Infectivity of EFSF6 promastigotes in vitro and in vivo. (A-B) Susceptibility of BMDM to infection with the EFSF6 isolate. Macrophages were infected with stationary-phase promastigotes of L. (V.) braziliensis reference strain (MHOM/BR/75/M2903) (A) or with the EFSF6 isolate (B) using a ratio of 15 parasites/macrophage for 3 hours at 33°C and 5% CO2. The infection was maintained for 6 days under these conditions (magnification 1000×). (C-J) Infectivity of EFSF6 promastigotes to mice. (C-D) Lesion progression after inoculation of 5 × 106 EFSF6 promastigotes in the hind footpad of BALB/c (C) or C57BL/6 (D) mice. Parasites were from the 2nd (solid line) or 6th day of culture (dashed line). Results shown are the mean and standard deviation of lesion size in groups of 6 mice. Lesion size is the difference between the thickness of infected and non-infected contralateral footpads. (E-H) Histopathological analysis of the uninfected (E) or EFSF6-infected (F-H) ear of a BALB/c mouse. Tissue was fixed 3 weeks after the inoculation of 1 × 105 EFSF6 promastigotes. Magnification: 40× (E-F) and 1000× (G-H). (I-J) Transmission electron microscopy showing ear fragments from mice infected with EFSF6. Arrows point to amastigotes surrounded by tight parasitophorous vacuoles; the thick arrow indicates a kinetoplast and the thin arrow shows the amastigote rudimentary flagellum. (A) amastigote, (N) nucleus, (E) eosinophil.