Titration and Inoculation of Borrelia burgdorferi

B. burgdorferi strain B31-A3 organisms were kindly provided by Dr. Jenifer Coburn (Medical College of Wisconsin). Low-passage (<10) spirochetes were grown in modified Barbour-Stoenner-Kelly (BSK)-H complete medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) at 32 °C until they reached a concentration of 10⁶ microbes/mL before being stored at −80 °C in 1.5 mL screw-top tubes. Aliquots were incubated in BSK medium for approximately 5 days, washed of the media, and, as described previously [11 (link)], resuspended in phosphate-buffered saline (PBS) supplemented with heat-inactivated normal mouse serum (NMS) at a concentration of 0.2%. Groups of wild-type and DEREG BALB/c mice were anesthetized with isoflurane and then injected subcutaneously between the scapulae with B. burgdorferi at doses ranging from 1 × 10² to 1 × 10⁵ organisms in a volume of 50 µL of PBS supplemented with NMS. Uninfected mice were injected with PBS supplemented with NMS alone. The motility of B. burgdorferi was confirmed using dark-field microscopy. Organisms were enumerated using a Petroff-Hausser counting chamber.
In a preliminary study, we infected wild-type mice with 1 × 10³, 1 × 10⁴, or 1 × 10⁵ B. burgdorferi organisms in PBS supplemented with NMS, or with PBS supplemented with NMS alone, to determine the dose of infection to be used in our Treg cell depletion studies. We measured tibiotarsal joint swelling (described below) over the course of 5 weeks as an indication of pathology. Based on this, we used a range of doses of infection (1 × 10²–5 × 10³ organisms) that would lead to edematous changes of the joint such that any anticipated increases in swelling due to Treg cell depletion would still be apparent.