Proceedings of the 10th International Colloquium on Paratuberculosis

stored at -20ºC until fur<strong>the</strong>r analysis. The antigen specific IFN-� production in supernatants
were determined by an in-house ELISA as described elsewhere (Mikkelsen et al., 2009). The level
<strong>of</strong> IFN-� (pg/ml) was calculated using linear regression on log-log transformed reading from <strong>the</strong>
two-fold dilution series <strong>of</strong> a reference standard with known IFN-� concentration.
Samples were excluded if <strong>the</strong> IFN-� value in <strong>the</strong> SEB-stimulated sample was below 1500 pg/ml or
<strong>the</strong> PBS-stimulated value was higher than 250 pg/ml. The correlations between IFN-� responses
in <strong>the</strong> three types <strong>of</strong> samples and on different sampling dates were <strong>the</strong>n investigated.
RESULTS
In Figure 1 <strong>the</strong> IFN-� (pg/ml) responses to PPDj-stimulation in fresh samples and day-old samples
+ IL-12 from all 3 sampling dates are compared. The greatest correlation was observed at <strong>the</strong>
first sample date (R 2 = 0.72) whereas lower correlations were observed at <strong>the</strong> second (R 2 =
0.16) and third (R 2 = 0.21) sample date.
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Figure 1. Correlations between IFN-� responses to PPDj stimulation in fresh samples and day-old
samples + IL-12 potentiation. Results represent data from 28 samples at sampling 1 and 27 samples
at sampling 2 and 3, each from 30 animals, since some samples were excluded in accordance with
<strong>the</strong> exclusion criteria.
In Figure 2 <strong>the</strong> correlations between IFN-� (pg/ml) responses to PPDj-stimulation in fresh
samples and day-old samples + anti-IL-10 antibodies form all 3 sampling dates are presented.
Again, <strong>the</strong> greatest correlation was observed at <strong>the</strong> first sample date (R 2 = 0.67), and lower
correlations at <strong>the</strong> second (R 2 = 0.09) and third (R 2 = 0.35) sample date.
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IFN-g response (pg/ml) to PPDj in fresh samples vs. day-old + anti-IL-10
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Figure 2. Correlations between IFN-� responses to PPDj stimulation in fresh samples and day-old
samples + anti-IL-10 antibodies. . Results represent data from 27 samples at all three sampling dates,
since some samples were excluded in accordance with <strong>the</strong> exclusion criteria.
The highest correlations were observed between fresh samples and day-old samples
potentiated with IL-12, but <strong>the</strong>re was little difference between addition <strong>of</strong> IL-12 and anti-IL-10
antibodies. The between-sample day variation was high for both types <strong>of</strong> day-old samples
irrespective <strong>of</strong> treatment type. We have previously observed fluctuating IFN-� responses in
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