Genome-Wide Analysis of Dynamic RNA Profiles During Toxoplasma gondii Infection in the Felines- SI_3
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Parasite Strain, Induction of Infection, and Sample Collection The T. gondii Prugniuad (Pru) strain (genotype II) was maintained in our laboratory and passaged in Kunming mice. Female Kunming mice, aged 6-8 weeks, were purchased from the laboratory animal center of LVRI. All the mice had libitum access to sterile food and water, and were raised in a spacious cage at ±25℃. The infected mice were humanely euthanized for collecting the T. gondii tissue cysts from the brain, following which the brain tissues were homogenized and the tissue cysts of T. gondii were counted microscopically. Fifteen domestic cats (Chinese Li Hua breed, 7-9 months old) were purchased from a local breeder and raised in a spacious cage at ±25℃ with sufficient food and water. Their serum was tested with ELISA (Enzo) for ensuring that they were free from infections with the four feline viruses, namely, feline immunodeficiency virus, feline leukemia virus, feline calicivirus, and feline parvovirus, The modified agglutination test (MAT, cut-off 1:25) was additionally performed for confirming that the cats were free from T. gondii infection (31). All the cats were kept in mixed farming systems for one month for building a similar intestinal flora. The 15 cats were then randomly assigned to five groups, including one control group, three primary infection groups, and one secondary infection group, with three replicates in each group. The cats in the control group were inoculated with 0.9% normal saline, while the cats in the infection groups, including three primary infection groups and one secondary infection (SI) group, were separately challenged with 600 tissue cysts of the T. gondii Pru strain. The cats in the secondary infection group were rechallenged with 600 cysts of Pru strain at two weeks after the infected cats had stopped shedding oocysts. All the cats were humanely euthanized for collection of tissue samples. The ileal part of the small intestines of the control and primary infection groups were harvested at 6, 10, and 14 days post infection (DPI). The small intestinal tissues of the secondary infection group were harvested at 30 DPI, following rechallenge with 600 cysts at 27 DPI. The collected intestinal tissues were extensively washed with PBS for removing the intestinal contents. The small intestinal epithelium was collected using a cell lifter, frozen in liquid nitrogen, and stored at -80°C until use. The remaining small intestinal tissues were fixed and stained with H&E.