong>the</strong> same CD patient were analyzed. Individuals without Map infection are 17-times less likely to have IBD than Map-infected individuals (Scanu et al., 2007), which is in accordance with our Map-negative non-IBD control group. Since this is a preliminary study, all IS900-positive specimens are still to be confirmed by sequencing. In conclusion, our findings contribute to <strong>the</strong> current knowledge about Map being <strong>the</strong> infectious agent proposed to play <strong>the</strong> role in <strong>the</strong> etiology <strong>of</strong> CD, also in Slovenian CD patients. REFERENCES Baksh FK, Finkelstein SD, Ariyanayagam-Baksh SM, Swalsky PA, Klein EC, Dunn JC, 2004. Absence <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in <strong>the</strong> microdissected granulomas <strong>of</strong> Crohn’s disease. Mod Pathol, 17, 1289 -1294. 70
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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
Page 20: Diagnostics and Genotyping Convenor
Page 23 and 24: #229 Detecting Johne’s Disease he
Page 25 and 26: Kalis CHJ, Hesselink JW, Barkema HW
Page 27 and 28: MATERIALS AND METHODS Sampling For
Page 29 and 30: To better determine the</st
Page 31 and 32: #107 Multilocus Short Sequence Repe
Page 33 and 34: Fig. 1. Dendogram (showing genetic
Page 35 and 36: #105 MIRU-VNTR genotyping o
Page 37: Diagnostics and Genotyping Poster A
Page 40 and 41: Missouri). Serum from a cow with do
Page 42 and 43: #7 Significance of
Page 44 and 45: RESULTS Of the 327
Page 46 and 47: Direct fecal nested Map PCR testing
Page 48 and 49: #16 Histopathological and immunohis
Page 50 and 51: #19 Development of
Page 52 and 53: #36 Genetic diversity of</s
Page 54 and 55: #60 Evaluation of
Page 56 and 57: #66 Comparison of
Page 58 and 59: (b) IS900 Nested PCR, using p90-p91
Page 60 and 61: (d) f57 Real Time PCR. The figure o
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Page 64 and 65: indexes, compared to each single an
Page 66 and 67: #108 Evaluation of
Page 68 and 69: #110 Detection and molecular confir
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Page 74 and 75: #131 Seroprevalence of</str
Page 76 and 77: could be partly attributed to a sma
Page 78 and 79: #146 Analysis of v
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Page 86 and 87: samples from the s
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Page 90 and 91: #196 Inter- and intra-subtype genot
Page 92 and 93: RESULTS In 601 (29%) of</st
Page 94 and 95: #211 Culture of my
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Page 100 and 101: Table 2. Paratuberculosis PCR resul
Page 102 and 103: #254 Programmes on Paratuberculosis
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Page 106 and 107: A bulk milk quality assurance progr
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Page 112 and 113: other projects and
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Page 116 and 117: Host Response and Immunology Keynot
Page 118 and 119: #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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#74 Economic analysis of</s
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#87 Evaluation of
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separately for each existing herd <
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#98 An inter-laboratory ring-trial
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#120 Paratuberculosis vaccine inter
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The statement provides: • A stand
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COMPLEMENTARY ASPECTS On-Farm Disea
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#168 Paratuberculosis in Iran: past
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#180 Behavioural change in owners <
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the fact that <str
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#182 An integrated risk based appro
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etween 0 to 10, depending on <stron
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#203 Milk quality assurance for par
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#234 Johne’s disease vaccine: a c
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Pathogenomics and MAP Biology Conve
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#45 Identification of</stro
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#117 Adsorption of
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#222 Atypical structural features <
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Pathogenomics and MAP Biology Persp
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#52 Is ‘Indian Bison Type’ Myco
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#69 In vitro susceptibility <strong
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#118 Cloning, expression and purifi
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#176 A reference proteomic pr<stron
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#213 Construction the</stro
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#230 Iron concentration dependent s
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Abstract not available at press tim
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#174 Sharing of
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#177 Associations between CARD15 po
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Perspectives Talk: Public Health My
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A total of 206 qua
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Feller, M., K. Huwiler, R. Stephan,
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In 2008, the Ameri
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#115 Crohn’s disease and ruminant
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International John
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#76 Towards improved reporting stan
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#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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