Figure 3
MPEC P4-96 strain is attenuated in mammary colonization, fitness and virulence. Mammary virulence of P4-NR and attenuation of P4-96 were demonstrated in lactating C3H/HeN mice 24 h after IMM challenge of L4 and R4 glands with 104 CFUs of bacteria. 24 h after challenge mammary tissues were harvested for bacterial counts (scatter plot in A), each data point represents a single gland, and the horizontal bars indicate the median of data from three or more independent experiments. Statistical significance was determined by non-parametric Mann–Whitney two-independent-samples test (A) using GraphPad Prism 6 (GraphPad Software, Inc.) and P value of 0.05 or less was considered significant. Mammary colonization by P4-96 was significantly reduced in C3H/HeN mice (A). Massive neutrophil recruitment into alveolar milk spaces was only elicited following challenge with P4-NR strain and is demonstrated in representative microscope images of H&E stained paraffin sections (B–C). Attenuated disease was also demonstrated using whole body imaging 24 h after IMM challenge with 104 CFUs P4-NR (D–E) or P4-96 (F) bacteria expressing the fluorescent reporter mCherry. Fluorescence and bioluminescence imaging was performed using IVIS Lumina Series III (PerkinElmer Inc., MA, USA) in live mice (D; top panels and E; left panels) and of exposed glands in euthanized mice (D; bottom panels and E; middle and right panels). Each image is displayed as a false-color photon-count image superimposed on a grayscale anatomic image. Mammary colonization by P4-NR is widespread and the fluorescence signal is correlated with the high bacterial counts in the mammary tissue (D; left panels and E; left and middle images). Mammary virulence of P4-NR was correlated with neutrophil recruitment to the mammary glands quantified by bioluminescence imaging of myeloperoxidase (MPO) upon systemic administration of luminol (D; right panels) and visible decrease in milk production in challenged glands (E; right bottom panel). P4-96 attenuation is demonstrated by focal colonization (F; left and middle images) and no visible decrease in milk production (F; right image). Representative images of ≥ 6 mice challenged by each bacterial strain. Scale bars; 100 µm (B) and 20 µm (C).