4.2. Babesia duncani Culture In Vitro

Babesia duncani strain WA1 (ATCC^® PRA-302™) was cultured in vitro in HaRGM medium as described by Abraham et al. (2018) with slight modifications [14]. Briefly, 500 μL of cryopreserved culture was used to infect hamsters through intraperitoneal injection and monitor blood parasitemia every day. When the parasitemia reached approximately to 10%, blood was collected by cardiac puncture after isoflurane anesthesia. Washed, infected RBCs (10 μL) were mixed with 90 μL of fresh hamster uninfected RBCs and resuspended in 1.2 mL warm HaRGM and then dispensed into a single well of a 24-well plate. Specifically, 1.2 mL of medium had a depth of 0.7 cm in the 24-well plate for generating a microaerophilic condition [54]. The plate was inoculated at 37 °C under an atmosphere of 5% CO₂ with 95% humidity. The overlying medium was removed and replaced with fresh HaRGM daily. When the RBCs laid on the bottom turned dark and black, 10 μL of infected RBCs of which parasitemia had been confirmed by Giemsa stain was mixed with 90 μL of fresh uninfected RBCs then cultivated into a new well of the 24-well plate.