Isolation and in vitro cultivation of Babesia venatorum (Apicomplexa: Babesiidae), a zoonotic hemoprotozoan from Ixodes persulcatus ticks in China

The emerging Babesia venatorum upsurges as a potential health threat occurring in China and other endemic countries. Few attempts to isolate and culture the Babesia species had been conducted in vitro. We collected the questing Ixodes persulcatus from identified endemic areas and allowed them to feed on the severe combined immunodeficiency (SCID) mice. The positive mice were chosen to provide positive erythrocytes with asexual B. venatorum for continuous culture in mouse or human erythrocytes in vitro, with RPMI 1640 medium and appropriate serum. With B. venatorum in the SCID mice, erythrocytes were cultured in vitro for confirmation by morphological observations with transmission electron microscopes. Sequences of B. venatorum were then identified by way of conventional PCR amplification. Parasitemia counts monitored the growth of B. venatorum on thin blood smears and real-time quantitative PCR in parallel. As expected, B. venatorum positive mice were harvested by sufficient attacks of I. persulcatus ticks. The erythrocytes of the infected mice were then inoculated and successfully cultured in donated erythrocytes from humans and mice in RPMI 1640 culture medium. Active growth of B. venatorum was well demonstrated in human erythrocytes with 3.3 times parasite-load when compared with a mouse under similar conditions. With the increased subcultures, a prolonged period of detectable parasitemia with much higher peak parasitemia and shorter time to reach peak parasitemia were observed in the following subcultures. A new strategy for isolation and in vitro culture of B. venatorum has been provided with a continuous supply of sufficient pathogens to satisfy human babesiosis's testings and clinical therapies.