(A–B) Confluent monolayers of HFFs were infected with either HhCatEth1 or TgVEG sporozoites, obtained via in vitro excystation at an MOI of 0.5. Infected monolayers were fixed at 1, 2, and 3 DPI and assayed for the number of parasites observed in each vacuole for infection with T. gondii and H. hammondi for a total of 2 replicates, Replicate 1 (A) and Replicate 2 (B). Vacuoles containing more than 16 parasites were binned at 16 as it was defined as the limit of confident detection. Bars represent mean ± SD. For both Biological Replicate 1 and 2, there was a significant difference between the number of parasites per vacuole on Days 1, 2, and 3 PI for TgVEG. For Biological replicate 1, there was no significant difference between the number of parasites per vacuole for HhCatEth1 on Days 1, 2 and 3 PI. For Biological Replicate 2, there was a significant difference in the number of parasites per vacuole between 1 and 3 DPI for HhCatEth1. Statistical significance was determined with a 2-Way ANOVA and Tukey's multiple comparison test. (C–D) There is no significant difference in sporozoite infectivity between HhEthCat1 and TgVEG. Graphs demonstrate the in vitro percent viability of excysted HhCatEth1 and TgVEG sporozoites 2 and 3 days, respectively, post excystation. Bars show the mean ± SD of three technical replicates. The viability assay was repeated with two biological replicates and there was no significance in viability between species within experiments, determined via unpaired t-test with Welch's correction.