Establishment of Continuous In Vitro Culture of Babesia gibsoni by Using VP-SFM Medium with Low-Concentration Serum

ABSTRACT The establishment of in vitro culture methods has greatly facilitated the research of Babesia . However, the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum, which intensively limits the culture and is unable to satisfy the demands of long-term studies. In this study, AlbuMAX I (2 mg/mL) and 2.5% dog serum (vol/vol) were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD (2.5%), and the effectiveness of this medium was assessed by the growth of B. gibsoni . The results showed that VP-SFMAD (2.5%) could support the continuous growth of the parasite, and the parasitemia has no difference with the cultivation in RPMI 1640 with 20% dog serum. In contrast, either a low concentration of dog serum or absence of AlbuMAX I will significantly lower the parasite growth or fail to maintain B. gibsoni growth in the long term. The strategy of reducing the hematocrit was also evaluated, and VP-SFMAD (2.5%) improved the parasitemia to over 50% within 5 days. The high parasitemia is helpful for larger numbers of parasite collection, which is valuable for studying the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites. In addition, VP-SFMAD (2.5%) medium was successfully used for monoclonal parasite screening, which obtained monoclonal strains with parasitized erythrocytes about 3%, which is similar to RPMI-1640D (20%) medium that obtains monoclonal strains on the 18th day. Those results showed that VP-SFMAD can be applied to B. gibsoni continuous long-term, expansion culture, and subclone culture. IMPORTANCE The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia . Importantly, several technical problems impeding such studies have been overcome.