Proceedings of the 10th International Colloquium on Paratuberculosis

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#19 Development <strong>of</strong> a multiplex PCR assay targeting IS900 and F57 genes in <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis Bhupendra N. Tripathi, R B Bind, Ganesh G Sonawane, S K Munjal, Karen Stevenson, Shirish Narnaware, Biplab Debroy Indian Veterinary Research Institute, Izatnagar India; Central Sheep and Wool Research Institute, Awikanagar, Rajsthan, India The objective <strong>of</strong> <strong>the</strong> study was to develop a PCR simultaneously amplifying two genes IS900 and F57 for identifying and confirming <strong>the</strong> presence <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) in clinical samples from animals. This could avoid subsequent confirmation <strong>of</strong> PCR products in light <strong>of</strong> <strong>the</strong> questionable specificity <strong>of</strong> <strong>the</strong> IS900 gene. Two specific primer sets; one from IS900 and o<strong>the</strong>r from F57 genes were used for <strong>the</strong> amplification <strong>of</strong> 314 bp and 439 bp PCR products, respectively. The DNA samples used in <strong>the</strong> multiplex PCR were extracted from tissues and MAP isolates maintained in <strong>the</strong> Johne’s disease laboratory (GD Lab.) using standard protocols. The PCR was applied to high quality DNA from 52 MAP, 5 non-MAP isolates from various hosts and geographic origins and 45 fresh intestinal tissue samples from infected sheep and goats and suspected cases <strong>of</strong> bovine paratuberculosis. All MAP isolates (100%) were positive for both genes and all 5 non-MAP isolates were negative for both genes. Twenty six out <strong>of</strong> 30 goat tissue samples and 6 <strong>of</strong> 8 sheep samples yielded amplification <strong>of</strong> both genes, whereas only IS900 was amplified from 4 goat and 2 sheep samples and one <strong>of</strong> 7 suspected cases <strong>of</strong> bovine paratuberculosis. These samples probably contained low numbers <strong>of</strong> MAP which could be detected only by PCR directed against a multicopy gene such as IS900. The results <strong>of</strong> <strong>the</strong> present study suggested that while multiplex PCR could be used for identification and confirmation <strong>of</strong> MAP isolates, a minority <strong>of</strong> clinical samples may be positive for only <strong>the</strong> IS900 gene and additional tests may be required to confirm a diagnosis <strong>of</strong> Johne’s disease in <strong>the</strong>se animals. #29 Conductometric biosensor for diagnosis <strong>of</strong> Johne’s disease Chika C Okafor, Daniel L Grooms, Evangelyn C Alocilja, Steven R Bolin Michigan State University, USA The development <strong>of</strong> non-laboratory based assays would support more frequent testing <strong>of</strong> animals for Johnes disease (JD) and could improve its control. Newly developed conductometric biosensors combine immunomigration technology with electronic signal detection and has been recently developed for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratubercolosis (MAP) IgG. Optimization <strong>of</strong> this biosensor is needed to support its usefulness in diagnosis. In this study, two aspects <strong>of</strong> <strong>the</strong> previously developed conductometric biosensor were optimized for its application in JD diagnosis. After optimization, a pilot study on <strong>the</strong> biosensor’s use in JD diagnosis was conducted, using samples whose JD status where unknown. A commercially available antibody detection ELISA was used as a gold standard for comparison. The biosensor’s sensitivity was (71.43%) and specificity (70%) when compared to <strong>the</strong> antibody detection ELISA. There was a moderate strength <strong>of</strong> agreement (Kappa = 0.41) between <strong>the</strong> two assays. Findings from this study support <strong>the</strong> continued development <strong>of</strong> conductometric biosensors for use in <strong>the</strong> diagnosis <strong>of</strong> JD. Fur<strong>the</strong>r optimization <strong>of</strong> <strong>the</strong> biosensor and validation with a larger sample size could support its use in JD control programs. 49
