CONTINUOUS IN-VITRO CULTURE OF BABESIA DIVERGENS IN A SERUM-FREE MEDIUM

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplement ed with 10 % human serum (RPMI-10 % HS) with a high percentage of para sitized erythrocytes (PPE) (greater than or equal to 40 %). Standardiz ation of in vitro tests, purification of exoantigens, biochemical stud ies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3 % routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The Supplementation of basal medium with lipidic carrier (Albumax I o r bovine serum albumin-Cohn's fraction V) promoted the growth of B. di vergens with high PPE (> 30 %) close to those obtained in RPMI-10 % HS . Neither protein nor lipid fractions alone were able to restore the g rowth of B. divergens. Nevertheless, the whole lipid fraction from ser um or Albumax I added to delipidated albumin partially restored the gr owth (7 % PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Alb umax I can replace human serum offering the advantages of safety, stan dardization for chemosensitivity tests, and exoantigen purification.