USE OF A FLUORESCENT-PROBE TO ASSESS THE ACTIVITIES OF CANDIDATE AGENTS AGAINST INTRACELLULAR FORMS OF ENCEPHALITOZOON MICROSPORIDIA

Microsporidia are obligate intracellular protozoan parasites. Three sp ecies of the genus Encephalitozoon are among the microsporidia that in fect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study des cribes a method that uses the fluorescent probe calcein and confocal m icroscopy to detect drug-induced effects in Encephalitozoon-infected g reen monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swellin g and gross morphological changes of parasite developing stages in sit u; (iii) killing of developing parasite stages in situ, detected by th eir uptake of the fluorescent probe; and (iv) reduction in the viabili ty of the host cell population, assessed by the loss of the probe. Ver apamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporid ial activity, albendazole and fumagillin, caused all three types of pa rasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin tha t caused swelling and disorganization of parasite developing stages we re 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidat e antimicrosporidial agents in infected cultured cells.