Proceedings of the 10th International Colloquium on Paratuberculosis

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#60 Evaluation <strong>of</strong> 2 extraction methods and 2 PCR assays for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues Kelly S Anklam, Marie Pinkerton, Elizabeth J Manning, Michael T Collins School <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Wisconsin, Madison, USA Objectives: To evaluate and compare two methods for extracting DNA from formalin-fixed paraffin-embedded (FFPE) tissues plus two PCRs for detecting extracted Mycobacterium avium subsp. paratuberculosis (MAP) DNA. Procedure: Sections (24 mm slices) were sliced from 31 formalin-fixed paraffin-embedded tissue blocks collected from cases with ante-mortem evidence <strong>of</strong> Johne’s (bovine, bison, caprine, swine) or Crohn’s disease (human). The number and types <strong>of</strong> tissues per block varied but in all cases <strong>the</strong> ileum or jejunum and/or <strong>the</strong> mesenteric lymph node was included. Five negative control tissue block slices from a known uninfected animal were included as well for extraction. Five mm sections from each block were stained (haematoxylin and eosin and by Ziehl-Neelsen) to classify <strong>the</strong> tissues for <strong>the</strong> presence/absence <strong>of</strong> microscopic lesions (granulomatous infiltration and giant cells) and quantity <strong>of</strong> acid-fast organisms (scale 0-4) as <strong>the</strong> gold standard. DNA was extracted from paired sections by a boil/freeze method, which involved 2 cycles <strong>of</strong> boiling and freezing followed by centrifugation, and by a commercial DNA extraction kit (WaxFreeTM DNA) specific for FFPE tissue samples a modified protocol was supplied by TrimGen. Each <strong>of</strong> <strong>the</strong> extracted samples was amplified by IS900 nested PCR and by hspX real-time PCR. Results: Two methods <strong>of</strong> DNA extraction and two PCR protocols using different targets were compared. None <strong>of</strong> <strong>the</strong> negative control tissues were PCR positive. When <strong>the</strong> PCR results were compared with <strong>the</strong> presence <strong>of</strong> acid-fast organisms in <strong>the</strong> tissues, <strong>the</strong> sensitivity <strong>of</strong> <strong>the</strong> detection <strong>of</strong> MAP in FFPE tissue blocks was 96.8% (one false negative result) when using <strong>the</strong> boil/freeze method <strong>of</strong> extraction with <strong>the</strong> hspX real-time PCR and 93.5% (one false negative and one false positive result) with <strong>the</strong> IS900 nested PCR. When using <strong>the</strong> commercial extraction kit with hspX real-time PCR <strong>the</strong> sensitivity was 90.3% (three false negative results) and with <strong>the</strong> IS900 nested PCR 54.8% (14 false positives). Conclusions: A simple and inexpensive method <strong>of</strong> DNA extraction <strong>of</strong> FFPE tissues is effective and easy to perform. Coupling <strong>the</strong> boil/freeze extraction method with <strong>the</strong> hspX real-time PCR yields a rapid and reliable method for detecting MAP in tissues processed in paraffin. 53
#62 Field and interlaboratory evaluation <strong>of</strong> four commercial ELISA kits for M. avium subsp. paratuberculosis antibody detection in bovine serum Nicola Pozzato, Norma Arrigoni, Sabrina Bonizzato, Mariagrazia Chiavegato, Annalucia Tondo, Katia Capello Istituto Zoopr<strong>of</strong>ilattico Sperimentale delle Venezie - Laboratorio di diagnostica clinica, Verona, Italy; Istituto Zoopr<strong>of</strong>ilattico Sperimentale della Lombardia e Emila-Romagna - Sezione di Piacenza, Piacenza, Italy; Istituto Zoopr<strong>of</strong>ilattico Sperimentale delle Venezie - Direzione Sanitaria, Legnaro, Italy Detection <strong>of</strong> antibodies by ELISA is an important tool for Johne’s disease control. The goal <strong>of</strong> this work was to evaluate four commercial ELISA tests. We determined diagnostic sensitivity and specificity on field samples and <strong>the</strong> robustness <strong>of</strong> <strong>the</strong> kits in an interlaboratory trial. To assess diagnostic sensitivity and specificity, 92 sera from fecal culture positive animals at different level <strong>of</strong> excretion and 32 from negative herds were analysed in one laboratory. All <strong>the</strong> kits demonstrated a specificity <strong>of</strong> 100.0%. Merging inconclusive and positive results, test sensitivity was 71.7% for kit A and B and 67.4 for kit C and D. Among infected animals, ELISA sensitivity did not varied significantly with Map excretion levels with all <strong>the</strong> kit used. For <strong>the</strong> interlaboratory trial, 30 coded samples composed by eight replicates <strong>of</strong> one negative sample and two replicates <strong>of</strong> 11 positive samples were selected and delivered with <strong>the</strong> kits to 10 laboratories throughout Italy. All <strong>the</strong> participants tested each sample in duplicates. Decoded results were analysed for reproducibility within and among laboratories and quantitative results were transformed into S/P values to compare analytical results. Kit A gave 100% <strong>of</strong> <strong>the</strong> expected results and Kit B gave almost <strong>the</strong> same outcome: just one laboratory obtained one inconclusive and one negative result in one replicate. Kit C gave <strong>the</strong> expected results for 9/11 positive samples and Kit D for 5/11 positive samples. Variations among replicates and laboratories were obtained with <strong>the</strong> remaining positive samples for <strong>the</strong>se two kits. Regarding <strong>the</strong> replicates on negative sample, 5 incorrect results distributed in three laboratories for kit C and one doubtful replicate in one laboratory for kit D were detected. According to <strong>the</strong>se results, two (A and B) <strong>of</strong> <strong>the</strong> four ELISA kits evaluated showed good performances and reproducibility within and among laboratories. 54
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Book of Abstracts
Page 4 and 5: Proceedings <stron
Page 6 and 7: 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 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
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
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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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#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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247
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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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