Proceedings of the 10th International Colloquium on Paratuberculosis

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(b) IS900 Nested PCR, using p90-p91 primers 4 for <strong>the</strong> first round followed by a second round with specific primers (see Table 1). The first round <strong>of</strong> Nested PCR was performed in 25 μl final volume with 0.1 U <strong>of</strong> TAQ (Qiagen, Milan, Italy) adding two μl <strong>of</strong> DNA. The second round was performed by adding two μl <strong>of</strong> <strong>the</strong> previous run in a final volume <strong>of</strong> 25 μl. The reaction was carried out in Mastercycler ep gradient s (Eppendorf); (c) IS900 TaqMan Real time PCR, performed adding two μl <strong>of</strong> DNA in 25 μl final volume, using TaqMan® Gene Expression Master Mix (Applied Biosystems, Milan, Italy) in StepOnePlus Real-Time PCR System (Applied Biosystems). Fluorescence intensity was normalized with ROX dye; (d) f57 SYBR Green Real Time PCR, performed adding two μl <strong>of</strong> DNA in 25 μl final volume using Power SYBR® Green PCR Master Mix (Applied Biosystems) in StepOnePlus Real-Time PCR System (Applied Biosystems). Fluorescence intensity was normalized with ROX dye. The amplification was followed by melting curve analysis, melting <strong>the</strong> amplicon from 60 °C to 95 °C and recording <strong>the</strong> fluorescence 3.3 times per degree with a temperature ramp rate <strong>of</strong> 2.2 °C/sec. Both <strong>the</strong> commercial PCR (a) and <strong>the</strong> TaqMan (c) contained an internal amplification control in order to discriminate between negative or inhibited samples, while <strong>the</strong> o<strong>the</strong>r test did not contain any internal amplification control. Primers sequences are listed in table 1. Table 1: Primers and probes sequences. Methods Oligo name Sequences Ta (°C) a Adiavet Kit primers unknown 62 b P90 5’-GAAGGGTGTTCGGGGCCGTCGCTTAGG-3’ P91 5’-GGCGTTGAGGTCGATCGCCCACGTGAT-3’ Nested Forward 5'-GACGACTCGACCGCTAATTG-3' Nested Reverse 5'-CCGTAACCGTCATTGTCCAG-3' 62 58 IS900 Forward 5’-CCGGTAAGGCCGACCATTA-3’ c IS900 Reverse 5’-ACCCGCTGCGAGAGCA-3’ 60 IS 900 Probe 6-FAM-CATGGTTATTAACGACGACGCGCAGC-TAMRA-3’ d f57 Forward f57 Reverse 5'-ATAGCTTTCCTCTCCTTCGTC-3' 5'-CAGGGCAACAACATATTCGG-3' 60 RESULTS Out <strong>of</strong> 37 milk samples tested we found only one positive sample (3%) using (a), (c) and (d) methods. The Nested PCR method (b) showed a total <strong>of</strong> eight positive samples (22%). No inhibited samples were found with (a) and (c) methods. (a) Adiavet PCR. Note <strong>the</strong> internal amplification control in all samples. Only one sample (4) was positive. M: Marker; NE: negative extraction control; NN: no template control; 1-9: milk samples. M NE 1 2 3 4 5 6 7 8 9 NN 57
(b) Nested PCR. All positive samples (4, 7, 9, 10, 13) showed a 99 bp amplicon with <strong>the</strong> same size as <strong>the</strong> positive control. M: 100 bp Marker; NE: negative extraction control; 1°NN: no template 1 st round PCR; 2°NN: no template 2 nd round PCR; +: positive control (DNA from MAP ATCC 19698); 1-13: milk samples. M NE 1°NN 1 2 3 4 5 6 7 8 9 10 11 12 13 + 2°NN (c) IS900 TaqMan Real Time PCR. Note <strong>the</strong> internal amplification control. DNA from MAP ATCC 19698 was used as positive control. Data were in duplicate. 58 Positive control Internal amplification control Positive sample
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
Page 8 and 9: Scott Wells - Planning Committee Ch
Page 10 and 11: Monday, August 10, 2009 - All sessi
Page 12 and 13: Kari Røste Lybeck, Anne Kristine S
Page 14 and 15: 1120-1140 #88: Influence of
Page 16 and 17: Concurrent Session B - Rm 175 Wille
Page 18 and 19: 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 60 and 61: (d) f57 Real Time PCR. The figure o
Page 62 and 63: • MIRU (3 loci); • VNTR-MIRU (7
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
Page 70 and 71: endoscopic criteria and histopathol
Page 72 and 73: Bull TJ, McMinn EJ, Sidi-Boumedine
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
Page 84 and 85: #191 Potentiating day-old blood sam
Page 86 and 87: samples from the s
Page 88 and 89: (MAA) (MAP87 and MAP3776) and 1 fro
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
Page 96 and 97: #242 Application and field validati
Page 98 and 99: #251 Diagnosis of
Page 100 and 101: Table 2. Paratuberculosis PCR resul
Page 102 and 103: #254 Programmes on Paratuberculosis
Page 104 and 105: Fig. 1. European countries specifyi
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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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#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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273
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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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