Figure 5
(A) The fluorescent image shows localization of BmSERA1, BmMCFRP1 and BmPiβS1 in B. microti infected mouse red blood cells (see results). Green channel (Alexa 488) images correspond to the ___location of B. microti parasite proteins. Red channel images are cell membrane stained by PKH26 and Blue channel shows DAPI stained nuclei. Merged images contain the overlay of green, red and blue channels. As observed, sera from mice immunized with Freund’s adjuvant only (negative control) shows DAPI stained nuclei demonstrating B. microti infected RBCs without a traceable green signal, confirming specificity of the assay. Sera collected from mice immunized with BmSERA1, BmMCFRP1 and BmPiβS1 in Freund’s Complete Adjuvant show specific localization of the B. microti proteins as membrane bound on the parasite surface (anti-BmSERA1) or on the surface of infected RBCs (anti-BmMCFRP1 and anti-BmPiβS1). Scale bar indicates 1 µm. (B) I. Free B. microti merozoite located near the uninfected RBC expressed BmMCFRP1 (arrow, green). Note that plasma membrane of B. microti and RBC were stained by PKH26 dye (red). Counterstaining by DAPI indicates B. microti nuclei (blue). II. The B. microti merozoite found inside RBCs showed negative staining for anti-BmMCFRP1. The apical region (entry site) of RBCs showed strong positive staining for BmMCFRP1 (arrow, green). In addition, the surface of infected and surrounding uninfected RBCs showed punctate pattern of BmMCFRP1 (arrowheads). Scale bar indicates 2 μm.
