1. Establishing the bradyzoite burden within tissue cysts using BradyCount 1.0.
Establishing the bradyzoite burden within tissue cysts using BradyCount 1.0. (A) A z-stack of a tissue cyst labeled with Dolichos lectin (DBA) and Hoechst (DNA) spanning the tissue cyst in eight optical sections. The central section (yellow box) was selected, and the diameter (yellow dashed line) was recorded. A DIC image of the specific cyst is adjacent to the fluorescent image. The DNA image is opened in the BradyCount 1.0 application where a screen grab with two panels reveals the DNA image (left) and the Otsu-transformed (thresholded) image (see Materials and Methods) (right). Sliders under the images allow for the adjustment of the thresholding level such that each discrete nucleus is outlined in the right panel. Clicking the count button (red asterisk) counts the nuclear profiles, which correspond directly to the number of bradyzoites. (B) Nuclear (bradyzoite) counts from 463 tissue cysts harvested at all time points plotted against the imaged volume (i.e., the widest optical slice) revealed a general pattern whereby larger tissue cysts tend to harbor more bradyzoites. However, for all size ranges, considerable heterogeneity in bradyzoite (nuclear) numbers are found (magenta box; green and cyan arrows marking the green and cyan boxes in the inset), indicating that tissue cyst size is not an accurate measure of bradyzoite number. Tissue cysts that have vastly different volumes can contain very similar bradyzoite burdens (red asterisks). (C) The mean bradyzoite burden (plus standard error [SE] [error bar]) for tissue cysts harvested at weeks 3 (n = 175), 4 (n = 30), 5 (n = 124), 6 (n = 37), and 8 (n = 97) postinfection reveal evidence for bradyzoite replication between weeks 3 to 6, after which the bradyzoite numbers appear to stabilize.
Elizabeth Watts et al. mBio 2015; doi: /mBio