A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass culture-derived exoantigens

The supernatants of in vitro cultures of Babesia divergens Rouen 1987 in human erythrocytes, obtained by using a semidefined medium based on human high-density lipoproteins, were fractionated by gel filtration chromatography into four fractions, F1 to F4. The crude supernatant as well as each fraction adjuvanted with Quil-A protected gerbils from mortality due to a homologous infectious challenge. Analysis of the humoral response of the 10 protected gerbils with fraction F4, containing major proteins with molecular masses lower than 50 kDa, showed that a few antigens (from 50 to 17 kDa) could be important candidates for an improved vaccine against B. divergens babesiosis. As an immunodominant response was directed against the 37-kDa antigen (Bd37) in two different B. divergens strains tested, a polyclonal antibody directed against Bd37 was produced in a rabbit. In an immunofluorescence assay, the anti-Bd37 antiserum strongly labelled small internal vesicles of the merozoites and the cell surface was diffusely labelled after fixation, whereas on live merozoites, this labelling was not observed. 3H]glucosamine-radiolabelling experiments demonstrate that Bd37 is a glycoprotein. The Bd37 protein can also be labelled with 14C]palmitate but not with 3H]myristic acid. In Triton X-114 temperature phase partitioning of B. divergens-infected erythrocyte extracts, Bd37 was exclusively found into the detergent phase, indicating that the palmitoylated Bd37 protein was in the membrane fraction. In the in vitro supernatant, the glycoprotein Bd37 was found in a nonpalmitoylated form, indicating excretion and/or release of the glycoprotein from the merozoite.