Proceedings of the 10th International Colloquium on Paratuberculosis

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#108 Evaluation <strong>of</strong> colostrum as a diagnostic sample for M. a. paratuberculosis antibody detection via ELISA Hea<strong>the</strong>r Cushing, Elizabeth J.B. Manning, Michael T. Collins University <strong>of</strong> Wisconsin, Madison Cattle infected with Mycobacterium avium ss. paratuberculosis (MAP) produce antibodies detectable in biological samples such as serum, plasma and milk. Antibody to MAP is also present in colostrum. The goal <strong>of</strong> this study was to compare antibody detection rates in colostrum vs. serum using an NVSL check-test validated non-commercial ELISA (“JTC-ELISA”). Colostrum was collected within 1-2 days <strong>of</strong> calving from 44 pregnant cows in a known infected dairy herd. Serum and fecal samples from <strong>the</strong>se cows were <strong>the</strong>n collected two to three weeks after calving. Serum and colostrum was frozen before processing and were tested by JTC-ELISA at dilutions 1:50 and 1:2 respectively. The fecal samples were processed fresh by <strong>the</strong> MGIT method for isolation <strong>of</strong> MAP. Thirty-nine cows were fecal culture positive. Of <strong>the</strong>se, 21/39 were both serum and colostrum JTC-ELISA positive (54%); 11/39 were colostrum ELISA positive but serum ELISA negative (28%) and 7/39 were ELISA negative for both sample types (18%). Five cows were both fecal culture and serum JTC-ELISA negative. Colostrum from two <strong>of</strong> <strong>the</strong>se cows was interpreted as JTC-ELISA positive. Antibody was thus detected in colostrum from 34 <strong>of</strong> <strong>the</strong> 44 cows tested from this infected herd. The range <strong>of</strong> optical density ELISA results were statistically similar for serum and colostrum samples (i.e., 0.00 – 1.13). This preliminary trial demonstrated that colostrum may be a useful sample for MAP infection surveillance. The JTC-ELISA was more sensitive with colostrum than serum samples. Factors needing fur<strong>the</strong>r study to limit false-positives and establish assay specificity include ELISA interpretation algorithms for colostrum, optimization <strong>of</strong> sample collection timing, and trials with herds <strong>of</strong> varied (and zero) infection prevalence as previously established by fecal culture, serum ELISA and/or milk ELISA. 65
#109 A sample and efficient method for DNA extraction from Mycobacterium avium subsp. paratuberculosis cultured in an automated broth culture system Miguel Angel Salgado, Robert Söderlund, Göran Bölske, David Herthnek, Anna Aspán, Juan Kruze Universidad Austral de Chile, Chile; National Veterinary Institute (SVA), Sweden Liquid culture <strong>of</strong> MycobacteriuM. avium subsp. paratuberculosis (Map) has shown advantages over culture on solid media to detect Map infection. PCR represents a rapid and efficient means <strong>of</strong> confirming Map in broth culture. There are several commercial kits and proposed methods <strong>of</strong> harvesting Map DNA and removal <strong>of</strong> inhibitors, most <strong>of</strong> which are costly, laborious and time consuming. This study proposes a simple and efficient method <strong>of</strong> harvesting DNA from Map cultured in liquid medium (MGIT). A lysis buffer and proteinase K, toge<strong>the</strong>r with mechanical cell disruption, was used as cell lysis followed by ethanol and differential centrifugation for removing inhibiting substances from <strong>the</strong> liquid medium. The method was evaluated comparing its performance on clinical samples with a commercial DNA extraction kit based on bead beating, proteinase K, and column purification and a protocol described in <strong>the</strong> literature, also based on mechanical disruption and phenol chlor<strong>of</strong>orm purification. Moreover, spiked samples in serial dilutions were used for confirming <strong>the</strong> shortest time to detection (TTD) by <strong>the</strong> MGIT reader for a positive sample. Finally, analytical sensitivity <strong>of</strong> <strong>the</strong> method was calculated spiking clean MGIT tubes with a manually counted bacterial concentration <strong>of</strong> approximately 1,000 bacteria/ml <strong>of</strong> medium. The proposed method could accurately confirm more samples than <strong>the</strong> commercial kit and <strong>the</strong> published protocol by real-time PCR. In addition, it showed lower Ct values and minimal inhibition. At different concentrations <strong>of</strong> spiked MGIT medium <strong>the</strong> proposed extraction protocol confirmed a positive signal 12 to 17 days after culture. The analytical sensitivity was assessed to less than 104 Map/ml medium. These results indicate that <strong>the</strong> new extraction method combines reliability and efficiency with simplicity. The lack <strong>of</strong> inhibition and <strong>the</strong> high analytical sensitivity suggest an effective Map DNA harvest and an effective removal <strong>of</strong> inhibitors from both <strong>the</strong> specimen and <strong>the</strong> MGIT medium components. 66
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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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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 64 and 65: indexes, compared to each single an
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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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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