#31 Case study: Identification <strong>of</strong> a non-classical (bison) strain <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in Cervus elaphus subsp. nelsoni (Rocky Mountain Elk) Suelee Robbe-Austerman, Philip E. Dykema, Bruce V. Thompsen, Rob Bildfell National Veterinary Services Laboratories, USA; College <strong>of</strong> Veterinary Medicine, Oregon State University, USA Tissues from an emaciated, hunter-killed elk harvested on public land in Harney County, Oregon were submitted to <strong>the</strong> Oregon State University in <strong>the</strong> fall <strong>of</strong> 2008. No o<strong>the</strong>r abnormalities were noted at necropsy. Histologically <strong>the</strong>re were large numbers <strong>of</strong> macrophages filled with numerous acid fast bacilli expanding <strong>the</strong> lamina propria <strong>of</strong> <strong>the</strong> small intestine, and granulomas containing acid fast bacteria in <strong>the</strong> liver. Paraffin imbedded intestinal tissue was submitted to NVSL for PCR analysis, and frozen tissues were submitted for general mycobacterial culture, including bovine tuberculosis and Johne’s disease. PCR on formalin fixed tissue was negative for IS6110 (M. tuberculosis complex) and positive for IS900 (Mycobacterium avium subsp. paratuberculosis). All general mycobacterial media without mycobactin J (MJ), and Herrold’s Egg Yolk media with MJ did not support mycobacterial growth, whereas Trek ESP ® media supplemented with 2ml <strong>of</strong> additional egg yolk signaled positive in 8 days. Middlebrook 7H10 media supplemented with MJ and 250ml <strong>of</strong> egg yolk/L also supported growth <strong>of</strong> <strong>the</strong> isolate after 7 weeks <strong>of</strong> incubation. The isolate failed to grow when subcultured directly onto HEY media w/MJ. Biochemically <strong>the</strong>se growth patterns are consistent with sheep strains <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP). Confirmation <strong>of</strong> this isolate as MAP was done using commercially available MAP PCR reagents (Tetracore ® and Ambion ® ). However, <strong>the</strong> IS1311 restriction enzyme analysis pattern did not match sheep or cattle isolates, but was instead similar to a MAP strain isolated from bison in Montana in 2001. Therefore, this constitutes <strong>the</strong> first known report <strong>of</strong> a “bison” strain <strong>of</strong> MAP in elk in North America. #32 Laboratory performance <strong>of</strong> fecal culture and direct PCR measured by pr<strong>of</strong>iciency testing Suelee Robbe-Austerman, Beth Harris National Veterinary Services Laboratories, USA National Veterinary Services Laboratories administers a Johne’s Disease Fecal Pr<strong>of</strong>iciency Panel annually to diagnostic and research laboratories. In 2008, 118 panels were sent to 71 laboratories. Collectively, <strong>the</strong>se data provide insight into <strong>the</strong> performance <strong>of</strong> fecal culture and direct PCR methods currently used throughout veterinary diagnostic laboratories. While some laboratories correctly classified all 25 samples for each method, interlaboratory variation within methods differed significantly: 24 <strong>of</strong> 32 (75%) laboratories passed using liquid culture; 35 <strong>of</strong> 50 (70%) passed using direct PCR; and 20 <strong>of</strong> 36 (55%) passed using solid media. Liquid media culture systems were significantly more accurate at classifying samples as positive or negative than solid media (odds ratio 2.1, 95% CI = 1.5-3.0) and more accurate than direct PCR (OR = 1.8, 95% CI= 1.3-2.4). While not significant, direct PCR tends to be more accurate than solid media (OR=1.2 95%CI=0.95-1.5). Lack <strong>of</strong> sensitivity and incorrect identification <strong>of</strong> negative samples as positive were <strong>the</strong> most common causes <strong>of</strong> misclassification. Confirmatory PCR on solid and liquid media had a false positive rate <strong>of</strong> 2.7%, whereas direct PCR had a false positive rate <strong>of</strong> 0.6%. Classification <strong>of</strong> an animal’s shedding status (low, moderate, high) is currently based on colony counts from solid media. Recent research suggest that clinically important “supper shedders” may not be easily identified by colony counts on traditional solid media. Although not routinely reported by laboratories, continuous results supplied by direct PCR and liquid culture systems may have potential to classify shedding levels that are more clinically relevant. In summary, liquid culture systems currently <strong>of</strong>fer <strong>the</strong> highest sensitivity across laboratories; however, strict procedures must be in place to prevent false positives during confirmatory PCR. Increased use <strong>of</strong> direct PCR and liquid media systems highlights <strong>the</strong> need to establish guidelines for reporting fecal shedding status for <strong>the</strong>se methods. 50
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 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 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
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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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