Proceedings of the 10th International Colloquium on Paratuberculosis

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indexes, compared to each single analysis. However, MLSSR, an expansive and more complex technique, also when applied as single analysis, showed higher DI values than <strong>the</strong> combination <strong>of</strong> <strong>the</strong> o<strong>the</strong>r techniques used in this study. Finally, MLSSR coupled with MLVA showed <strong>the</strong> best discriminatory power. REFERENCES 1. Bull TJ, Hermon-Taylor J, Pavlik I I, El-Zaatari F, Tizard M. 2000. Characterization <strong>of</strong> IS900 loci in mycobacterium avium subsp. paratuberculosis and development <strong>of</strong> multiplex PCR typing. Microbiology.,146 (Pt 12),3285. 2. Cousins DV, Williams SN, Hope A, Eamens GJ. 2000. DNA fingerprinting <strong>of</strong> Australian isolates <strong>of</strong> Mycobacterium avium subsp paratuberculosis using IS900 RFLP. Aust Vet J.,78,184-90. 3. Motiwala AS, Stro<strong>the</strong>r M, Amonsin A, Byrum B, Naser SA, Stabel JR, Shulaw WP, Bannantine JP, Kapur V, Sreevatsan S. 2003. Molecular epidemiology <strong>of</strong> Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification <strong>of</strong> unique targets for diagnosis. J Clin Microbiol.,41,2015-26. 4. Feizabadi MM, Robertson ID, Hope A, Cousins DV, Hampson DJ. 1997. Differentiation <strong>of</strong> Australian isolates <strong>of</strong> Mycobacterium paratuberculosis using pulsedfield gel electrophoresis. Aust Vet J.;75:887-9 5. Bull TJ, Sidi-Boumedine K, McMinn EJ, Stevenson K, Pickup R, Hermon-Taylor J. 2003. Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from o<strong>the</strong>r species <strong>of</strong> <strong>the</strong> Mycobacterium avium complex. Mol Cell Probes.,17,157-64. 6. Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F. 2007. New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol.,45,2404-10. 7. Overduin P, Schouls L, Roholl P, van der Zanden A, Mahmmod N, Herrewegh A, van Soolingen D. 2004. Use <strong>of</strong> multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol.,42,5022-8. 8. Amonsin A, Li LL, Zhang Q, Bannantine JP, Motiwala AS, Sreevatsan S, Kapur V. 2004. Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains. J Clin Microbiol.,42,1694-702. 9. Hunter PR, Gaston MA. 1988. Numerical index <strong>of</strong> <strong>the</strong> discriminatory ability <strong>of</strong> typing systems: an application <strong>of</strong> Simpson's index <strong>of</strong> diversity. J Clin Microbiol.,26,2465-6. 10. Möbius P, Luyven G, Hotzel H, Köhler H. 2008. High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination <strong>of</strong> IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing. J Clin Microbiol.,46,972-81. 63
#94 A simple liquid culture method for <strong>the</strong> assessment <strong>of</strong> paratuberculosis status in dairy cattle herds Elisabetta Stefani, Caterina Dal Ben, Jacek Gwozdz, Anna Valente, Vanessa Benini, Paola Fiorini, Nicola Pozzato Istituto Zoopr<strong>of</strong>ilattico Sperimentale delle Venezie - Laboratorio di biologia molecolare, Verona, Italy Automated liquid systems for culture <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) are more sensitive and rapid than culture on solid media, but <strong>the</strong>y are expensive and require specialised equipment. In this study an inexpensive, non-automated culture method using <strong>the</strong> liquid 7H9 medium was compared with <strong>the</strong> conventional method on Herrold’s medium for Map detection in dairy cattle manure. Infected herds A and B and negative herds C and D were recruited. The seroprevalence <strong>of</strong> paratuberculosis in herds A and B was 10% and 1%, respectively. A mix <strong>of</strong> manure was collected from <strong>the</strong> floor <strong>of</strong> <strong>the</strong> lactating-cow feeding area on 3 occasions at weekly intervals. Three replicates <strong>of</strong> each sample were processed for a direct realtime-PCR test, conventional Herrold’s culture, and non-automated liquid culture. In <strong>the</strong> latter