Figure 5.

The dual luciferase assay quantitates known patterns of B. burgdorferi transcript expression in nymphs. (A) Groups of eight fed nymphs per B. burgdorferi clone were surface sterilized and crushed in 250 μl of PBS and the in vivo tick luciferase assay performed with 100 μl of tick extract and 700 μM D-luciferin or 3.5 mM coelenterazine. The data are presented as the mean relative Fluc_Bb units per 10⁸ relative Rluc_Bb units ± standard deviation (n = 3; B. burgdorferi carrying pCFA803 n = 2). (B) Relative Rluc_Bb luciferase activity is reflective of spirochete numbers in extracts from fed infected nymphs. 1 μl of fed nymph extract was plated for CFUs in solid BSKII containing RPA cocktail and 50 μg/ml streptomycin. The number of CFUs/100 μl of tick extract (CFUs) was plotted against the relative Rluc_Bb units for the same extract. The red dotted line indicates the established LoQ for relative Rluc_Bb units. Black symbols represent extracts with quantifiable relative Rluc_Bb units and red symbols represent extracts with non-quantifiable Rluc_Bb units. A nymph extract with no detectable CFU (CFU = 0) is represented as the data point on the Y-axis. A significant positive correlation was detected between CFU and relative Rluc_Bb units (Pearson coefficient, r = 0.8022, p = 0.0017).