Fig. 4

Cyst formation in vitro and virulence assay in mice. a Double-knockout strain and Δku80 were cultured in alkaline medium at pH 8.2 to induce cyst formation. Four days after induction, both DBA and BAG1 were detected in Δmca1mca2 by IFA (both top and bottom panels). b The rate of bradyzoite formation of Δmca1Δmca2 was significantly higher than that of Δku80 when cultured in alkaline medium at pH 8.2. Total 100 PVs with anti-GAP45 serum labeling parasites were counted in each strain and the number of PVs with DBA positive parasites was counted to calculate the rate of bradyzoite formation. c BALB/c mice were injected intraperitoneally with 100 tachyzoites of Δmca1Δmca2, Δmca1, Δmca2, or Δku80 (n = 5), and three independent experiments were performed with similar outcomes. Mice infected with Δmca1Δmca2 survived longer time than those infected with Δmca1, Δmca2 or Δku80, and a total of five mice of each of the three independent experiments (total 15 mice) were alive at 32 days post-infection. d Brain tissue of three surviving mice at 32 days post-infection was tested for the specific surface antigen protein BAG1 in bradyzoites by western blot. The brains of mice orally infected with PRU cysts were used as a positive control and mice injected intraperitoneally with PBS as a negative control. e The cyst was clearly observed in the brains of mice infected with tachyzoites of Δmca1Δmca2 intraperitoneally at 32 days post-infection under the microscopy. Scale bar is 20 μm