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Immunization with irradiated-attenuated malaria sporozoites has been shown to protect both rodents and humans against a homologous sporozoite challenge. Irradiated-attenuated sporozoites retain their capacity to invade hepatocytes and transform into trophozoites without undergoing complete schizogony. As a result, the minute size of these trophozoites (4-8 microns) makes their detection by conventional microscopy difficult. An additional problem lies in obtaining sufficient quantities of exoerythrocytic stages of attenuated parasites in vivo to study their antigenic repertoire and the sequence of events that occur after immunization of hosts. We have used a previously described method of inoculating Plasmodium berghei sporozoites directly into specific liver lobes (HPBI = hepatic portal branch inoculation) to improve parasite yields. Comparing HPBI with tail vein inoculation of sporozoites in Brown Norway rats and C57BL/6 mice revealed up to a 6-fold increase in hepatic parasite yields by HPBI method. The inoculation of 3 x 10(6) irradiated sporozoites via HPBI yielded 139 +/- 2 and 69 +/- 2 exoerythrocytic parasites per cm2 of liver in Brown Norway rats and C57BL/6 mice, respectively. The HPBI method therefore not only facilitates visualization of a large number of irradiated hepatic stage parasites within the defined lobes of the liver but also provides ample numbers of parasites for immunization and for immunological analysis.

Protection conferred to mice by Plasmodium berghei sporozoites increased significantly when the time interval between 60Co-irradiation of the infected mosquitoes and harvest of sporozoites increased. One thousand sporozoites conferred no protection against challenge if harvested on the day of irradiation, but protected 60% of recipient mice when harvested 28 days postirradiation. When the time between feeding of mosquitoes and irradiation was varied, sporozoites from mosquitoes irradiated 3 days after feeding were infective for mice. Sporozoites from mosquitoes irradiated on day 10 postfeeding were not infective, but were immunogenic. In all experiments a decline occurred in the number of recoverable sporozoites over a 28-day period postirradiation to less than 10% of the yield on the day of irradiation.