Figure 4.

Live-imaging of luciferase-expressing B. burgdorferi strain N40D10/E9. In vitro assay was first performed to select the most suitable solvent to be used. (A) d-Luciferin substrate was diluted either in PBS or in methanol in Eppendorf tube to select the best solvent to detect bioluminescence. (B) Examination of two luciferase-expressing N40 concentrations added to the Eppendorf tubes, 10⁴ and 10⁶ spirochetes, with the substrate. In vivo assay: (C) Measurement of luciferase-expressing B. burgdorferi ss N40 strain injected in C3H/HeN mice at 7 days post-infection (natural peak of Borrelia multiplication-3 mice were selected) or 90 days (clobetasol reactivation—mouse#4) post-infection. The intradermal injection site of luciferase-expressing Borrelia is marked by an asterisk. (D) Quantification of luciferase-expressing N40 strain in the skin of C3H/HeN mice at day 55: the site of Borrelia inoculation was always at the lower back of mice, but the clobetasol reactivation was either “on site” of inoculation or “at distance”. Infected but non-reactivated mice were included as controls. The skin was collected either from the back (site of inoculation) or from the ear (distant skin from the inoculation site). Different groups were compared by a Mann–Whitney test and p values relative to control mice from left to right for back are: 0.01 and 0.01, and for ear are: 0.2 and 0.01. (E) The study was completed by culture of other tissues at different time points: heart, right joint, skin at the site of inoculation (back) and skin at a distant site (ear). The clobetasol application site is marked by an ‘R’ for reactivated mice (Day 55 and 95) or ‘NR’ for infected skin but ‘not reactivated’. “%” positive in this table was calculated using the number of mouse organ from which Borrelia could be recovered by culture with respect to the total number of samples cultured and examined.