Figure 5
Resistance to complement is an essential virulence trait in mammary pathogenicE. coli. Loss and rescue of mammary virulence were demonstrated in normal and decomplemented C3H/HeN mice, respectively. Normal mice (left and middle columns in A and panel B) or mice decomplemented by IP injection of cobra venom factor (CVF; right column in panel A and panel C) well challenged by IMM infusion of L4 and R4 glands with 104 CFUs of complement-sensitive rough P4-NR∆galU and P4-NR∆galE strains. 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. Loss of virulence (B) and rescue (C) were also demonstrated using whole body imaging 24 h after challenge for bacterial growth (mCherry fluorescence; middle columns in B and C), neutrophil recruitment (luminol bioluminescence; left columns in B and C) and milk in the glands (autofluorescence; right columns in B and C). Fluorescence and bioluminescence imaging was performed using IVIS Lumina Series III (PerkinElmer Inc., MA, USA) in live mice (top panels in B and C) and of exposed glands in euthanized mice (bottom panels in B and C). Each image is displayed as a false-color photon-count image superimposed on a grayscale anatomic image. Bacterial growth, neutrophil recruitment and reduced milk were not visible following challenge with galU mutant strain (B). Mammary colonization (A and C) and virulence (C) of P4-NR∆galU bacteria were regained in decomplemented mice demonstrated by bacterial counts and whole body imaging. Representative images of ≥ 6 mice challenged by each bacterial strain.