Fig 4. The effect of bbK32 deletion in an infectious strain background on vascular transmigration and clearance in Cd1d ^-/- mice.

A) GFP-expressing B. burgdorferi strains, infectious (GCB966), a bbk32 deletion strain (GCB971) and a high-passage non-infectious strain (GCB706) were injected into the tail vein of Cd1d ^-/- mice (n = 6/group, 4x10⁸ spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated or Alexa Fluor 555-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05). B) Concentrations of B. burgdorferi in mouse plasma after iv inoculation. Cd1d ^-/- mice were injected with B. burgdorferi through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05).