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United States Department of Agriculture

Agricultural Research Service

Title: Flow Cytometric Intracellular Ionized Calcium Analysis of Immune Cells in Periparturient Dairy Cattle

Authors
item Kimura, Kayoko - IOWA STATE UNIV., AMES
item Goff, Jesse
item Reinhardt, Timothy
item Harp, James
item Pesch, Bruce
item Kehrli Jr, Marcus - PFIZER, TERRE HAUTE, IN
item Stabel, Judith

Submitted to: American Dairy Science Association Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: June 20, 1999
Publication Date: N/A

Technical Abstract: Impaired immune function in periparturient dairy cows contributes to the increased susceptibility of dairy cows to infectious disease. We established a flow cytometric method using a combination of the Ca indicators, Fluo-4 and Fura-Red, to analyze changes in [Ca2+]i following stimulation of peripheral blood mononuclear cells (PBMC), CD8 positive cells, and polymorphonuclear cells (PMN) of cattle. Fluo-3/Fura-Red ratios have been used in humans and rodents as a sensitive index of [Ca2+]i changes. Fluo-4 is an analog of Fluo-3 with higher fluorescent intensity than Fluo-3. To test the method, we used anti-CD3 mAb (murine igG1) in combination with anti-murine Ab 92nd Ab) to stimulate bovine PBMC, and we were able to measure a 2.0 +/- 0.2 fold increase in Fluo-4/Fur ratio in PBMC and a 2.7 +/- 0.2 fold increase in CD8 positive cells from nonpregnant, nonlactating (NPNL) dairy cows (n=5). We then applied this assay to immune cells in periparturient Jersey cows (n=8) in comparison with age matched NPNL Jersey cows (n=9). We saw a decrease in peak response (fold change from baseline to Fluo-4/Fura-Red ratio) in PBMC in periparturient cows compared to NPNL cows stimulated by anti-CD3 mAb + 2nd Ab (2.57 +/- 0.12 in NPNL vs. 1.84 +/- 0.07 in periparturient cows). Similar decreases were seen in PBMC, PMN, and CD8 positive cells when stimulated by H2O2 and other stimulants. Periparturient immunosuppression is associated with reduced ability to increase [Ca2+]i in response to stimulation by a variety of stimulants. This may represent impaired signal transduction in immune cells. Intravenous infusion of Ca solution into milk fever cows (n=3) restored function of immune cells showing increase in [Ca2+]i above that of NPNL in response to various stimuli.

Last Modified: 10/21/2014
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