Proceedings of the 10th International Colloquium on Paratuberculosis

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#187 Comparison <strong>of</strong> a quantitative and a semi-qantitative method to investigate survival <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages Rebecca M. Mitchell, Nicole S. Gollnick, Srinand Sreevatsan, David G. Russell, Ynte H. Schukken College <strong>of</strong> Veterinary Medicine, Cornell University, USA; Clinic for Ruminants, University <strong>of</strong> Munich, Germany; College <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Minnesota, USA Intracellular growth and survival <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) is dependent on <strong>the</strong> bacterial genotype. Until recently we lacked methods to differentiate MAP strains beyond <strong>the</strong> level <strong>of</strong> host specificity. Techniques such as multilocus short sequence repeat (MLSSR) strain typing enable more precise differentiation between host-specific isolates. This allows investigating <strong>the</strong> characteristics <strong>of</strong> MAP in in vitro infections. In this study we report <strong>the</strong> comparison <strong>of</strong> two quantitations systems (fluorescent microscopy and real-time quantitative PCR (qPCR)) evaluating in vitro survival and growths <strong>of</strong> MAP in bovine monocyte-derived macrophages (MDMs) by using data from a single experimental setup. MDMs for <strong>the</strong>se experiments were obtained from Johne’s disease-positive (n = 3) and age and stage <strong>of</strong> lactation matched Johne’s disease-negative (n = 3) multiparious cows from <strong>the</strong> same farm. MDMs were challenged in vitro with four MAP strains <strong>of</strong> different host specificity (bovine, ovine) and were harvested at 2 hours, 2 days, 4 days and 7 days following infection. For each time point we measured simultaneously <strong>the</strong> number <strong>of</strong> both host cells and bacteria applying a semi-quantitative approach by using fluorescence microscopy and a quantitative approach using qPCR. Overall, <strong>the</strong>re was a high level <strong>of</strong> agreement between <strong>the</strong> bacterial and cell numbers assessed by <strong>the</strong> two divergent methods: Host infection status (test-positive or test-negative) did not influence bacterial survival irrespective <strong>of</strong> MAP strain used while bovine-specific MAP strains showed significantly better survival in bovine MDMs compared to ovine-specific strains. Throughout <strong>the</strong>se analyses compared to <strong>the</strong> fluorescence microscopy assays qPCR proved to be more consistent between samples. It may be concluded that both methods are appropriate to investigate MAP infections in vitro. For <strong>the</strong> investigation <strong>of</strong> larger data sets <strong>the</strong> qPCR is preferred not only because <strong>of</strong> <strong>the</strong> higher precision <strong>of</strong> results but also to reduce <strong>the</strong> work load. 81
#188 The utility <strong>of</strong> fecal culture, direct fecal real-time PCR, and direct fecal nested PCR in <strong>the</strong> determination <strong>of</strong> Mycobacterium avium subsp. paratuberculosis infectivity Ching Ching Wu, Tsang Long Lin, Gilles R. G. Monif Purdue University, USA; Infectious Diseases Incorporated, USA The present study was conducted to evaluate <strong>the</strong> utility <strong>of</strong> fecal culture, direct fecal real-time PCR, and direct fecal nested PCR in determining <strong>the</strong> status <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) infectivity in dairy herd. Eight hundred and twenty-seven (827) fecal samples were collected from two dairy herds participating in Johne’s Disease Demonstration Herd Program. Fecal culture was carried out by using Trek ESP system with IS900 PCR confirmation. Direct fecal Map real-time PCR uses heat shock protein gene (hsp) as <strong>the</strong> target in PCR (Tetracore VetAlert TM Johne’s Real-Time PCR, Rockville, MD, USA). Direct fecal Map nested PCR is based on IS1311 (FecaMap ® , Infectious Diseases Incorporated, Bellevue, NE, USA). The percentages <strong>of</strong> fecal samples positive for Map were 13.5% by culture, 11.6% by real-time PCR, and 21.8% by nested PCR. Using positivity by direct fecal culture as <strong>the</strong> gold standard, 35 samples were positive for Map by real-time PCR with 83.3% accuracy and 51positive for Map by nested PCR with 77.3% accuracy. Using positivity by culture or both PCR as <strong>the</strong> gold standard, 112 samples were positive for Map by culture with 97.3% accuracy, 49 positive for Map by real-time PCR with 85.0% accuracy, and 65 positive for Map by nested PCR with 78.8% accuracy. Using positivity by culture or direct fecal Map real-time PCR or direct fecal Map nested PCR as <strong>the</strong> gold standard, 112 samples were positive for Map by culture with 81.7% accuracy, 96 positive for Map by real-time PCR with 76.8% accuracy, and 180 positive for Map by nested PCR with 87.0% accuracy. The results indicated that using positivity for Map by culture or both PCR as <strong>the</strong> gold standard is a more accurate tool in determining <strong>the</strong> status <strong>of</strong> Johne’s disease infectivity in dairy herd. 82
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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
Page 31 and 32: #107 Multilocus Short Sequence Repe
Page 33 and 34: Fig. 1. Dendogram (showing genetic
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Page 52 and 53: #36 Genetic diversity of</s
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Page 56 and 57: #66 Comparison of
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Page 64 and 65: indexes, compared to each single an
Page 66 and 67: #108 Evaluation of
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Page 70 and 71: endoscopic criteria and histopathol
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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
Page 80 and 81: #162 Comparison of
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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
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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Page 110 and 111: #276 Elimination of</strong
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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
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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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