Phenotypic and Gene Expression Changes among Clonal Type I Strains of Toxoplasma gondii

Plaque formation assay using different type I strains. (A) Example of plaques formed by RH-ERP2009 and GT1 when grown on HFF monolayers in six-well plates. On the left is a low-magnification image of one entire well. Scale bar, 1 cm. The image on the right was obtained by microscopic examination and shows a single plaque formed by GT1. Scale bar, 0.2 mm. (B) Quantification of the area of plaques developed by RH-ERP2009 and other type I strains. Plaques developed by RH-ERP77, RH-ERP88, and RH-ERP2009 were significantly (P ≤ 0.005) larger than other type I strains including from RH-JSR. Values are means ± the SEM (n = three separate experiments, 50 plaques/experiment) for RH-ERP77, RH-ERP88, RH-ERP2009, GT1, and GT1-LP, and means ± the standard deviation (SD) for each of two experiments, for remaining isolates.

Comparison of intracellular growth and extracellular survival between strains of T. gondii. (A) Comparison of intracellular growth rate between RH-ERP2009 and GT1. Monolayers of HFF cells were infected with parasites, grown for 30 h, fixed, and stained with MAb DG52 to detect surface antigen SAG1. The average numbers of parasites present per vacuole was determined by microscopic examination and counting of 50 vacuoles/coverslip. GT1 contained significantly more vacuoles with 16 parasites/vacuole (*, P ≤ 0.05), while the number of vacuoles with 32 parasites was significantly higher in RH-ERP (**, P ≤ 0.005). Values indicate means ± the SEM (n = three separate experiments with three coverslips each). (B) Comparison of extracellular survival between RH-ERP2009, RH-JSR, and both low- and high-passage GT1. The number of tachyzoites surviving after natural egress was determined by incubation of parasites for at 37°C with CO₂ in complete medium and subsequent plaque assay. The viability of RH-ERP2009 was higher at the outset and viability dropped by 50% over the next 24 h (**, P ≤ 0.005, comparison of 2 h versus 24 h). The initial viability of GT1 was significantly lower at 0 h after egress (**, P ≤ 0.005), and this dropped further to almost negligible levels by 12 h after egress (**, P ≤ 0.005, comparison of 2 h versus 12 h). There was also a significant difference in the viability of low passaged GT1-LP and RH-JSR isolates versus GT1 even at early time points (*, P < 0.05). Values are means ± the SEM (n = three experiments).

Differentiation of tissue cysts after in vitro culture. Parasites were induced to differentiate by high-pH culture for 7 days (“Induced”), and cyst development was detected by staining with fluoresceinated lectin DBL (green), followed by MAb to the parasite surface protein SAG1 conjugated to Alexa 594 (red). GT1 readily formed cysts under these conditions, whereas RH-ERP2009 did not. Neither strain converted to tissue cysts under normal growth conditions used for the plaque assay (“Normal”). All pictures shown were recorded under similar optical conditions, imaged with the same exposure time, and processed identically. Scale bars: 15 μm in the top panel and 40 μm in the remaining panels.

Acute virulence of RH-ERP2009 and GT1 strains as monitored by infection in outbred mice. (A) All animals inoculated with RH-ERP2009 died within 12 days, regardless of dose. (B) Animals injected with 4 or 40 tachyzoites of GT1 also died rapidly. Most of mice injected with 0.4 GT1 tachyzoites survived; however, surviving mice were not infected, as shown by a negative serological response in Western blot (*). The results shown are the combination of two experiments with five mice per group each. Inocula have been adjusted for viability based on efficiency of plaque formation (see Materials and Methods).