Normal sporozoite production. Sporozoites had normal motility, cell traversal capacity and infectivity to hepatocytes in vitro.Equal numbers of WT-F or ZIPCO-F salivary gland sporozoites were injected into mice intradermally (ID) or intravenously (IV), and pre-patent periods of infection were assessed by Giemsa-stained blood smears. ZIPCO-F blood-stage parasites emerged with a 34 day delay compared to WT-F, independently of the sporozoite injection route. Since the multiplication in the blood stages is normal, these results indicated a approximately 10(3) loss in infectivity for the mutant during pre-erythrocytic development. A similar delay was observed with ZIPCO sporozoites after IV injection.Although numbers of WT-F and ZIPCO-F EEFs were counted in vitro by fluorescence microscopy at 24 hpi, significantly fewer ZIPCO-F than WT-F EEFs were observed at 48 hpi (43%) and 65 hpi (30%). Most notably, the average size of ZIPCO-F EEFs was significantly smaller at all time points, being approximately 65, 80 and 78% that of WT-F EEFs at 24, 48 and 65 hpi, respectively.EEF development was also examined by intra-vital fluorescence microscopy at 24, 48 and 65 after IV of sporozoites. Like in vitro, ZIPCO-F EEFs were significantly smaller than the WT-F, displaying a approximately 90% reduction in size at 48 hpi. At 65 hpi, WT-F EEFs were no longer observed, having liberated merozoites, while a few ZIPCO-F EEFs were still detected. These data indicated that mutant EEFs were affected both in growth and viability, resulting in reduced and delayed release of merozoites into the blood. At 66 hpi, at the peak of merosome release in the WT-F EEFs, approximately 1,900 times fewer ZIPCO-F than control merozoites were counted.