GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE
Location: Infectious Bacterial Diseases Research Unit
Title: Assessment of Different Strategies to Determine MAP-specific Cellular Immune Responses in Cattle
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 9, 2009
Publication Date: August 9, 2009
Citation: Bridger, P.S., Bulun, H., Akineden, O., Seeger, T., Bannantine, J.P., Waters, W.R., Davis, W.C., Bauerfeind, R., Menge, C. 2009. Assessment of Different Strategies to Determine MAP-specific Cellular Immune Responses in Cattle [abstract].
Assessment of cellular immunity in cattle against Mycobacterium avium ssp. paratuberculosis (MAP) by established methods remains unsatisfactory for diagnostic purposes. Recent studies conclude that analysis of T-cell subset responsiveness may improve diagnostic outcome. Aim of 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) of MAP, M. avium ssp. avium, and M. phlei for up to 6 days. Flow cytometry was used to quantify IFN-gamma production in CD4+ and CD8+ T-cells and to quantify CD25 and CD26 expression on CD4+ and CD8+ memory T-cells (CD45RO+), gamma-delta-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-' 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-gamma content after incubation with WCS-MAP. IFN-gamma production by CD8+ PBMC hardly differed between the groups. CD4+/CD45RO+ PBMC from MAP-positive cows responded to WCS-MAP with a significantly higher expression of CD25 and CD26. gamma-delta-T-cells (CD2+/-) from MAP-positive cows reacted similarly with a higher CD25 expression. CD8+ and NK-cells from MAP-positive but not of MAP-negative cows responded with an enhanced CD25 expression independent of the origin of mycobacterial antigen. In conclusion, CD4+ T-cells from MAP-positive cows responded most sensitively and specifically to WCS-MAP with regard to both, IFN-gamma production and CD25/CD26 expression. Because early stages of MAP-infection are dominated by cellular immunity, quantifying the responsiveness of CD4+ T-cells ex vivo may be a useful approach to improve early MAP-diagnosis in cattle.