Bacterial strain
A single B. bacilliformis strain (ATCC#35685) and a recently isolated strain from an acute phase patient (IMTAVH#00032) was used in this study. B. bacilliformis stocks were grown on 10% sheep blood Columbia Agar flasks at 29°C. Bacteria were harvested in a laminar flow hood, scraping colonies off the agar surface into brain heart infusion (BHI) broth media. The cells were collected by centrifugation and suspended in phosphate-buffered saline solution (PBS). Colony-forming units (CFU) of harvested Bartonella cultures were estimated using the Mc Farland turbidity scale (0.5 mL of tube 0.5 of Mc Farland scale ~1.5 × 108 CFU/mL).
Inoculations
B. bacilliformis was prepared as described above, and thawed on ice prior to inoculation. Forty six BALB/c mice (10–12 weeks old) were anaesthetized with ketamine and administered live B. bacilliformis by the routes indicated as described below. Additionally, forty six BALB/c control mice were inoculated with sterile saline.
For intraperitoneal (i.p.), intradermal (i.d.) and subcutaneous (s.c.) immunization, mice were dosed with volumes of 200 – 500 μL containing around 1.5, 3, or 4.5 × 108 CFU of B. bacilliformis administered using a tuberculin syringe and needle. For oral immunizations, mice were dosed by using a 22-gauge gavages (force feeding) needle attached to a 1 mL syringe. For intranasal (i.n.) immunizations, mice were dosed with volumes of 10–50 μL containing around 1.5 × 108 CFU of B. bacilliformis administered into the nostrils using a micropipette fitted with a 200 μL tip. Following immunizations, mice were observed daily for signs of illness.
Animals were sacrificed at different times up to day 60 post-infection. Sections of formalin-fixed liver, spleen, lung, kidney, brain, and lymph node tissue were stained with hematoxylin and eosin.
Detection of B. bacilliformis in vivo
From all mice, bacterial loads in liver, spleen, lung, kidney, brain and lymph nodes were determined by plating of 10-fold serial dilutions of organ homogenates on Columbia sheep blood agar. In addition, quantitative cultures of blood specimens were performed at the time of necropsy. Before samples were plated on Columbia agar, erythrocytes were lysed either by freezing and thawing or by vigorous mixing with distilled water. All cultures were incubated at 27°C for at least 4 weeks before being recorded as negative for growth.
Additionally, 4 i.d. immunized mice were subjected to intradermal skin testing with B. bacilliformis (using the harvested ATCC#35685 strain in 2 mice and the harvested IMTAVH#00032 strain in the other 2), to monitor delayed swelling reactions as an indication of specific immune induction. The mice were boosted i.d. with 3 × 108 CFU on day 50 after primary i.d. inoculation. One week after the boost, each mouse was challenged in the right dorsal skin with the same number of bacteria. The injection site was monitored at 24, 48, and 72 hours for swelling responses and compared to the control skin as an external negative control. Swelling was determined from three readings using a caliper.