Characterization of MPEC P4-NR O15:H21H54 and sub strain P4-96 in the murine mastitis model. Mammary virulence of P4-NR and attenuation of P4-96 are demonstrated in lactating C57BL/6 mice following IMM challenge of L4 and R4 glands with ~1000 CFUs of bacteria. P4-NR and P4-96 were inoculated as single strains or co-inoculated at a 1:1 ration in competition study. 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. Mammary colonization by P4-96 was significantly reduced in C57BL/6 (A) mice and reduced fitness of strain P4-96 is demonstrated after co-challenge studies in C57BL/6 mice where P4-NR outcompeting P4-96 in the mammary gland (B), competition index significantly < 1. Massive neutrophil recruitment into alveolar milk space is demonstrated in representative microscope images of H&E stained paraffin sections (P4-NR; top panels in C, P4-96; lower panels in C). Neutrophil recruitment was also quantified by measuring the relative expression of Ly6G gene in mammary tissues using qPCR (black markers for P4-NR and blue markers for P-96 in D). Inflammation was further quantified by measuring the relative expression of the following genes in mammary tissues; KC, MIP2, TNFα, IL-1β, IL-10 and iNOS (D). Glands with high bacterial counts (> 106 CFUs/gr tissue; filled markers in D) show consistently higher expression of inflammatory markers than glands with low counts (< 106 CFUs/gr tissue; open markers). Relationships between bacterial counts and inflammatory variables, which include cytokines, chemokines and Ly6G, were further visualized using non-metric multidimensional scaling (E). Scale bars; 100 µm (left panels in C) and 20 µm (right panels in C).