Iscove s modified dulbecco medium imdm

The E. bovis strain H used in the present study was originally isolated in the field in northern Germany and has been maintained since then by passages in parasite-free Holstein Friesian male calves. For oocyst production, calves (n = 3) were orally infected at the age of 10 weeks with 3 × 10⁴ sporulated E. bovis oocysts. Experimental infections were conducted under the Institutional Ethics Commission of the Justus Liebig University of Giessen, Germany (allowance no.: JLU 589_AZ). Excreted oocysts were isolated from the feces at 18 days p. i. according to the method of Jackson (1964). Sporulation of oocysts was achieved by incubation in a 2% (w/v) potassium dichromate (Merck) solution at room temperature (RT). Sporulated oocysts were stored in this solution at 4°C until further use. Sporozoites were excysted from sporulated oocysts as previously described (Hermosilla et al., 2002 (link)). Free sporozoites were washed three times in sterile phosphate-buffered solution (PBS), suspended in complete Iscove’s modified Dulbecco medium (IMDM; Gibco), and counted in a Neubauer hemocytometer as described elsewhere (Hermosilla et al., 2008 (link)). Sporozoite viability was determined by trypan blue exclusion tests according to Lang et al. (2009) (link).