Culturing and Infecting Protozoan Parasites

RH, GT1, Prugniaud (Pru), VEG strain T. gondii; and Nc1 [75] (link), Nc2 [76] (link) and NcLiv strain N. caninum were maintained by serial passage in human foreskin fibroblast (HFF) monolayers as described previously [77] (link). RH stably expressing mCherry were obtained from Dr. Anita Koshy [78] (link). Primary bovine fibroblasts were provided by Dr. Kenneth John McLaughlin (Penn). Cryopreserved bone marrow cells recovered from Myd88^−/−, Myd88/Trif^−/−, and Tlr2/Tlr4^−/− mice were differentiated into macrophages as described previously [79] (link), and infected with a 1∶1 MOI of parasites for QPCR and microarray experiments at 16–22 hours post-infection. Ifnar1^−/− and Tlr3^−/− mice were provided by Drs. Hao Shen and Yongwon Choi (Penn), respectively. For mouse infections, WT and Ifnar1^−/− mice received either control IgG or anti-IFN-γ (clone XMG-1.2, 1mg I.P., BioXCell) on day 0 and again at 4 days post-infection. Mice were infected with 3×10⁶ NcLiv strain Neospora caninum by I.P. injection on day 0. All mice were maintained at the University of Pennsylvania in accordance with Institutional Animal Care and Use Committee guidelines. For cytospins, 200,000 cells were spun onto glass slides using a Shandon Cytospin and stained with Diff-Quik, dehydrated, mounted with Permount (Sigma Aldrich), and images captured on a Nikon E600 upright microscope.