Characterisation of erythrocyte invasion by Babesia bovis merozoites efficiently released from their host cell after high-voltage pulsing

Apicomplexa are a phylum of obligate intracellular parasites critically dependent on invasion of a host cell. An in vitro assay for erythrocyte invasion by Babesia bovis was established, employing free merozoites obtained after the application of high-voltage to the parasitised erythrocytes. The invasion proceeds efficiently in phosphate-buffered saline solution without the requirement for any serum or medium components. The kinetics of invasion can be measured over a time span of 5-60 min after which invasion is completed at an average efficiency of 41%. The fast kinetics and high efficiency exceed those of most previously established apicomplexan invasion assays. The manipulation of intracellular calcium concentration inhibits invasion. Preincubation of merozoites at 37 degrees C also reduces invasion, possibly by the premature secretion of protein. Proteins that are shed into the environment during invasion were directly detectable by protein staining after 2-D gel electrophoresis. The limitations posed by the immunological detection of proteins released during in vitro invasion by other apicomplexan parasites can, therefore, be avoided by this method. A unique feature of the assay is the reversible uncoupling of invasion and intracellular development, the latter taking place only under serum-rich medium conditions. In addition, host cell attachment is uncoupled from invasion by cytochalasin B.