Proceedings of the 10th International Colloquium on Paratuberculosis

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#66 Comparison <strong>of</strong> efficacy <strong>of</strong> conventional and a quantitative real-time polymerase chain reactions (qRT-PCR) on tissues <strong>of</strong> pathologically characterized ovine paratuberculosis Ganesh G Sonawane, Bhupendra N. Tripathi Central Sheep and Wool Research Institute, Awikanagar, India; Indian Veterinary Research Institute, India A quantitative real-time PCR assay (SYBR green chemistry) employing IS900 gene specific primers <strong>of</strong> Mycobacterium avium subsp. parartuberculosis (MAP) was developed on a MAP culture (IVRI/C-132), which subsequently served as a basis for detection <strong>of</strong> MAP genome and its quantification (in copy number) in <strong>the</strong> intestinal and mesenteric lymph node (MLN) tissues <strong>of</strong> 38 paratuberculous sheep (multibacillary-23 and paucibacillary-15). The sensitivity <strong>of</strong> qRT-PCR was found to <strong>the</strong> standard curve. The qRT-PCR was compared be 34 copies <strong>of</strong> IS900 gene. The number <strong>of</strong> copies in <strong>the</strong> test samples was extrapolated from with conventional PCR targeting IS900 and 251 genes on tissue samples <strong>of</strong> <strong>the</strong>se sheep. Among <strong>the</strong> multibacillary group, IS900 PCR detected 19(82.6%), 251 PCR detected 21 (91.3%) and qRT-PCR detected all 23 (100%) sheep positive for MAP in intestine and lymph nodes <strong>of</strong> <strong>the</strong>se animals. Among <strong>the</strong> paucibacillary group, IS900 PCR detected 2 (13.3%), 251 PCR detected 4 (27%) and qRT-PCR detected all 15 (100%) sheep positive for MAP in <strong>the</strong> intestines and lymph nodes <strong>of</strong> <strong>the</strong>se animals. When results <strong>of</strong> both groups were taken toge<strong>the</strong>r, IS900 PCR detected 21(55.2%), 251 PCR detected 25 (65.7%) and qRT-PCR detected all 38 (100%) animals positive for MAP genome ei<strong>the</strong>r in <strong>the</strong> intestine or MLN tissues. The 251 PCR detected greater number <strong>of</strong> cases but it was not significant in comparison to IS900 PCR. The qRT-PCR results were significant in comparison to both PCR targeting IS900 and 251 genes. Though, <strong>the</strong> rates <strong>of</strong> detection <strong>of</strong> both MAP genes (IS900 and 251) were greater in <strong>the</strong> intestinal than <strong>the</strong> MLN tissues, <strong>the</strong> differences were statistically non-significant. Tissues from control sheep were negative for all three PCRs. Based on <strong>the</strong> results <strong>of</strong> <strong>the</strong> present study, it was concluded that qRT-PCR was a highly sensitive test in comparison to conventional PCR for <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis on infected tissues especially from paucibacillary sheep. #84 Genetic diversity in Mycobacterium avium subsp. paratuberculosis from cattle herds in <strong>the</strong> Ne<strong>the</strong>rlands Kimm van Hulzen, Henri Heuven, Mirjam Nielen, Jeroen Hoeboer, Wiebren Santema, Ad Koets Faculty <strong>of</strong> Veterinary Medicine, University Utrecth, The Ne<strong>the</strong>rlands The objective <strong>of</strong> this study was to investigate <strong>the</strong> genetic diversity <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) within and between herds in <strong>the</strong> Ne<strong>the</strong>rlands. Earlier research showed that MAP has a very low level <strong>of</strong> heterogeneity and it is difficult to investigate epidemiological relationships between isolates <strong>of</strong> infected herds or infected ruminant species. Recently developed PCR-based methods consisting <strong>of</strong> variable-number tandem repeats (VNTRs) <strong>of</strong> genetic elements called mycobacterial interspersed repetitive units (MIRUs) provided <strong>the</strong> possibility to study genetic variation in a simple and rapid way. For 52 isolates originating from 36 commercial dairy herds from different regions <strong>of</strong> <strong>the</strong> Ne<strong>the</strong>rlands, MIRU-VNTR was applied at 11 loci. Within <strong>the</strong> group <strong>of</strong> 52 isolates, 19 different genotypes were detected. One genotype was found in 28 isolates, one genotype in 4 isolates, one genotype in 3 isolates, one genotype in 2 isolates and 15 genotypes were found only once. Eleven herds provided multiple isolates. In 5 herds a single genotype was found whereas in 6 herds more than one genotype was found. In conclusion, this study shows that between dairy farms in <strong>the</strong> Ne<strong>the</strong>rlands as well as within dairy farms in <strong>the</strong> Ne<strong>the</strong>rlands, infected animals may shed different MAP genotypes. Although 53% <strong>of</strong> <strong>the</strong> MAP isolates were typed within one genotype, <strong>the</strong>y originated from different geographical regions in <strong>the</strong> Ne<strong>the</strong>rlands. Fur<strong>the</strong>r research will be done to study <strong>the</strong> possible association between <strong>the</strong> MAP genotype and host disease phenotype. 55
