A BspR-deficient strain is hyper-virulent against cultured mammalian cells.

<p>(<b>A</b>) L2 cells were infected with the indicated <i>B. bronchiseptica</i> strains at an MOI of 20 for 20 min. Infected cells were subjected to Giemsa staining and were analyzed under a light microscope. (<b>B</b>) HeLa cells were infected with the indicated <i>B. bronchiseptica</i> strains at an MOI of 10. The amount of LDH released into the extracellular medium from infected cells was quantified at 0, 60, 120, and 180 min post-infection, and the relative cytotoxicity (%) was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038925#s4" target="_blank">Materials and Methods</a> section. The data for each sample are presented as the mean ± SE of three independent experiments. ***, P<0.001. (<b>C</b>) RBCs were tightly pelleted with the indicated <i>B. bronchiseptica</i> strains by centrifugation and incubated for 30 min. After incubation, the release of hemoglobin from the RBCs into the extracellular buffer was calculated by measuring the absorbance at 492 nm. The presented values are percentages of Triton X-100-lysed RBCs after subtraction of the measured lysis of untreated cells. The data for each sample are presented as the mean ± SE of three independent experiments. *, P<0.05; NS, no significance. (<b>D</b>) C57BL/6J mice were infected intranasally (5×10⁶ CFU/mouse) with a wild-type or Δ<i>bspR</i> strain. The survival of the mice (n = 4 per group) was monitored for 8 days after infection. *, P<0.05 using the log-rank test.