method, each replicate sample was inoculated into 2 tubes <strong>of</strong> 7H9 medium that were subsequently tested for growth <strong>of</strong> Map using <strong>the</strong> realtime-PCR at weekly intervals over 8 weeks. Map was detected in each sample from herd A by each method applied. Positive samples from herd B varied according to <strong>the</strong> method used: 8/9 with Herrold’s, 5/9 with direct realtime-PCR and 18/18 with realtime-PCR on liquid cultures. Realtime-PCR confirmed <strong>the</strong> presence <strong>of</strong> Map at <strong>the</strong> 2nd week on 7H9 in 14/18 tubes for herd A and at <strong>the</strong> 5th week or before in 16/18 tubes for herd B. Herds C and D gave negative results in all replicates for all <strong>the</strong> methods applied. Our results demonstrated that nonautomated liquid culture on herd manure increases sensitivity and reduce <strong>the</strong> time with respect to traditional culture. A protocol based on direct realtime-PCR followed by liquid culture on negative samples, confirmed at 5 weeks and 8 weeks by realtime-PCR, can be used for a semiquantitative assessment <strong>of</strong> paratuberculosis status in dairy cattle herds. #99 Rapid assessment <strong>of</strong> <strong>the</strong> impact <strong>of</strong> heat treatments on viability <strong>of</strong> Mycobacterium avium subsp. paratuberculosis using an optimized phage assay Antonio Foddai, Chris T Elliott, Irene R Grant, Queen’s University Belfast, United Kingdom Thermal inactivation studies were carried out to demonstrate <strong>the</strong> utility <strong>of</strong> a recently optimized phage amplification assay to enumerate viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk. UHT milk was spiked with 10 6 -10 7 CFU MAP/ml and dispensed in 100 µl aliquots into thin-walled 200µl PCR tubes. A Primus 96 advanced <strong>the</strong>rmal cycler (Peqlab, Erlangen, Germany) was used to achieve <strong>the</strong> following time/ temperature treatments: a) 63°C for 3, 6 and 9 min; b) 68°C for 20, 40 and 60 s; and c) 72°C for 5, 10, 15 and 25 s. Milk samples (non-heated (time 0) and heat-treated) were centrifuged (16,000 x g for 15 min) to remove milk components inhibitory to <strong>the</strong> phage assay, and <strong>the</strong> pellet resuspended in 1 ml 7H9 broth plus 10% OADC and 2 mM CaCl 2 . After overnight incubation <strong>of</strong> <strong>the</strong> samples at 37 o C, appropriate dilutions <strong>of</strong> each sample were processed through an optimized phage assay (Foddai et al. (2009) AEM). Plaques were counted (PFU/ml) <strong>the</strong> next day and <strong>the</strong>rmal D values calculated for MAP in milk at 63, 68 and 72 o C; D values <strong>of</strong> 63.7 s, 10.4 s and 2.9 s, respectively, were obtained enabling calculation <strong>of</strong> a z value for MAP <strong>of</strong> 6.8°C. The plaque counts obtained using <strong>the</strong> optimized phage assay were similar to colony counts on HEYM, carried out in parallel. In contrast, phage assay results were available within 48 h ra<strong>the</strong>r than after a minimum <strong>of</strong> 6-8 weeks for conventional culture on HEYM. The D and z values obtained in this study are similar to previously published D and z values for MAP obtained using HEYM culture, so we have clearly demonstrated that <strong>the</strong> optimized phage assay enables rapid and accurate assessment <strong>of</strong> <strong>the</strong> impact <strong>of</strong> heat treatments on MAP viability. 64
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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
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 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 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
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
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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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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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