Proceedings of the 10th International Colloquium on Paratuberculosis

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#36 Genetic diversity <strong>of</strong> Mycobacterium avium subspecies paratuberculosis bovine isolates in <strong>the</strong> United Kingdom Karen Stevenson, Ian Heron, Joyce McLuckie, Franck Biet, Margaret McCall, George Caldow Moredun Research Institute, United Kingdom; INRA, France; Scottish Agricultural College, United Kingdom The objective <strong>of</strong> this study was to investigate <strong>the</strong> genetic diversity <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (Map) bovine isolates in <strong>the</strong> United Kingdom (UK). A panel <strong>of</strong> 204 isolates from dairy and beef (commercial and pedigree) cattle in <strong>the</strong> United Kingdom (UK) was assembled. The isolates were initially cultured from faeces samples using <strong>the</strong> ESP TREK liquid culture system and <strong>the</strong>n subcultured onto Middlebrook 7H11 agar supplemented with mycobactin J. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI using <strong>the</strong> standardised procedure developed at <strong>the</strong> Moredun Research Institute (www.moredun.ac.uk/PFGE-mycobacteria) and by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis as described by Thibault et al. (J. Clin. Microbiol. 2007 45:2404-2410). A total <strong>of</strong> 15 multiplex PFGE pr<strong>of</strong>iles and 13 MIRU-VNTR pr<strong>of</strong>iles were detected. Using <strong>the</strong> combined typing data from 178 isolates, <strong>the</strong> Simpson’s Index <strong>of</strong> Diversity was calculated to be 0.729. There were two predominant strain types represented by 45% and 26% <strong>of</strong> <strong>the</strong> isolates, which were widely distributed throughout <strong>the</strong> UK. The relative virulence <strong>of</strong> <strong>the</strong>se strain types has yet to be investigated. #38 Comparison <strong>of</strong> isolation <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) with <strong>the</strong> use <strong>of</strong> specific culture media Masoud Haghkhah, Hormat Aghajani Maleki Department <strong>of</strong> Pathobiology, School <strong>of</strong> Veterinary Medicine, Shiraz University, Iran The aim <strong>of</strong> this study was <strong>the</strong> comparison <strong>of</strong> isolation <strong>of</strong> MAP with <strong>the</strong> use <strong>of</strong> specific culture media in sheep and goats herds in Shiraz, Fars Province, Iran. A total <strong>of</strong> 50 fecal samples from sheep and goats with clinical signs suspected to Johne's disease were prepared. Only 10 out <strong>of</strong> 50 were positive with Ziehl-Neelsen staining method. The samples were streaked on four specific media including Middlebrook 7H9 agar, Middlebrook 7H11 agar, modified Lowenstein-Jensen (MLJ) and Herrold's egg yolk agar with mycobactin J (2 slopes) and without mycobactin (1 slope). No growth on three media (Middlebrook 7H9 agar, Middlebrook 7Hll agar and modified Lowenstein-Jensen) was observed, but 5 Herrold's cultures were positive. As a result, it seems that <strong>the</strong> growth <strong>of</strong> MAP on Herrold's egg yolk agar is more efficient than <strong>the</strong> o<strong>the</strong>r culture media. 51
#51 A new marker IS1311 PCR-REA assay for rapid differentiation <strong>of</strong> ‘Indian Bison type’ Mycobacterium avium subspecies paratuberculosis isolates Jagdeep Singh Sohal, Shoor Vir Singh, Pravin Kumar Singh, Ajay Vir Singh, M C Sharma Indian Council <strong>of</strong> Agricultural Research, India; Central Institute for Research on Goats, India Objective: Studies in last two years identified a unique molecular signature in some loci <strong>of</strong> IS1311 element capable <strong>of</strong> distinguishing native ‘Indian Bison type’ Mycobacterium avium subspecies paratuberculosis (MAP) from o<strong>the</strong>r non-Indian MAP isolates. Objective <strong>of</strong> <strong>the</strong> study was to design a specific test based on unique molecular signature for <strong>the</strong> specific detection and differentiation <strong>of</strong> native ‘Indian Bison type’ MAP. Materials and Methods: Bioinformatics tools were applied on <strong>the</strong> IS1311 sequences <strong>of</strong> native MAP and IS1311 sequences retrieved from GenBank database including MAP K10 sequences to