Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/28/2007
Publication Date: 10/28/2007
Citation: Karcher, E.L., Beitz, D.C., Stabel, J.R. 2007. Modulation of Cytokine Expression and Lymphocyte Subsets During the Periparturient Period in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis [abstract]. Abstract No. 1A-01, p. 35.
Technical Abstract: On-farm observations suggest that dairy cows infected with Mycobacterium avium subsp. paratuberculosis (MAP) may demonstrate increased signs of clinical disease during the weeks following parturition. To date, limited research is available characterizing host immunity in periparturient dairy cows infected with MAP or the potential impact of periparturient immunosuppression. Therefore, the objective of this study was to characterize cytokine gene expression and secretion in dairy cows naturally infected with MAP during the periparturient period as compared with healthy control cows. Twenty-two multiparous Holstein cows were placed into 3 groups consisting of 5 noninfected healthy cows, 13 subclinical cows, and 4 clinical cows. Blood was collected from the jugular vein at -21, -14, -7, +1, +7, +14, +21, and +28 days relative to calving. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h with and without concanavalin A (ConA) throughout the periparturient period. Real-time PCR was performed on each sample to evaluate the expression of IFN-gamma, IL-12, IL-10, TGF-ß, IL-4 and ß-actin. RT-PCR data was analyzed using 2-ddCt values calibrated to dCt value at +1 d for each animal. Additional PBMCs were incubated for 24 h with and without ConA or MAP whole cell sonicate (MPS). Cell culture supernatants were collected and ELISA assays performed to evaluate secretion of IFN-gamma, IL-10, TGF-ß, and nitric oxide (NO). All of the animals displayed concentrations of progesterone, 17ß-estradiol, and IGF-1 that are consistent with values reported in the literature for periparturient dairy cows (parturition effect, P <0.001). Regardless of infection group, expression of IFN-gamma from non-stimulated (NS) PBMCs declined as parturition approached and did not recover during the postpartum period (P <0.03). IL-12 and TGF-ß expression for all groups remained relatively stable during the 3 wks before calving and the 4 wks after calving. Expression of IL-4 by ConA-stimulated PBMCs isolated from infected cows, declined between -21 d and +1 d (P <0.05). IL-10 expression by NS PBMCs, for both infection groups, declined as parturition approached (P <0.05). However, during the postpartum period, there was an increase in expression of IL-10 by control cows (P <0.05). Production of IFN-gamma by ConA-stimulated PBMCs, tended to be greater in infected cows compared with the controls (P <0.12). Parturition did not have an effect on TGF-ß secretion by either NS or MPS-stimulated PBMCs and effects due to infection status were minimal. When subclinical and clinical cows were combined into one group, secretion of IL-10 tended to be greater for infected cows compared with control cows at +1 d and during the postpartum period (P < 0.10). Stimulating PBMCs with MPS resulted in a 7.7, 9.7, and 12.0-fold increase in IL-10 secretion for control, subclinical, and clinical cows, respectively, compared with secretion from NS PBMCs. MPS-stimulated PBMCs from clinical cows tended to have greater NO production compared to the control (P <0.09) and subclinical cows (P <0.15). In addition to characterizing cytokines, peripheral blood CD4+, CD8+, and gamma-delta T-cells and B-cell percentages were analyzed with flow cytometry. Cell populations were further delineated by staining for CD5, a marker for T and B-cell activation. Compared to the prepartum period, the percent of CD4+ T-cells increased at parturition for clinically infected cows (P <0.08), whereas healthy control cows showed a gradual decline in CD4+ T-cells from -21 to -7 d. Subclinical cows expressed an overall greater percentage of both CD8+ and gamma-delta T-cells (P <0.05) throughout the periparturient period. Clinical cows expressed a lower percentage of CD4+/CD5bright and CD8+/CD5bright compared with control cows, but greater percentages of CD5dim cells for all l