The ultra-thin sections prepared for TEM examination revealed a large parasitophorous vacuole (PV) containing the parasite inside the cytosol of infected cardiomyocytes ( Fig. 2C). The results of T. theileri attachment assays in BHK,

H9c2, SVEC and RAW 264.7 cells revealed 64 ± 20, 72 ± 12, 19 ± 4 and 84 ± 6 parasite-attached cells/100 cells, and 115 ± 37, 209 ± 49, 24 ± 5 and 197 ± 21 attached parasites/100 cells, respectively ( Table 1). The numbers of parasite-attached cells and attached parasites were significantly higher in BHK, H9c2 and RAW 264.7 cells as compared with SVEC cells ( Table 1, P < 0.001). In addition, the invasion assays showed 19 ± 7, 27 ± 16, 12 ± 3 and 22 ± 11 parasite-invaded cells/100 cells, respectively ( Table 1). The invasion rate was significantly higher in H9c2 and RAW 264.7 cells than in SVEC cells (P < 0.001 and 0.05). Selleckchem MEK inhibitor To determine whether T. theileri entry underlies membrane rafts of host cells, H9c2 cells were labeled with CTX-B. GM1 (green) enrichment was observed at the entry site of the plasma membrane around infected TCT ( Fig. 3A and B, arrowheads). In contrast, GM1 enrichment was not observed in non-infected INCB018424 control H9c2 cells ( Fig. 3A and B, upper right panel). According to a recent

study, the CATL sequence fragment is highly suitable as the target for phylogenetic analysis in T. theileri ( Rodrigues et al., 2010). We therefore sequenced this gene of Taiwan T. theileri isolates as previously described ( Cortez et al., 2009). Phylogenetic analyses revealed the sequence of TWTth1 trypanosomes was clustered within the clade of the T. theileri lineage TthIB genotype. Its sequence was completely identical with NCBI GenBank accession numbers of genes: GU299391.1, GU299394.1, GU299397.1, GU299399.1, and GU299400.1, with length 477, identities 477/477 (100%) and gaps 0/477 (0%), among the TthIB lineage. After TCTs were added to H9c2 cells, the gelatinolytic activity was significant increased and accumulated in the body of T. theileri and at the attachment site of the cell membrane ( Fig.

4B–D, arrowheads). In contrast, gelatinolytic activity was only present at the adjacent cell junction in the control ( Fig. 4A). Gelatin gels were incubated with TCT lysates of T. theileri, unwashed parasites ( Fig. 4E, lane 1) and washed twice with PBS ( Fig. 4E, lane 2). The gel contained prominent activity revolving Digestive enzyme in a molecular mass of approximately 65 kDa, the only visible activity on the gel ( Fig. 4E). Lyso-Tracker Red pre-stained H9c2 (Fig. 5A–C) or SVEC cells (Fig. 5D and E) were inoculated with TCTs (Fig. 5A, upper right panel) and amastigotes (Fig. 5D, upper right panel) for 24 h. Confocal laser scanning images corresponding to the stack of serial confocal sections and a larger magnification of one single-plane section is shown. TCTs stain as two dots, corresponding to the nucleus and kinetoplast in Hoechst stain, and were co-localized with lysosomes in Lyso-Tracker pre-stained H9c2 cells (Fig.