#85 Comparison <strong>of</strong> four different PCR methods for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in milk Ricchi M 1 , Manini F 1 , Cammi G 1 , Donaghy J 2 and Arrigoni N 1 1 Istituto Zoopr<strong>of</strong>ilattico Sperimentale della Lombardia e dell’Emilia Romagna, Piacenza, Italy 2 Food Microbiology Branch, Agri-Food & Biosciences Institute, Belfast, Nor<strong>the</strong>rn Ireland ABSTRACT Emerging evidence suggests a role <strong>of</strong> Mycobacterium avium subs. paratuberculosis (MAP) in <strong>the</strong> development <strong>of</strong> human pathologies like Crohn’s disease and type I diabetes. For this reason, <strong>the</strong> need for rapid and robust tools to detect <strong>the</strong> presence <strong>of</strong> MAP in food is increasing. Polymerase Chain Reaction (PCR) techniques are able to give rapid and specific results, but <strong>the</strong>ir sensitivity is generally lower than traditional culture methods. The aim <strong>of</strong> this study was to compare four different PCR methods to detect MAP on cow bulk milk samples collected from presumably infected herds. MAP DNA was extracted by Adiapure kit (Adiagene, France). The PCR were: (a) IS900 Commercial end-point PCR (kit Adiavet, Adiagene, France); (b) IS900 Nested PCR; (c) IS900 TaqMan Real time PCR; (d) f57 housemade Sybr Green Real Time PCR. Both <strong>the</strong> commercial PCR (a) and <strong>the</strong> IS 900 Real Time PCR TaqMan (c) contained an internal amplification control in order to discriminate between negative or inhibited samples. 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 eight positive samples (22%). Although we used bulk milk samples coming from presumably infected herds, <strong>the</strong> prevalence <strong>of</strong> direct detection <strong>of</strong> MAP is low in all <strong>the</strong> “one round” PCR methods. As expected, <strong>the</strong> most sensitive method is Nested PCR, although it remains difficult to apply as regards to possibility <strong>of</strong> cross contaminations. BACKGROUND Emerging evidence suggests a role <strong>of</strong> Mycobacterium avium subs. paratuberculosis (MAP) in <strong>the</strong> development <strong>of</strong> human disorders like Crohn’s disease and Type I diabetes 1, 2 . For <strong>the</strong>se reasons, <strong>the</strong> request for rapid and robust tools to detect <strong>the</strong> presence <strong>of</strong> MAP in food, especially milk, is increasing 3 . Molecular biology techniques, such as Polymerase Chain Reaction (PCR), are able to give rapid and specific results. These methods are also useful tools when <strong>the</strong> matrices are not appropriately conserved (compromised bacterial viability), or in presence <strong>of</strong> fastidious strains. PCR assays developed for <strong>the</strong> detection <strong>of</strong> MAP have high specificity, while <strong>the</strong>ir sensitivity is generally lower than traditional culture methods. These assays amplify some typical MAP regions, such as f57 (one copy per genome <strong>of</strong> MAP 4 ) and IS900 (12-18 copies per genome <strong>of</strong> MAP 4 ). While <strong>the</strong> discovery <strong>of</strong> some IS900-like elements could make not conclusive a test based on IS900 targeting, <strong>the</strong> f57 sequence is highly specific for MAP 5, 6 . However, due to <strong>the</strong> higher number <strong>of</strong> copies, IS900 PCR tests are generally more sensitive than those based on f57 4 . In order to compare <strong>the</strong> sensitivity <strong>of</strong> PCR tests, we applied a commercial end-point PCR, a Nested PCR and two Real Time PCR assays to bulk milk samples, previously positive to serological ELISA test. MATERIALS AND METHODS We tested about 3000 milk samples from different Italian regions using ELISA test (ID Screen® Paratuberculosis Indirect, Confirmation Test, ID Vet, MontPellier, France). Out <strong>of</strong> 82 positive samples, we extracted <strong>the</strong> DNA from 37 positive samples using Adiapure kit (Adiagene, France). The PCRs were: (a) IS900 Commercial end-point PCR (kit Adiavet, Adiagene, France), performed according to <strong>the</strong> kit manufacturing procedures. The reaction was carried out in Mastercycler ep gradient s (Eppendorf, Milan, Italy); 56
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Book of Abstracts
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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 54 and 55: #60 Evaluation 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 80 and 81: #162 Comparison of
Page 82 and 83: #187 Comparison of
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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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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#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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