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Title: Comparative evaluation of assays for the measurement of bovine neutrophil oxidative burst activity

Author
item RINALDI, MANUELA - UNIVERSITY OF MILAN
item MORONI, PAOLO - UNIVERSITY OF MILAN
item Paape, Max
item Bannerman, Douglas

Submitted to: Conference Research Workers Disease Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/30/2006
Publication Date: 12/3/2006
Citation: Rinaldi, M., Moroni, P., Paape, M.J., Bannerman, D.D. 2006. Comparative evaluation of assays for the measurement of bovine neutrophil oxidative burst activity. Proceedings of the 87th Annual Meeting of the Conference of Research Workers in Animal Diseases. p. 118, Dec. 3-5, 2006, Chicago, IL

Interpretive Summary:

Technical Abstract: During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. Phorbol 12-myristate, 13-acetate (PMA) and zymosan were used to induce neutrophil activation and corresponding ROS production. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.