Live imaging in vitro demonstrated AMCase dependent degradation of cysts as shown using both a chitinase inhibitor and AMCase2/2 macrophages. Although there is no evidence of an active chitinase produced by T. gondii (ToxoDB), the similar cyst survival times observed for AMCase-null macrophages and chitinase-inhibited macrophages exclude the possibility that bradyzoites are the source of enzymatic activity and are breaking out of the cyst. The chitin dependent induction of chitinase activity implies that macrophages have access to the chitin in the cyst wall Figure 6 . AMCase2/2 mice have a higher parasite burden in the brain and succumb to infection during the chronic stage. (A-F) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii. Serum was isolated from whole blood samples at days 3, 7 and 14 post infection and analyzed for (A) IFN-c, (B) IL-6, (C) MCP-1, (D) TNF-a, (E) IL-12p70 (F) At day 7 DNA was isolated from the lungs and analyzed for parasite burden using qPCR. Results are displayed as parasites per mg tissue. (G-K) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii and sacrificed at 5 weeks following infection. Brains were harvested and analyzed for cyst burden, cellular composition and histology. (G) Cyst counts were obtained from homogenized whole brain samples. (H) Cyst area, 20 cysts from each mouse were photographed microscopically and cyst area was determined using ImageJ software. (I) DNA from brains of WT and AMCase2/2 was isolated and analyzed for parasite burden using qPCR. (J) Whole brains were fixed, frozen and stained for H&E to examine cyst burden and pathology. (K) BMNCs were isolated and analyzed for expression of CD4+ T cells, CD8+ T cells, macrophages (CD45 hi /CD11b+) and microglia (CD45 hi /CD11b+) by flow cytometry. Significance was determined using log rank test with p = 0.0177. Data are representative of at least 2 individual experiments with a minimum of n = 4 and are represented as mean 6 SEM, ns = not significant, * p,0.05, ** p,0.01. (L) Survival data from C57Bl/6 (squares, n = 7) and AMCase2/2 (triangles, n = 7). Data are representative of 4 individual experiments with C57Bl/6 (n.40) and AMCase2/2 (n = 40) and significance tested using Logrank (Mantel-Cox) and Gehan-Breslow Wilcoxan test * p,0.05. doi:10.1371/journal.ppat.1002990.g006 prior to chitinase-mediated cyst destruction. The prevailing view from ultrastructure studies is that cysts remain intracellular within neurons [15, 86, 87] (A. Koshy and J. Boothroyd personal communication) yet analysis of cyst burden over time shows a reduction in cyst numbers implying some form of effector mechanism in place [16] . Several studies have demonstrated perforin dependent control of cyst burden during chronic infection [17, 18] . We suggest that instead of a direct effect of perforin on cysts, it is most likely that perforin production by CD8+ T cells may initiate this process by lysing the cyst infected cell, thus exposing the cyst wall to chitinase activity from macrophages. This model would explain the many observations of macrophages in close association with rupturing cysts [8, 17, 19] . Of note, we found that BALB/c macrophages that are more easily alternatively activated had enhanced cyst lysis activity when compared to C57Bl/6 ( Figure S7 ). This may be one explanation for the increased resistance to toxoplasmic encephalitis in BALB/c mice [30, 88] .