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. 2022 Jan 17;11:773938. doi: 10.3389/fcimb.2021.773938

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Copyright © 2022 Wherry, Dassanayake, Casas, Mooyottu, Bannantine and Stabel

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nitrite production by monocyte-derived macrophages (MDMs) co-cultured with peripheral blood mononuclear cells (PBMCs) from naturally infected dairy cattle (clinical n=7, subclinical n=7) or noninfected controls (n=9). Peripheral blood mononuclear cells (PBMCs) isolated from whole blood were cultured 5-6 days to generate MDMs, pre-treated with vitamin D₃ presented in methods, then incubated 24 hrs with live MAP (10:1 MOI) ± 25(OH)D₃ or ± 1,25(OH)₂D₃. Nitrite concentrations were determined from cell culture supernatants by competitive binding of sulfanilamide and NED solutions in the Griess assay. Absorbance was measured at 540 nm on a SpectraMax microplate reader. Data are presented as the mean ± SE and significance is defined by *< 0.05, **< 0.01, ***< 0.001.