Proceedings of the 10th International Colloquium on Paratuberculosis

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#98 An inter-laboratory ring-trial evaluating molecular and culture detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in dairy products: Outcome <strong>of</strong> an EU ParaTBTools workpackage John Anthony Donaghy, Michael Rowe, Anne Fisher, Jan Rademaker, Pat Murphy, Beatrice Blanchard, Geertrui Vlaemynck, Hans-Georg Walte, Philipp Hammer Food Microbiology Branch, Agri-Food & Biosciences Institute, Belfast, N. Ireland, United Kingdom; NIZO food research, Ede, The Ne<strong>the</strong>rlands; Moorepark Food Research Centre, Ireland; Adiagene, France; ILVO, Belgium; Max Rubner-Institute, Federal Institute <strong>of</strong> Nutrition and Food, Kiel, Germany There is a need for standardization and harmonization <strong>of</strong> culture and molecular methods for <strong>the</strong> detection/isolation/recovery <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) from dairy matrices. The objective <strong>of</strong> this study was to perform a dairy products ring-trial <strong>of</strong> culture and molecular methods across European laboratories. The prescribed molecular method used was a Real Time IS900-based TaqMan assay in combination with <strong>the</strong> commercial Map DNA extraction kit (Adiapure ® ). The prescribed culture method used was HEYM agar supplemented with PANTA, nisin and penicillin. The ‘blind’ ring-trial was completed according to <strong>the</strong> NordVal validation method: eleven participant laboratories, one Map strain, three contamination levels, duplicates for each level and five dairy matrices. The dairy matrices analysed were Map-infected faecally-contaminated milk, artificially-contaminated Gouda, Danish Blue and Munster cheeses and yogurt. The contamination levels <strong>of</strong> cheeses and yogurt were 5 x 103 cfu/ g(high) and 50 cfu/g (low) and 1.8 x 103 cfu/g(high) and 18 cfu/g(low) for faecally-contaminated milk. In addition to <strong>the</strong> prescribed methods assessed, 12 alternate molecular and 14 culture ‘own laboratory’ detection protocols were employed on test samples. For <strong>the</strong> prescribed molecular method, average sensitivities across all laboratories, for combined high and low contamination levels, were 95%, 96%, 100%, 84% and 96% for faecally-contaminated milk, yogurt, Gouda, Danish Blue and Munster cheese respectively. Specificities for all matrices were in <strong>the</strong> range 95-100%. Overall accordance (repeatability) and concordance (reproducibility) values were 0.975 and 91.7% respectively for <strong>the</strong> molecular method assessed. Culture methods employed showed lower sensitivities than molecular methods, Map was recovered more frequently at ‘high’ inoculum levels. The reproducibility <strong>of</strong> a commercial Map DNA extraction kit used in combination with a Real Time TaqMan assay was demonstrated in this ring-trial. The extraction and assay protocol was applicable to a range <strong>of</strong> dairy matrices whereas <strong>the</strong> trial demonstrated <strong>the</strong> idiosyncrasies associated with Map culture methods. 215
#106 A gap in <strong>the</strong> bio security fence. Bought in beef bulls in dairy herds in Ireland (2004-2008) Peter C. Mullowney, Damien Barrett, Kevin Kenny, Richard Fallon, Martin Blake, Margaret Good, Department <strong>of</strong> Agriculture, Ireland; Teagasc Beef Research Centre, Grange, Ireland Beef breeds are <strong>of</strong>ten used as bulls in dairy herds once a sufficient number <strong>of</strong> cows have been bred to dairy AI sires to ensure adequate supply <strong>of</strong> replacement dairy stock. The introduction and use <strong>of</strong> such bulls curtails <strong>the</strong> potential genetic improvement <strong>of</strong> <strong>the</strong> herd but it also leads to an increased bio-security risk especially when source herds are not participating in a disease monitoring/control programme. Diseases such as Johne’s disease may be introduced into dairy herds in this way. A total <strong>of</strong> 942,000 calves were born to dairy cows in 2008. 51% <strong>of</strong> <strong>the</strong>se had a dairy sire, 45% <strong>of</strong> which were by AI, and 49% a beef sire, 32% <strong>of</strong> <strong>the</strong>se by a stock bull. If a Johne’s control programme in dairy herds is not extended to <strong>the</strong> producers <strong>of</strong> <strong>the</strong>se beef bulls <strong>the</strong>n <strong>the</strong>se bulls may be <strong>the</strong> vehicle for <strong>the</strong> organism to enter <strong>the</strong> dairy herd. While a control programme would be much more difficult to implement in a beef herd due to <strong>the</strong> impossibility <strong>of</strong> separating calves from adult animals, a certified free programme in closed herds would be possible. A previous study <strong>of</strong> Irish beef herds had examined animal movements in to and out <strong>of</strong> case herds infected with Johne’s Disease (faecal positive) compared with control herds that were not infected.(negative on a serum survey) [1]. In <strong>the</strong> current study a total <strong>of</strong> 478 bulls were purchased by dairy herds from beef case herds, 96 <strong>of</strong> <strong>the</strong>se bulls subsequently moved to a second herd. The average number <strong>of</strong> bulls sold from case herds was eleven. A total <strong>of</strong> 931 bulls were purchased by dairy herds from 167 <strong>of</strong> <strong>the</strong> 277 beef control herds, 176 <strong>of</strong> <strong>the</strong>se bulls subsequently moved onwards to o<strong>the</strong>r herds. The average number <strong>of</strong> bulls sold from control herds was seven. [1] Mullowney,P.C., Barrett,D., Egan,J., Fallon,R., Blake,M., Good,M ;Cattle movements in to and out <strong>of</strong> Johne’s infected beef suckler herds in Ireland. <strong>Proceedings</strong> 9th <strong>International</strong> <strong>Colloquium</strong> on Paratuberculosis, Tsukuba, Japan, 29 October – 2 November 2007. 216
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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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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#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
Page 166 and 167: #22 The Prevalence of</stro
Page 168 and 169: CONCLUSION IS1311-based nested Ma/M
Page 170 and 171: #42 Seroprevalence survey o
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Page 176 and 177: REFERENCES 1) Chiodini RJ, Van Krui
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Page 182 and 183: Latium Region Viterbo districts RES
Page 184 and 185: #144 The suitability of</st
Page 186 and 187: #178 Age structure of</stro
Page 188 and 189: A statistically significant lower f
Page 190 and 191: #183 Sero-prevalence of</st
Page 192 and 193: #218 Johne’s disease in t
Page 194 and 195: #244 A survey to estimate t
Page 196 and 197: Control Programs Keynote Lecture Re
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Page 200 and 201: #141 Control of pa
Page 202 and 203: #198 Use of small
Page 204 and 205: Control Programs Poster Abstracts 1
Page 206 and 207: RESULTS Loss of al
Page 208 and 209: #40 Managing Bovine Johnes Disease
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Page 212 and 213: #87 Evaluation of
Page 214 and 215: separately for each existing herd <
Page 218 and 219: #120 Paratuberculosis vaccine inter
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Page 238 and 239: Pathogenomics and MAP Biology Conve
Page 240 and 241: #45 Identification of</stro
Page 242 and 243: #117 Adsorption of
Page 244 and 245: #222 Atypical structural features <
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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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