ABSTRACT. Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O 2 , 5% CO 2 and 93% N 2 ). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO 2 in air, with parasitemia ranging from 8-10%. -KEY WORDS: Babesia caballi, in vitro cultivation.
Babesia caballi cultured continuously in equine erythrocytes was examined by transmission electron microscopy. The use of cultured B. caballi permitted examination of a large number of parasitized cells with various stages of intra erythrocytic development. The piriform merozoites of B. caballi were composed of an outer membrane and an inner double-membrane complex. Numerous micronemes and three rhoptries were found in the pellicle of the merozoite, and a spherical body was seen in the anterior part of the merozoite which usually lay adjacent to the nucleus and the pellicle. These findings were similar to those for merozoites of bovine Babesia parasites such as B. bigemina. The trophozoites were surrounded by a single membrane, were continuously changing their body shape with extension and retraction of the pseudopod. A long pseudopod extended far into the host cell cytoplasm, and was finally completely enclosed in a cell, but did not have hemozoin pigment, the breakdown product of hemoglobin digestion.
In-vitro-propagated Babesia caballi parasites were examined by scanning and transmission electron microscopy. Many small pores were observed over the entire surface of infected erythrocytes on scanning electron microscopy, and on transmission electron microscopy these small pores were found to be openings of tubular structures. By the examination of a number of infected cells the tubular structures were found to be connected with the parasite, and this observation might indicate that the tubular structures arose the edge of the parasite and terminated at an Invagination on the surface of the erythrocyte. These findings suggest that intraerythrocytic stages of B. caballi come into direct contact with culture medium.
Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.
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