OspC-deficient B. burgdorferi persists within the I. scapularis gut but fails to invade the salivary gland during feeding. (a) Detection of B. burgdorferi mRNA within guts of unfed or engorged I. scapularis which were microinjected with wild-type spirochetes (lane 1), OspC-deficient B. burgdorferi (lane 2), or OspC-deficient spirochetes that were complemented with a plasmid expressing ospC (lane 3). Injected unfed nymphs were analyzed by RT-PCR for the detection of viable B. burgdorferi at 7 days after injection or 24–72 hours after the onset of feeding. Equal amounts of total RNA from ticks (25 ticks/group) were converted to cDNA with reverse transcriptase, subjected to PCR with flaB primers, and analyzed on a 1.5% agarose gel. I. scapularis β-actin was used as a control to confirm equal loading of total RNA isolated from infected ticks. An aliquot of prepared RNA from each group was subjected to RT-PCR in the absence of reverse transcriptase to confirm the absence of genomic DNA (data not shown). (b) Distribution of microinjected wild-type B. burgdorferi (WT), OspC-deficient B. burgdorferi (OspC-deficient), and OspC-deficient B. burgdorferi complemented with a plasmid carrying the ospC gene (OspC-complemented) within I. scapularis gut at 24 or 72 hours of feeding. The spirochetes (arrows) were stained with FITC-labeled goat anti–B. burgdorferi (shown in green), and the nuclei of the gut epithelial cells were stained with propidium iodide (shown in red). Images were obtained at ×400 magnification and are presented as merged images for clarity. (c) Migration of microinjected spirochetes from feeding gut, as shown in b, to salivary glands at 24–72 hours of feeding. Salivary glands were invaded by wild-type and OspC-complemented B. burgdorferi (arrows); no spirochetes were detected within the salivary glands of ticks that were microinjected with OspC-deficient B. burgdorferi. Images were obtained at ×400 magnification and are presented as merged images for clarity (n = 3).