Proceedings of the 10th International Colloquium on Paratuberculosis

#4 NOD2 and mycobacterial recognition
Marcel Behr McGill University, Canada
Objective: To determine <strong>the</strong> effect <strong>of</strong> NOD2 polymorphisms on mycobacterial recognition in vivo. Materials and
Methods: We obtained Nod2-/- mice that manifest <strong>the</strong> same loss-<strong>of</strong>-function phenotype during ex vivo stimulation
with peptidoglycan (PGN) as is observed with Crohn’s patients that are homozygous for <strong>the</strong> 3020insC
mutation. Mice were challenged in vivo challenge with M. tuberculosis and M. bovis, looking at early time-points
(pathology and adaptive immunity) and late-points (bacterial CFU and survival). In a second study, macrophages
from Nod2-/- mice were stimulated ex vivo with mycobacteria wild-type and disrupted for namH, responsible
for <strong>the</strong> modification <strong>of</strong> PGN to produce N-glycolyl muramyl dipeptide (MDP). Results: Nod2-/- mice generated
a diminished inflammatory response, with less pathology, less IL-12 production and reduced adaptive immunity
one month after infection. After 6 months, Nod2-/- mice had higher bacterial burdens and succumbed to infection
sooner than wild-type controls. Using bacterial genetics and syn<strong>the</strong>tic chemistry, we found that NOD2 activation
depends on <strong>the</strong> presence <strong>of</strong> <strong>the</strong> mycobacterial namH and that <strong>the</strong> mycobacterial PGN (N-glycolyl MDP)
is at least 10-times more potent at NOD2 activation than <strong>the</strong> commonly encountered N-acetyl MDP. Conclusion:
NOD2 is important and specific for recognition <strong>of</strong> mycobacterial infection, in vitro and in vivo.
#27 Assessment <strong>of</strong> different strategies to determine MAP-specific cellular immune responses
in cattle
Philip S. Bridger, Hakan Bulun, Oemer Akineden, Torsten Seeger, John P. Bannantine, W. Ray Waters, William
C. Davis, Rolf Bauerfeind, Christian Menge
Institute for Hygiene and Infectious Diseases <strong>of</strong> Animals, Justus-Liebig University Giessen, Germany; Institute for
Food Science, Justus-Liebig University Giessen, Germany; Clinic for Ruminants and Swine, Justus-Liebig University
Giessen, Germany; National Animal Disease Center, USDA-ARS, Ames, Iowa, USA; Washington State University,
Pullmann, USA
Assessment <strong>of</strong> cellular immunity in cattle against Mycobacterium avium ssp. paratuberculosis (MAP) by established
methods remains unsatisfactory for diagnostic purposes. Recent studies conclude that analysis <strong>of</strong> T-cell
subset responsiveness may improve diagnostic outcome. Aim <strong>of</strong> this study was to identify T-cell subsets which
respond most specifically to MAP antigens. Peripheral mononuclear cells (PBMC) from 7 MAP-positive and 5
MAP-negative cows (defined by herd status, serology, fecal culture, PCR) aged 2 - 5 years were incubated in
medium supplemented with whole cell sonicates (WCS) or purified protein derivatives (PPD) <strong>of</strong> MAP, M. avium
ssp. avium, and M. phlei for up to 6 days. Flow cytometry was used to quantify IFN-γ production in CD4 + and
CD8 + T-cells and to quantify CD25 and CD26 expression on CD4 + and CD8 + memory T-cells (CD45RO + ), γδ-
T-cells (TcR1-N24 + /CD2 +/- ), and NK-cells (CD335 + /CD2 + ). Different gating strategies were applied to analyze
lymphocyte and lymphoblast populations as well as IFN-γ and CD markers individually or in combination. WCS
preparations induced more specific responses compared to PPD throughout. Compared to MAP-negative animals,
CD4 + lymphoblasts from MAP-positive cows responded with a significantly higher IFN-γ content after incubation
with WCS-MAP. IFN-γ production by CD8 + PBMC hardly differed between <strong>the</strong> groups. CD4 + /CD45RO +
PBMC from MAP-positive cows responded to WCS-MAP with a significantly higher expression <strong>of</strong> CD25 and
CD26. γδ-T-cells (CD2 +/- ) from MAP-positive cows reacted similarly with a higher CD25 expression. CD8 + and
NK-cells from MAP-positive but not <strong>of</strong> MAP-negative cows responded with an enhanced CD25 expression
independent <strong>of</strong> <strong>the</strong> origin <strong>of</strong> mycobacterial antigen. In conclusion, CD4 + T-cells from MAP-positive cows responded
most sensitively and specifically to WCS-MAP with regard to both, IFN-γ production and CD25/CD26
expression. Because early stages <strong>of</strong> MAP-infection are dominated by cellular immunity, quantifying <strong>the</strong> responsiveness
<strong>of</strong> CD4 + T-cells ex vivo may be a useful approach to improve early MAP-diagnosis in cattle.
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