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Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

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#98 An inter-laboratory ring-trial evaluating molecular and culture detecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobacterium<br />

avium subsp. paratuberculosis in dairy products: Outcome <str<strong>on</strong>g>of</str<strong>on</strong>g> an EU ParaTBTools<br />

workpackage<br />

John Anth<strong>on</strong>y D<strong>on</strong>aghy, Michael Rowe, Anne Fisher, Jan Rademaker, Pat Murphy, Beatrice Blanchard, Geertrui<br />

Vlaemynck, Hans-Georg Walte, Philipp Hammer<br />

Food Microbiology Branch, Agri-Food & Biosciences Institute, Belfast, N. Ireland, United Kingdom; NIZO food<br />

research, Ede, The Ne<str<strong>on</strong>g>the</str<strong>on</strong>g>rlands; Moorepark Food Research Centre, Ireland; Adiagene, France; ILVO, Belgium; Max<br />

Rubner-Institute, Federal Institute <str<strong>on</strong>g>of</str<strong>on</strong>g> Nutriti<strong>on</strong> and Food, Kiel, Germany<br />

There is a need for standardizati<strong>on</strong> and harm<strong>on</strong>izati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> culture and molecular methods for <str<strong>on</strong>g>the</str<strong>on</strong>g> detecti<strong>on</strong>/isolati<strong>on</strong>/recovery<br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobacterium avium subsp. paratuberculosis (Map) from dairy matrices. The objective <str<strong>on</strong>g>of</str<strong>on</strong>g><br />

this study was to perform a dairy products ring-trial <str<strong>on</strong>g>of</str<strong>on</strong>g> culture and molecular methods across European laboratories.<br />

The prescribed molecular method used was a Real Time IS900-based TaqMan assay in combinati<strong>on</strong><br />

with <str<strong>on</strong>g>the</str<strong>on</strong>g> commercial Map DNA extracti<strong>on</strong> kit (Adiapure ® ). The prescribed culture method used was HEYM agar<br />

supplemented with PANTA, nisin and penicillin.<br />

The ‘blind’ ring-trial was completed according to <str<strong>on</strong>g>the</str<strong>on</strong>g> NordVal validati<strong>on</strong> method: eleven participant<br />

laboratories, <strong>on</strong>e Map strain, three c<strong>on</strong>taminati<strong>on</strong> levels, duplicates for each level and five dairy matrices. The<br />

dairy matrices analysed were Map-infected faecally-c<strong>on</strong>taminated milk, artificially-c<strong>on</strong>taminated Gouda, Danish<br />

Blue and Munster cheeses and yogurt. The c<strong>on</strong>taminati<strong>on</strong> levels <str<strong>on</strong>g>of</str<strong>on</strong>g> cheeses and yogurt were 5 x 103 cfu/<br />

g(high) and 50 cfu/g (low) and 1.8 x 103 cfu/g(high) and 18 cfu/g(low) for faecally-c<strong>on</strong>taminated milk. In additi<strong>on</strong><br />

to <str<strong>on</strong>g>the</str<strong>on</strong>g> prescribed methods assessed, 12 alternate molecular and 14 culture ‘own laboratory’ detecti<strong>on</strong> protocols<br />

were employed <strong>on</strong> test samples.<br />

For <str<strong>on</strong>g>the</str<strong>on</strong>g> prescribed molecular method, average sensitivities across all laboratories, for combined high and<br />

low c<strong>on</strong>taminati<strong>on</strong> levels, were 95%, 96%, 100%, 84% and 96% for faecally-c<strong>on</strong>taminated milk, yogurt, Gouda,<br />

Danish Blue and Munster cheese respectively. Specificities for all matrices were in <str<strong>on</strong>g>the</str<strong>on</strong>g> range 95-100%. Overall<br />

accordance (repeatability) and c<strong>on</strong>cordance (reproducibility) values were 0.975 and 91.7% respectively for <str<strong>on</strong>g>the</str<strong>on</strong>g><br />

molecular method assessed. Culture methods employed showed lower sensitivities than molecular methods,<br />

Map was recovered more frequently at ‘high’ inoculum levels.<br />

The reproducibility <str<strong>on</strong>g>of</str<strong>on</strong>g> a commercial Map DNA extracti<strong>on</strong> kit used in combinati<strong>on</strong> with a Real Time TaqMan<br />

assay was dem<strong>on</strong>strated in this ring-trial. The extracti<strong>on</strong> and assay protocol was applicable to a range <str<strong>on</strong>g>of</str<strong>on</strong>g> dairy<br />

matrices whereas <str<strong>on</strong>g>the</str<strong>on</strong>g> trial dem<strong>on</strong>strated <str<strong>on</strong>g>the</str<strong>on</strong>g> idiosyncrasies associated with Map culture methods.<br />

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