Effect of age and freezing on development of Eimeria adenoeides and E. tenella sporozoites in cell culture.

Eimeria adenoeides and E. tenella sporozoites, that were (1) young, unfrozen, (2) young, frozen, or (3) old, frozen, were inoculated into cell cultures of bovine embryonic kidney. At 5 hr, more unfrozen sporozoites were intracellular than were those that had been frozen; of those frozen, more young than old were intracellular. At 48 hr, the percentages of developmental stages from young, frozen sporozoites were (1) similar to those from young, unfrozen sporozoites and (2) greater than those from sporozoites that were old when frozen. Freshly excysted sporozoites of Eimeria meleagrimitis, obtained from oocysts that were 5 to 34 weeks old, survived and developed better in cell culture than those from oocysts that were 54 and 60 weeks old (Doran and Vetterling, 1969b). When freshly excysted or frozen E. meleagrimitis sporozoites of the same age were inoculated into cell culture (Doran, 1969a), more unfrozen than frozen sporozoites were found in cells at 5 hr, but the percentage of development at 48 hr was similar. This report concerns the influence of age and freezing on development of E. adenoeides and E. tenella sporozoites in cell culture. MATERIALS AND METHODS Oocysts were made bacteria-free by treatment with undiluted Clorox (Jackson, 1964). They were stored in Ringer's solution at 3 to 6 C until used. Sporocysts were released from oocysts by grinding (Doran and Vetterling, 1968b) and treated with trypsin-bile solution (Doran and Vetterling, 1967) at 43 C until at least 85% of the sporozoites had excysted. Oocysts from which E. adenoeides sporozoites were obtained for freezing were 4 and 36 weeks old; those of E. tenella, 3 and 54 weeks old. Unfrozen E. adenoeides sporozoites were 4 to 7 weeks old; those of E. tenella, 3 to 5 weeks old. Frozen sporozoites were stored as before (Doran and Vetterling, 1968a, 1969a) for 1 to 4 weeks. They were frozen in 3-ml amounts under the conditions previously found most favorable for sporozoite survival (Doran, 1969b). Each tube contained 2.5 to 3.0 million sporozoites/ml. Cell cultures were of bovine embryonic kidney (BEK). They were on 10by 35-mm cover glasses in 16by 150-mm Leighton tubes and were prepared from a frozen supply of cells that had been serially passed 21 times prior to freezing. Only cultures that were confluent and did not contain Received for publication 20 June 1969. cell aggregates were used. The growth and maintenance media were the same as before (Doran and Vetterling, 1968b). Freshly excysted sporozoites were concentrated by centrifugation and resuspended in 95% Medium 199 with Hank's balanced salt solution + 5% chicken serum. This medium was also used to dilute the suspension of previously frozen sporozoites so that the dimethyl sulfoxide content was less than 1%. The concentration of sporozoites in each of the 3 suspensions was adjusted so that 1.0 ml contained the desired inoculum. As before (Doran, 1969b), only those sporozoites that were refractive to the microscope light were counted. After adjusting the suspending medium to pH 7.0 to 7.2 and thorough agitation, 1.0 ml of each suspension was pipetted into 6 Leighton tubes. In Experiment 1 the inoculum was either 150,000 E. adenoeides or 100,000 E. tenella; in Experiments 2 and 3, 300,000 of either species. At 4 hr, the medium used to inoculate the sporozoites was replaced with 5 ml of maintenance medium (pH 7.0 to 7.2). Cultures were kept in a walk-in incubator with a regulated temperature cycle as previously described (Doran and Vetterling, 1968b). At 5 and 48 hr, cover glasses were removed from 3 tubes in each set of inoculated cultures. They were fixed and stained as before (Doran and Vetterling, 1968b). Counts of sporozoites, trophozoites, and schizonts were made at 645 X. They represent the total number of each found by examining 4 equally spaced "rows" across the length of each of 3 cover glasses. Differentiation of sporozoites as Types A and B was made on the basis of size, intensity of staining, and nuclear morphology. As previously mentioned (Doran and Vetterling, 1968b), a Type B sporozoite was larger, more intensely stained with a nucleus containing a large, prominent nucleolus.