Rabbit serum

Two Borrelia species, B. burgdorferi and B. garinii, were tested in their three morphological forms: spirochetes, rounded forms, and biofilm. As Borrelia sp. are aero‐tolerant anaerobes, they were cultured stationary in the presence of 5% CO₂ in screw‐capped tubes. Low passage isolates of the B31 strain of B. burgdorferi and CIP103362 strain of B. garinii were obtained from the American Type Culture Collection (Manassas, VA). Stocks of both species were cultured in commonly applied conditions, i.e. Barbour‐Stoner‐Kelly H (BSK‐H) medium, supplemented with 6% rabbit serum (Sigma, St. Louis, MO) without antibiotics at 33°C with 5% CO₂, in sterile screw‐capped 15 ml polypropylene test tubes with or without gentle shaking. B31 strain is an isolate from Ixodes dammini, whereas CIP103362 strain is an isolate from Ixodes ricinus.

4^′,6-Diamidino-2-phenylindole (DAPI) (Cat#D9542), rat serum (Cat#R9759), and rabbit serum (Cat#R9133) were purchased from Sigma-Aldrich. Aqua live/dead was from ThermoFisher Scientific (Cat#L3495), UV Zombie was from BioLegend (Cat#423107), and Ammonium-Chloride-Potassium (ACK) lysis buffer was from Lonza (Cat#10-548E).

Samples on filter papers were examined by a microscopic agglutination test (MAT) [44 (link)]. Eight serovars of Leptospira interrogans sensu lato (Lisl) (Grippotyphosa, Icterohaemorrhagiae, Bratislava, Canicola, Sejroe, Sorex jalna, Pomona, and Pyrogenes), belonging to serogroups Grippotyphosa, Icterohaemorrhagiae, Australis, Canicola, Sejroe, Javanica, Pomona, and Pyrogenes, respectively, represent the most prevalent Leptospira serovars in Europe [6 (link)]. These serovars were stored long-term in liquid nitrogen at a temperature of −196 °C. Before use, ampoules with Leptospira cultures were thawed and cultured at 28 °C in commercial media (Ellinghausen–McCullough–Johnson–Harris or HIMEDIA Leptospira HiVeg Medium Base, Korthof Modified, REF MV457Z, Test Line, Brno, Czech Republic), with the addition of 10% rabbit serum (Sigma Aldrich, Prague, Czech Republic), to the concentration of approximately 2 × 10⁸ leptospires per ml. The density of Leptospira cultures was determined using a Petroff–Hausser counting chamber after 5–7 days of cultivation. Cultures in concentrations of approximately 2 × 10⁸ leptospires per ml were used for MAT. The degree of agglutination (reaction of antigen with antibodies) was evaluated by dark-field microscopy. Samples were marked positive if more than 50% of Leptospira appeared to be agglutinated. Samples with titres ≥100 were considered positive.

B. burgdorferi strain B31 was cultured in BSK-H medium (HiMedia Laboratories Pvt. Ltd.) with 6% rabbit serum (Sigma-Aldrich, St. Louis, MO, USA). All culture medium was filter-sterilized by 0.2 μm filter. Cultures were incubated in sterile 50 ml conical tubes (BD Biosciences, CA, USA) in microaerophilic incubator (33°C, 5% CO₂) without antibiotics.

All B. burgdorferi s.l. strains used for panels I and II were cultured at LCM, Sweden. The strains were kindly provided by Professor Sven Bergström, Umeå University, Sweden. One mL of each strain was cultured in 14 mL Barbour-Stoenner-Kelly (BSK) II medium [22 (link)] supplemented with 6% rabbit serum (Sigma Aldrich, St. Louis, Missouri, US). B. afzelii Lu81 was cultured at 35°C for 8 days, B. garinii Lu59 at 37°C for 7 days and B. burgdorferi s.s. B31 at 35°C for 6 days. The spirochetes were counted by phase-contrast microscopy. A 10-fold dilution series ranging from 2000 to 0.2 spirochetes μL^-1 was prepared in RNase-free water (GE Healthcare Life Science, Chicago, Illinois, US) for each strain. These dilutions were used to spike samples in panels I and II. The samples were spiked and aliquoted immediately after the dilution series was prepared. The samples for panel I were extracted as described below, and the samples for panel II were placed at -80°C.