Objective: Toxoplasma gondii is a highly successful parasite of veterinary and medical importance. Disease outcome can depend on many factors, including host background and virulence of the infecting strain. This work aimed to compare infection dynamics between mouse virulent and non-virulent strains of T. gondii in different host cells, and examine host and parasite responses during infection using transcriptomics.

Methods: Intestinal epithelial cells (IEC) from mice and humans were infected with 2 × 10⁵ tachyzoites from 5 different T. gondii strains of differing mouse virulence (virulent TgCatBr71 and TgCkStK9; non-virulent TgCatBr60, TgWildBrMG6 and TgRhHmBr1), and cells were either fixed at 24 hpi for microscopy (invasion assay) or DNA was extracted for parasite quantification at 24, 48, 72 and 96 hpi (proliferation assay). Dual RNA-Seq of T. gondii-infected mouse and human IECs were sequenced by DNBSeq (BGI) at 24 and 72 hpi. DNA was extracted from TgCkStK9, TgRhHmBr1, and TgWildBrMG6 for short-read WGS by DNBSeq, and long-read WGS by Pacbio HiFi sequencing.

Results: The invasion assay demonstrated a statistically significant interaction between parasite strain and host (p=0.005), with the highest number of infection events being recorded for TgCkStK9. The level of parasite invasion for each strain was comparable between mouse and human hosts, with the exception of TgCkStK9, which showed statistically significantly higher invasion of human cells (p=0.033). The proliferation assay demonstrated statistically significant effects of strain and host (p< 0.001), with TgRhHmBr1 estimated to replicate at the fastest rate compared to the other strains when averaging across human and mouse cells. RNA-Seq analysis revealed that hundreds of genes were differentially expressed (DEGs) between strains. Long-read sequences of TgCkStK9, TgRhHmBr1, and TgWildBrMG6 were assembled and polished. The comparison revealed that while TgCkStK9 and TgWildBrMG6 exhibited no structural variants compared to the RH88 reference genome, TgRhHmBr1 displayed a structural variant.

Conclusions: This study highlights the role of host background in parasite virulence and indicates that the virulence of T. gondii in mice may not correlate with that in humans. RNA-Seq revealed DEGs between strains. High-quality precision genome assemblies were produced to aid in future research, which includes screening for potential virulent markers and conducting a comparative genome analysis.