Proceedings of the 10th International Colloquium on Paratuberculosis

#191
Potentiating day-old blood samples for detection <strong>of</strong> interferon-gamma
responses following infection with Mycobacterium avium subsp.
paratuberculosis
Heidi Mikkelsen 1,2 , Søren Saxmose Nielsen 2 , Gregers Jungersen 1
1 National Veterinary Institute, Technical University <strong>of</strong> Denmark, Copenhagen, Denmark;
2 Faculty <strong>of</strong> Life Sciences, University <strong>of</strong> Copenhagen, Frederiksberg, Denmark,
ABSTRACT
The interferon gamma (IFN-�) test measuring specific cell-mediated immune responses in
whole blood can be used for diagnosis at an early stage <strong>of</strong> Mycobacterium avium subsp.
paratuberculosis (MAP) infection. A major obstacle for <strong>the</strong> practical use <strong>of</strong> IFN-� testing is <strong>the</strong>
recommended maximum 8 hour time interval from blood sampling to culture.
The objective <strong>of</strong> <strong>the</strong> study was to assess options for use <strong>of</strong> day-old blood samples for early-stage
diagnosis <strong>of</strong> MAP infections. Bovine interleukin 12 (IL-12) can induce, and IL-10 reduce, IFN-�
production. Therefore, addition <strong>of</strong> recombinant IL-12 and anti-IL-10 antibodies could result in
enhanced production <strong>of</strong> IFN-� in samples exposed to MAP antigens.
Whole blood samples were collected from heifers in a Danish dairy herd known to be
infected with MAP. The samples were collected on three sample dates. On each date <strong>the</strong>
blood samples were stimulated with johnin purified protein derivative (PPDj) and recombinant
antigens as fresh samples, as day-old samples potentiated with bovine IL-12, and as day-old
samples treated with anti-bovine IL-10 antibody. 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. The highest
correlations were seen between fresh samples and day-old samples added anti-IL-10 antibodies.
There was little difference between addition <strong>of</strong> IL-12 and anti-IL-10 antibodies. However,
between-sample day variation was high for both types <strong>of</strong> day-old samples, irrespective <strong>of</strong>
treatment type. Optimisation <strong>of</strong> <strong>the</strong> IFN-� test on day-old samples for diagnosing MAP
infected herds would facilitate <strong>the</strong> large scale use <strong>of</strong> this diagnostic test.
INTRODUCTION
Early MAP diagnosis can be achieved by measurement <strong>of</strong> specific cell-mediated immune responses
to MAP antigens by <strong>the</strong> IFN-� test. The IFN-� test is a proliferation assay in which whole blood
samples are cultured with MAP antigens and released IFN-� is measured in <strong>the</strong> supernatant by
enzyme linked immunosorbent assay (ELISA) (Wood et al., 1989). The large scale use <strong>of</strong> <strong>the</strong>
IFN-� test for routine diagnostics is limited by <strong>the</strong> short timeframe recommended <strong>of</strong> less than
8 hours from blood sampling at <strong>the</strong> farm to culture with antigens in <strong>the</strong> laboratory. To
circumvent this short time frame, <strong>the</strong> use <strong>of</strong> day-old blood samples potentiated with
recombinant bovine IL-12 have previously been investigated (Jungersen et al., 2005).
Potentiation with IL-12 demonstrated to rescue antigen specific IFN-� responses <strong>of</strong> day-old
samples. Ano<strong>the</strong>r study demonstrated that <strong>the</strong> IFN-� response can be enhanced by adding anti-IL-
10 antibodies to whole blood samples stimulated with PPDj (Buza et al., 2004). Here, we fur<strong>the</strong>r
explored <strong>the</strong> use <strong>of</strong> day-old blood samples in <strong>the</strong> IFN-� test and addition <strong>of</strong> ei<strong>the</strong>r <strong>of</strong> IL-10
antibodies or recombinant bovine IL-12.
MATERIALS AND METHODS
Blood samples were collected 3 times with 4 and 5 week intervals from <strong>the</strong> same 30 heifers
15-24 months <strong>of</strong> age in a Danish dairy herd known to be infected with MAP. On each sample
date <strong>the</strong> whole blood samples incubated for 20-22 hours at 37ºC in 5% CO2 with PPDj, a
negative (PBS) and a positive control (Staphylococcal enterotoxin B; SEB). All samples were
incubated with antigens as fresh samples, as day-old samples potentiated with recombinant
bovine IL-12, and as day-old samples treated with anti-bovine IL-10 antibody (MCA2110,
AbD Serotec, UK). The day-old samples were stored overnight at 4ºC from <strong>the</strong> day <strong>of</strong> blood
collection, but o<strong>the</strong>rwise followed <strong>the</strong> same protocol as <strong>the</strong> fresh samples. Following
overnight culture, <strong>the</strong> culture plates were centrifuged and <strong>the</strong> supernatants collected and
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