The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) (> or = 40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies 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 or bovine serum albumin-Cohn's fraction V) promoted the growth of B. divergens with high PPE (> 30%) close to those obtained in RPMI-10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

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