identify a restriction enzyme capable <strong>of</strong> differentiating two sets <strong>of</strong> IS1311 sequences from ‘Indian Bison type’ MAP and ‘non-Indian’ MAP strinas based on its restriction pr<strong>of</strong>ile. Criteria <strong>of</strong> selection <strong>of</strong> restriction enzyme was ei<strong>the</strong>r loss or gain <strong>of</strong> new restriction site due to presence <strong>of</strong> unique molecular signature in IS1311 sequences <strong>of</strong> native ‘Indian Bison type’ MAP compared to ‘non-Indian’ MAP. IS1311 sequences from MAP ‘Indian Bison type’ isolates recovered from different geographical regions and different host species <strong>of</strong> <strong>the</strong> country were amplified and processed for PCR-REA using <strong>the</strong> restriction enzyme selected in <strong>the</strong> above step. Sequencing <strong>of</strong> PCR products was also done in order to confirm <strong>the</strong> presence/ absence <strong>of</strong> molecular signature. Results and Conclusions: Based on <strong>the</strong> criteria described above BsaJI enzyme was selected for PCR-REA studies. PCR-REA was successfully optimized based on this enzyme capable <strong>of</strong> distinguishing native ‘Indian Bison type’ MAP from ‘non-Indian’ MAP. Results revealed that all ‘Indian Bison type’ MAP isolates carried molecular signature in <strong>the</strong>ir IS1311 sequences despite <strong>the</strong>ir origin from different species. Sequencing also confirmed results <strong>of</strong> PCR-REA. Thus PCR-REA optimized in this study may be used for rapid differentiation <strong>of</strong> ‘Indian Bison type’ isolates. These findings may give new directions to <strong>the</strong> paratuberculosis research in <strong>the</strong> country. #58 Environmental fecal samples for <strong>the</strong> detection <strong>of</strong> MAP in small cattle herds Johannes Lorenz Khol, Matthias Vill, Martina Mattes, Walter Baumgartner Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University <strong>of</strong> Veterinary Medicine Vienna, Austria Detection <strong>of</strong> MAP in environmental fecal samples to determine <strong>the</strong> paratuberculosis herd status <strong>of</strong> large dairy herds has recently been described. Two studies were performed at <strong>the</strong> Clinic for Ruminants at <strong>the</strong> University <strong>of</strong> Veterinary Medicine in Vienna to evaluate this new approach for <strong>the</strong> assessing <strong>of</strong> <strong>the</strong> paratuberculsois herd level in small farms. Austrian cattle herds with and without known paratuberculosis status were sampled for MAP periodically by collecting <strong>of</strong> environmental fecal samples. Environmental samples were taken from different sites within <strong>the</strong> stable and manure storage and tested for MAP by bacterial culture and PCR (Polymerase Chain Reaction). The correct MAP infection status was assigned in almost 70 % <strong>of</strong> <strong>the</strong> herds by fecal culture <strong>of</strong> <strong>the</strong> environmental samples. When 3 consecutive environmental samples were tested for MAP, 80 % <strong>of</strong> <strong>the</strong> farms could be assigned <strong>the</strong> correct herd infection status. All farms with a high within herd prevalence <strong>of</strong> MAP or an increasing frequency <strong>of</strong> clinical cases <strong>of</strong> Paratuberculosis could be detected by environmental fecal sampling. These results indicate that environmental fecal samples could be used to detect <strong>the</strong> paratuberculosis herd status <strong>of</strong> small farms as a cheap alternative to o<strong>the</strong>r testing schemes. Repeated testing for MAP by bacterial culture seems to be <strong>the</strong> most effective way to determine paratuberculosis herd status according to <strong>the</strong>se results and might be more accurate and economic than o<strong>the</strong>r screening methods. 52
Page 1 and 2: 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 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 96 and 97: #242 Application and field validati
Page 98 and 99: #251 Diagnosis of
Page 100 and 101: Table 2. Paratuberculosis PCR resul
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#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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