Eclipse e600 darkfield microscope

Treponema pallidum subsp. pallidum (Nichols strain) was propagated in male specific pathogen-free (SPF) New Zealand White rabbits as described elsewhere^{26 (link)}. The animal study was reviewed and approved by the local institutional review board at the University of Victoria under protocol 2020-024, and was conducted in strict accordance with standard accepted principles as set forth by the Canadian Council on Animal Care, National Institutes of Health and the United States Department of Agriculture in a facility accredited by the Canadian Council on Animal Care and the American Association for the Accreditation of Laboratory Animal Care. For extraction, treponemes were harvested from the testes of rabbits approximately 10–12 days after infection in sterile saline (0.9% w/v NaCl, pH 7.0) in the presence or absence (refer to method development section below for details) of 10% normal rabbit serum (NRS). Extractions were performed in an anaerobic chamber (Coy Laboratories, Grass Lake, MI, USA) at room temperature in an atmosphere of 1.5–3% O₂ and 5% CO₂, balanced with N₂. Rabbit cells and debris were separated and removed as described below. Treponemes in suspension were enumerated using a Nikon Eclipse E600 darkfield microscope (Nikon Canada, Mississauga, ON, Canada).

Micrographs were captured with a Nikon Eclipse E600 darkfield microscope (Nikon Canada, Mississauga, Ontario, Canada) with a ×40 objective (×400 magnification) or a Cytation 5 Imaging Reader (BioTek) with a ×20 objective (×200 magnification). As previously described (Lithgow et al., 2020 (link)), for T. pallidum adhesion assays, samples were blinded and the numbers of endothelial nuclei (DAPI; 10–60 μm) and T. pallidum cells (FlaA; 2–8 μm) were measured from each field of view with the Cytation 5 software using size exclusion quantification settings (BioTek). Mean fluorescence intensity of VE-cadherin was quantified using the Cytation 5 Imaging Reader (BioTek) with a ×20 objective at five random fields of view per well.

Naïve control rabbits were sedated and bled 10 days before challenge to confirm negative VDRL reactivity (Supplementary Table 2). At 25 days after the final immunization and 10 days after control animals were bled, the rabbit's backs were shaved, cleansed with 70% ethanol and injected intradermally with 0.1 ml of 1 × 10⁷T. pallidum subsp. pallidum (Nichols strain) per ml in 0.9% saline, in each of 10 spots. Challenge sites were monitored daily for erythema, induration and painless ulceration and were measured daily to assess lesion diameter. Challenge sites from control and immunized animals were evaluated for the presence of motile T. pallidum 10 days PC by syringe aspiration using a 26-gauge needle; aspirates were examined in a blinded manner for the presence of T. pallidum using a Nikon Eclipse E600 darkfield microscope (Nikon Canada, Mississauga, ON, Canada).