Proceedings of the 10th International Colloquium on Paratuberculosis

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#242 Application and field validation <strong>of</strong> real-time PCR assay for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis from bovine fecal samples Yasuyuki Mori, Reiko Nagata, Kazuhiro Yoshihara National Institute <strong>of</strong> Animal Health, Japan Background: While <strong>the</strong> real-time PCR has been widely used for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map), a few issues, including <strong>the</strong> sensitivity, specificity, reproducibility and standardization <strong>of</strong> real-time PCR analysis, must be addressed before it’s routine usage in clinical laboratories. We investigated some <strong>of</strong> <strong>the</strong>se issues for <strong>the</strong> development <strong>of</strong> diagnostic system for Johne’s disease using quantitative realtime PCR (qPCR). Methods: Fecal DNA samples were prepared by commercial DNA extraction reagents with bead-beating. For specificity testing, 775 fecal samples were collected from farms which have been confirmed Map-free status by a routine culture and ELISA tests. Sensitivity and reproducibility were validated in collaborative studies with 23 prefectural animal health centers by performing <strong>the</strong> qPCR with <strong>the</strong> same reference fecal samples and purified Map DNA which were supplied from our lab. Fur<strong>the</strong>rmore, 3,782 fecal samples were collected from 23 prefectures and tested with both <strong>the</strong> qPCR and <strong>the</strong> culture. Results: In a validation <strong>of</strong> sensitivity and specificity <strong>of</strong> qPCR, it detected 0.001 picogram purified Map DNA and 2 copies <strong>of</strong> IS900, and no cross-reactions were observed with any o<strong>the</strong>r Mycobacterium species including closely rerated Mycobacterium sp. st.2333. Fecal DNA samples from Map-free farms were all negative in <strong>the</strong> qPCR. In reproducibility tests using reference fecal sample and Map DNA provided, <strong>the</strong> most <strong>of</strong> <strong>the</strong> results reported from collaborative centers were almost concordant with <strong>the</strong> values we have expected. Comparative analysis <strong>of</strong> 3,782 fecal samples <strong>of</strong> unknown Map status yielded results that 194 samples (5.1%) were positive in bacterial culture and 327 (8.6%) were positive in <strong>the</strong> qPCR. Of 194 samples with culture positive, 169 were also positive in <strong>the</strong> qPCR (a concordance <strong>of</strong> 87.1%). Conclusion: The combination <strong>of</strong> validated fecal DNA preparation and <strong>the</strong> qPCR is a reliable diagnostic tool in terms <strong>of</strong> rapid and appropriate JD-control measures. #243 Evaluation <strong>of</strong> a Johne’s disease bulk milk ELISA as a herd screening tool Hinrich Voges, Penny Back, Margaret Nash, Tracey Trotter LIC, New Zealand A genomic study for <strong>the</strong> Johne’s Disease Research Consortium requires extensive screening to identify New Zealand dairy cows with advanced MAP infection. We <strong>the</strong>refore need efficient tools to target and screen herds with higher Johne’s disease risks. One such tool may be a bulk milk ELISA test - which is simple and cheap to administer using dairy company vat samples. To investigate <strong>the</strong> performance <strong>of</strong> <strong>the</strong> ‘Pourquier Paratuberculosis ELISA screening kit’ as a vat test, we sampled 154 dairy herds (case herds) that have recorded Johne’s disease cows over several years on <strong>the</strong> LIC National Dairy Cow Database, as well as 278 random herds from across New Zealand as controls. A single vat test revealed a clear right shift <strong>of</strong> test results amongst <strong>the</strong> case herds with 14% > 0.1 S/P ratio compared to 1.4% <strong>of</strong> control herds. 64 (42 case and 22 control) herds across <strong>the</strong> range <strong>of</strong> vat test results (including most herds >0.1 S/P) were <strong>the</strong>n selected for pooled-individual ELISA testing <strong>of</strong> herd-test milk samples, targeting 2nd + lactation cows. Herds with S/P ratios up to 0.05, >0.05 – 0.10 and greater than 0.10 were grouped into low, mid and high vat test herds. The vat test result showed a very high correlation with individual cow reactor prevalence (R2 = 0.83). Mean apparent reactor prevalence differed significantly between low, mid versus high vat test herds (p
