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United States Department of Agriculture

Agricultural Research Service

Title: Assay for Proliferation of Porcine Pbmc with Oregon Green 488 and Its Application for Evaluation of T Cell Responsiveness to Swine Influenza Virus

item Kimura, Kayoko - IOWA STATE UNIVERSITY
item Vincent, Amy
item Lager, Kelly
item Kehrli Jr, Marcus

Submitted to: Journal of Immunology
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2006
Publication Date: April 1, 2006
Citation: Kimura, K., Vincent, A.L., Lager, K.M., Roth, J.A., Kehrli, Jr., M.E. 2006. Assay for proliferation of porcine PBMC with Oregon Green 488 and its application for evaluation of T cell responsiveness to swine influenza virus [astract]. Journal of Immunology Supplement. 176:S118. Paper No. 59.12.

Technical Abstract: Cell proliferation assays are a useful method to analyze cell-mediated immunity (CMI) to microbial antigens. In order to define T cell responsiveness to virus antigens in pigs, we have developed flow cytometric assays for porcine peripheral blood mononuclear cell (PBMC) proliferation using Oregon Green 488, an analog of carboxy-fluorescein diacetate, succinimidyl ester. Oregon Green 488 dye dilution and 5-bromo-2-deoxyuridine incorporation were very well correlated (r = 0.7307) after stimulation of porcine PBMC to undergo mitosis. We applied this technique together with intracellular IFN-gamma, and CD25 cell surface expression assays in order to determine recall responses of PBMC to swine influenza virus (H1N1) (SIV) antigen after vaccination (pigs primed and boosted with a killed virus vaccine, 3 weeks apart) and challenge (2 weeks after the second vaccination). All three parameters increased with in vitro virus stimulation of PBMCs obtained from vaccinated pigs at five days following live SIV challenge but not in unchallenged control pigs. Vaccinated and challenged pigs had the strongest CMI responses whereas pigs that were either vaccinated or challenged with live virus alone, had weaker responses. Pigs challenged without vaccination had the lowest expression of CD25 at five days after challenge. These techniques are useful to evaluate antigen specific CMI responses in pigs to understand pathogenesis and protective immunity after vaccination and/or infection with SIV.

Last Modified: 4/22/2015
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