In vitro infection of macrophages with transfected amastigotes. (A, B). Bone marrow derived macrophages from CBA mice were infected for (A) 24 hours or (B) 7 days with wild-type or transfected amastigotes at a ratio of 10 parasites per macrophage. Parasites were detected and counted by immunofluorescence. Between 100 and 200 macrophages were counted for each time-point. The χ2 test showed significant differences in the numbers of infected vs uninfected macrophages between wild-type and either alt-1 or alt-2 transfectants at both time points (p < 0.001). (C, D). In vitro infection of macrophages with L. mexicana expressing the cysteine protease inhibitor-2 (CPI-2) gene of B. malayi (C) after 24 hours and (D) after 7 days. (E, F) Infection of C57BL/6 mice with transfected L. mexicana. (E) Time-course of lesion development in mice. Groups of 8 female C57BL/6 mice were injected subcutaneously in the footpad with 3 × 106 stationary-phase wild-type L. mexicana, alt-1 transfectants or alt-2 transfectants. Lesion size was measured weekly during the course of infection with a dial micrometer and expressed as the difference in size between the infected footpad and the contralateral uninfected footpad. One of two replicate experiments with similar results is shown; because data are not normally distributed (see for example recovery of wild-type parasites in panel F), standard error values cannot be applied. (F) Recovery of parasites from footpads at 15 weeks post-infection. The number of parasites in the footpad was estimated by limiting dilution assay. Results are expressed as log10 of the highest dilution containing parasites.