Mycobacterium bovis AN5 strain induces caspase-3-independent DNA fragmentation in bovine macrophages. Bovine macrophages (1.2 × 106) were either infected (MOI 10:1) with M. bovis AN5 strain or camptothecin-treated (25 µg/mL) during 16 h. A After infection, DNA fragmentation was analyzed by TUNEL (BrdU-FITC), the experiment is a representative FACS plot of TUNEL result. The results are expressed as total cell counts versus FITC signal in log10 scale. B Quantification of DNA fragmentation in A, bars represent the mean fluorescence intensity (MFI) fold change of three independent experiments of M. bovis-infected and camptothecin-treated macrophages with respect to uninfected cells. One-way ANOVA showed significant variation of MFI in infected versus uninfected cells (P < 0.05). C Caspase activity was evaluated by a fluorogenic technique using a caspase three specific substrate (Ac-YVAD-AMC). Macrophages were incubated under each of the following conditions: camptothecin (25 µg/mL) (positive control), CRPMI medium (negative control), M. bovis (MOI, 10:1) 4, 8, and 16 h. The results are expressed as fluorescence emitted per minute during 15 min per 200 µg of protein and are mean ± standard deviation from two independent experiments, each with three internal replicas. There was a statistical significance difference (p ≤ 0.05) as early as 3 min after the substrate addition. Statistical difference persisted across the experiment.