Modulation of cytokine expression and lymphocyte subsets during the periparturient period in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

The objective of this study was to evaluate cytokine gene expression and populations of lymphocyte subsets in periparturient dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP). Blood was collected from noninfected, subclinical, and clinical MAP-infected dairy cows for 3 wks preto 4 wk post-calving. Expression of IFN-γ, IL-4, and IL-10 declined at calving compared with prepartum values in both control and infected cows. PBMCs isolated from infected cows had higher secretion of IFN-γ, IL-10, and TGF-β in the postpartum period compared with control cows. Flow cytometric analysis revealed that subclinical cows expressed a greater percentage of both CD8 and γδ T-cells compared with the clinical cows. The percentage of CD4 T-cells increased in clinical cows as parturition approached. Clinical cows expressed lower percentages of CD4CD5 and CD8CD5 compared with control cows, but greater percentages of CD5 cells for all lymphocyte subsets. These data suggest that parturition is a very dynamic time period for host immunity, with potential for altered immunity to hinder the ability of dairy cows to thwart infectious diseases. INTRODUCTION On-farm observations suggest that dairy cows infected with MAP may demonstrate increased signs of clinical disease during the weeks following parturition. Parturition has a major impact on the number of lymphocytes and monocytes in the peripheral blood of healthy cows and alterations in these percentages play a significant role in the ability of the animal to respond to infection. The transition from the subclinical to clinical stage of MAP infection is characterized by a shift from cell-mediated (Th1) immunity to an antibody-mediated (Th2) humoral response. To date, limited research is available characterizing detailed aspects of periparturient immunosuppression in the dairy cow. Further, it is not clear what impact this time period and its associated stressors may have on host immunity in cows with paratuberculosis. Therefore, the objectives of this study were 1) to characterize cytokine gene expression and secretion, and 2) to determine percentages of lymphocyte subsets in periparturient dairy cows naturally infected with MAP. MATERIALS AND METHODS Twenty-three Holstein dairy cows were grouped according to infection status. These three groups consisted of 5 non-infected healthy cows, 14 cows naturally infected with MAP, but asymptomatic, and 4 naturally infected cows with clinical Johne’s disease. Non-infected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3-5 year period and were negative on any serological assays (ELISA, IFN-γ) performed during this period. Blood was collected from the jugular vein in 2x acid-citrate-dextrose (1:10) at -21, -14, 7, +1, +7, +14, +21, and +28 days relative to parturition. Peripheral blood mononuclear cells (PBMCs) were isolated and cells were cultured at 1.4 x 10/mL in 48-well flat-bottomed plates with either medium alone (non-stimulated, NS), with concanavalin A (ConA; 10 μg/mL) or with MAP whole cell sonicate (MPS; 10 μg/mL) added to designated wells. Plates were incubated for 24 h at 39°C in 5% CO2 in a humidified atmosphere. After 24 h plates were removed and centrifuged at 400 x g for 5 min. Supernatants were removed and stored at Proceedings of 9ICP 2007 34 20°C prior to cytokine measurement. The secretion of IFN-γ, IL-10, and TGF-β by PBMCs in the cell culture supernatant was determined by ELISA assay. For flow cytometric analysis, PBMCs were resuspended in complete medium and 50 μL of the cell suspension was added to wells containing 50 μL of primary monoclonal antibody to CD4, CD8, γδ T-cells, B cells, and CD5. Analysis was conducted by gating on mononuclear cells based on forward and side scatter characteristics. RNA was extracted from NS and ConA-stimulated PBMCs using the standard protocol for Trizol reagent. Total RNA was converted to first strand cDNA. For RT-PCR analysis, SYBR Green PCR master mixture, template cDNA, and gene-specific primers for IFN-γ, IL12p35, IL-4, IL-10, and TGF-β were combined in a 20 μL reaction mixture . The β-actin gene was used as the control for calculation of dCt. RT-PCR was analyzed by using the 2 method. The mean +1 DRTC dCt value within treatment was used as the reference expression point. RESULTS AND DISCUSSION Due to sampling error, we were not able to evaluate the cytokine gene expression data for the clinical cows in this study. When comparing control and subclinically infected cows, subclinical MAP infection did not have an effect on the gene expression of IFN-γ or IL-12. However, ConA-stimulated PBMCs from subclinical cows (14.36 ng/ml ± 1.6) tended (P < 0.06) to secrete more IFN-γ compared with the control cows (8.30 ± 2.2). Previous work in our laboratory showed that subclinical JD cows had greater IFN-γ expression compared with clinical cows (Stabel, 2000). Parturition had a significant effect on IFN-γ expression with declines in IFN-γ expression by NS (P < 0.05) (Fig. 1A) and ConA-stimulated (P < 0.01) PBMCs from both infection groups as parturition approached. This is in agreement with previous studies reporting a decline in Th1 cytokines at parturition (Shafer-Weaver et al., 1999). Interleukin-12 expression was not effected by infection or parturition (data not shown).