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.
Division of Parasitology and Tropical Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, P.O. Box 80165, 3508 TD, Utrecht, The Netherlands.
Frits F J
FF
Franssen
Fasila R
FR
Gaffar
Ana P
AP
Yatsuda
Erik
E
de Vries
D000818:Animals; D016793:Babesia bovis / Q000254:growth & development* / Q000472:pathogenicity*; D001404:Babesiosis / Q000469:parasitology; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology; D003470:Culture Media; D004560:Electricity; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000469:parasitology*; D015800:Protozoan Proteins / Q000378:metabolism*
D003470:Culture Media; D015800:Protozoan Proteins