#246 Results from <strong>the</strong> 2008 Johne’s Serologic Pr<strong>of</strong>iciency Test and Update on <strong>the</strong> 2009 Johne’s Milk ELISA Pr<strong>of</strong>iciency Test Janet Gail Marquardt USDA/NCAH, USA The 2008 Johne’s Disease Serologic Pr<strong>of</strong>iciency Test panel consisted <strong>of</strong> 0.25 ml <strong>of</strong> sera from 21 animals. Selected serum samples were used in duplicate to constitute <strong>the</strong> total <strong>of</strong> 25 serum samples per test panel. The Prionics Parachek TM Test was successfully utilized by 44 laboratories and 51 individuals. Three laboratories failed <strong>the</strong> first round <strong>of</strong> testing, but passed <strong>the</strong>ir retests. The IDEXX Herdchek TM ELISA Test was successfully utilized by 53 laboratories and 63 individuals. Five laboratories failed <strong>the</strong> first round <strong>of</strong> testing, but four successfully completed <strong>the</strong>ir retests. The fifth laboratory failed <strong>the</strong> first retest using <strong>the</strong> IDEXX test and submitted results for <strong>the</strong>ir second retest using <strong>the</strong> Prionics test. This laboratory failed again by missing one critical sample. Of o<strong>the</strong>r participating laboratories using alternative testing methods, three laboratories utilized <strong>the</strong> Pourquier Test to complete <strong>the</strong> test with a passing score and one <strong>of</strong> <strong>the</strong>se laboratories also submitted a successful Complement Fixation Test. One laboratory failed using an in-house procedure, but had passed using <strong>the</strong> Prionics test. The 2009 Johne’s Disease Milk ELISA Pr<strong>of</strong>iciency Test has been sent to 39 laboratories with results due May 1, 2009. Participation in <strong>the</strong> Milk ELISA Pr<strong>of</strong>iciency Test has increased from last year when 36 laboratories participated. #248 A Simple Internal PCR Control for Mycobacterium avium subsp. paratuberculosis Constructed by PCR Techniques Ian Marsh, Martin McLoon, Sue Austin, Shayne Fell, Leslie Reddacliff, Richard Whittington New South Wales Department <strong>of</strong> Primary Industries, Elizabeth Macarthur Agricultural Institute, Australia; Faculty <strong>of</strong> Veterinary Science, University <strong>of</strong> Sydney, Australia Aims: Polymerase chain reaction (PCR) is routinely used to confirm <strong>the</strong> presence <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) in <strong>the</strong> diagnosis <strong>of</strong> ovine Johne’s disease (OJD). In a recent study to overcome specificity issues with <strong>the</strong> current PCR we also developed an internal amplification control (IAC) to monitor <strong>the</strong> integrity <strong>of</strong> individual reactions. This is important given <strong>the</strong> opportunity to introduce PCR inhibitory substances within samples. Unlike o<strong>the</strong>r internal controls that require cloning and o<strong>the</strong>r molecular manipulations, this IAC only requires PCR capability for its production. Method: The IAC was constructed in a two step PCR process that amplified a region <strong>of</strong> <strong>the</strong> Mycobacterium avium subsp. avium (MAA) genome that is not present in <strong>the</strong> MAP genome using composite primers made <strong>of</strong> an MAA region and an MAP region. The IAC was <strong>the</strong>n incorporated in to a multiplex PCR that included a new MAP specific target to increase specificity. The analytical sensitivity <strong>of</strong> <strong>the</strong> IAC and multiplex PCR was established prior to evaluation on DNA samples that had been previously examined for OJD. Results: The IAC had no adverse effects on <strong>the</strong> analytical sensitivity <strong>of</strong> <strong>the</strong> MAP specific multiplex PCR. The new PCR test was successfully used to determine <strong>the</strong> presence/absence <strong>of</strong> MAP in 25 faecal samples with known OJD status and simultaneously determine <strong>the</strong> integrity <strong>of</strong> each reaction. Conclusion: We present a new test multiplex PCR for MAP that incorporates an IAC. The procedure used to produce <strong>the</strong> IAC is simple and highly adaptable to o<strong>the</strong>r PCR-based diagnostic tests. 96
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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
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
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 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
Page 106 and 107: A bulk milk quality assurance progr
Page 108 and 109: Republic of Lithua
Page 110 and 111: #276 Elimination of</strong
Page 112 and 113: other projects and
Page 114 and 115: Woodbine KA, Schukken YH, Green LE,
Page 116 and 117: Host Response and Immunology Keynot
Page 118 and 119: #33 MERKAL AWARD LECTURE No interfe
Page 120 and 121: #75 Interleukin 17 and interleukin
Page 122 and 123: #173 Local and systemic roles for b
Page 124 and 125: #23 Role of Mycoba
Page 126 and 127: Host Response and Immunology Poster
Page 128 and 129: #28 Intestinal MAP Infection via Pe
Page 130 and 131: #73 Age susceptibility of</
Page 132 and 133: #95 Role of nitric
Page 134 and 135: #134 Analysis of <
Page 136 and 137: #157 Study of <str
Page 138 and 139: #163 Immunohistochemical expression
Page 140 and 141: #193 Development of</strong
Page 142 and 143: #209 Murine macrophages carrying an
Page 144 and 145: #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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