ABSTRACTThe establishment ofin vitroculture methods has greatly facilitated the research ofBabesia. However, the currentBabesia gibsoniin vitroculture 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 ofB. 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 maintainB. gibsonigrowth 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 ofBabesiaand 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 toB. gibsonicontinuous long-term, expansion culture, and subclone culture.IMPORTANCEThe VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuousin vitroculture ofBabesia gibsoniat 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 ofin vitroculture systems allows researchers to better understand the metabolism and growth patterns ofBabesia. Importantly, several technical problems impeding such studies have been overcome.