Proceedings of the 10th International Colloquium on Paratuberculosis

#147 Increasing <strong>the</strong> throughput <strong>of</strong> Johne’s testing with PARACHEK ® 2
Pascal Schacher, Daniel Zwald, Angela Zurfluh, Ram Krishnamurthy, Alex Raeber
Prionics AG, Switzerland
The PARACHEK ® is <strong>the</strong> original Johne’s absorbed ELISA. It is able to detect antibodies against M. paratuberculosis
in serum and milk prior to <strong>the</strong> onset <strong>of</strong> clinical signs. Prionics AG has now developed <strong>the</strong> PARACHEK ®
2 which is more user-friendly and enables automation. It contains a one component substrate and incubation
times were adapted for user-friendliness.
To evaluate <strong>the</strong> performance <strong>of</strong> both tests a set <strong>of</strong> negative and positive cattle and sheep serum samples
and a set <strong>of</strong> negative and positive milk samples from cattle were tested. The samples derived from animals
with a known fecal culture status for MAP. These samples were tested with both <strong>the</strong> PARACHEK ® and <strong>the</strong>
PARACHEK ® 2. The agreement is expressed by <strong>the</strong> Cohen’s Kappa coefficient and interpreted using <strong>the</strong>
Landis and Koch table. The agreement between <strong>the</strong> two assays is almost perfect with kappa values <strong>of</strong> 0.88
(ovine serum), 0.90 (bovine serum) and 0.91 (bovine milk).
The PARACHEK ® 2 also has two options for detection, a kinetic protocol for a high plate to plate reproducibility
or an end-point protocol enabling automation. The PARACHEK ® 2 was automated on a Beckman Coulter
Biomek ® FXP Laboratory Automation Workstation equipped with a 96-well plate washer and a plate reader
which allows for a throughput <strong>of</strong> up to 16 plates in one working day (8.5 hours) starting from serum or milk
samples. The results <strong>of</strong> <strong>the</strong> fully automated system were compared to manual processing <strong>of</strong> <strong>the</strong> samples. The
agreement is almost perfect with a kappa value <strong>of</strong> 0.96.
These results demonstrate that <strong>the</strong> PARACHEK ® 2 can be easily run on an automated system with <strong>the</strong>
same excellent performance as with manual processing <strong>of</strong> <strong>the</strong> samples using <strong>the</strong> original PARACHEK ® and
thus enabling laboratories to save time and freeing staff for o<strong>the</strong>r work.
#155 Comparative characterization from German isolates <strong>of</strong> Mycobacterium avium subsp.
paratuberculosis using SSR-, IS900-RFLP- and MIRU-VNTR-analysis
Isabel Fritsch, Heike Koehler, Petra Moebius
Institute <strong>of</strong> Molecular Pathogenesis, Friedrich-Loeffler-Institute, Jena, Germany
Objective: The objective <strong>of</strong> <strong>the</strong> present study was to detect <strong>the</strong> heterogeneity <strong>of</strong> a widespread panel <strong>of</strong> Map
isolates from German cattle herds and red deer using different techniques to be able to evaluate <strong>the</strong> methods
and to compare <strong>the</strong> results with genotypes originating from o<strong>the</strong>r countries <strong>of</strong> <strong>the</strong> world.
Material and Methods: A set <strong>of</strong> about 120 Map isolates were genotyped by restriction fragment length
polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion, by mycobacterial
interspersed repetitive unit – variable number tandem repeat (MIRU-VNTR) typing based on 8 markers, and
by short sequence repeat (SSR) analysis at locus 1 (G residue), 2 (G residue), 8 (GGT residue), and 9 (TGC
residue). The results were analyzed separately and in combination.
Results: Results <strong>of</strong> <strong>the</strong> three typing techniques were not associated, <strong>the</strong> discriminatory power was different.
A combined analysis <strong>of</strong> <strong>the</strong>se results enhances highly <strong>the</strong> discriminatory power <strong>of</strong> typing. Using all three
methods unique genotypes were detected for all studied isolates originating from epidemiological unrelated
herds. Different as well as identical genotypes were found inside <strong>of</strong> individual herds.
Conclusion: Results allow different epidemiological interpretations depending on <strong>the</strong> used typing technique.
SSR based on locus 1, 2, 8, and 9 can not replace <strong>the</strong> o<strong>the</strong>r techniques. The significance <strong>of</strong> single or
combined typing techniques concerning epidemiological conclusions will be fur<strong>the</strong>r clarified based on <strong>the</strong> randomly
selected large strain panel from Germany.
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