Cultivation of Eimeria bovis in Three Established Cell Lines and in Bovine Tracheal Cell Line Cultures

Abstract

Monolayer established cell line cultures of bovine kidney (Madin-Darby, 1958), human intestine (Intestine 407), and mouse fibroblasts (L cells), as well as bovine tracheal cell line cultures, were inoculated with E. bovis sporozoites and observed for a maximum of 21 days. Mature first-generation schizonts developed in each of the cell types, except for the L cells. In these, large numbers of sporozoites entered cells but only a few transformed into trophozoites; the most advanced stage seen was a trinucleate schizont. In the tracheal cells, relatively numerous schizonts developed and mature schizonts were first observed 8 days after inoculation. Thus, the rate of development of the schizont in the tracheal cells was unusually rapid, exceeding even that in calves. Some mature schizonts were relatively large; the maximum, 292 by 118 ,u, was observed in an 18-day culture. Intracellular merozoites and early schizonts of the second generation were found in 18and 19-day cultures of tracheal cells. Many schizonts occurred also in established kidney cell line cultures, but the rate of development was slower than that in tracheal cells, and the first mature schizonts were observed in a 14-day culture. The largest mature schizont seen (55 by 38 u) was in a 21-day culture. In the intestinal cells, relatively small numbers of schizonts developed; the rate of development was approximately the same as that in kidney cells. The largest mature schizont observed, 109 by 55 ,u, was seen in a 13day culture. Of the four cell types studied, the bovine tracheal cells appear to provide the best conditions for development of E. bovis. In earlier work (Fayer and Hammond, 1967), we found that sporozoites of E. bovis developed into mature first-generation schizonts in cell line cultures of bovine kidney, spleen, and thymus cells. Development only to binucleate and multinucleate schizonts occurred in cultures of bovine testicle and intestinal cells, respectively. Others (Patton, 1965; Strout et al., 1965; Doran and Vetterling, 1967a, b) have found that Eimeria species from chickens and turkeys undergo development in a variety of cells. These include established cell lines, as well as primary and cell line cultures, from species other than the normal host. In further work on the in vitro cultivation of E. bovis, we have attempted to obtain additional information about the variety of host cells in which sporozoites of this species will grow. In the present paper, we report the results of such work with bovine, human, and murine established cell lines, and with bovine tracheal cell line cultures. MATERIALS AND METHODS Three of the four cell types used in the experiments were obtained from the American Type CulReceived for publication 12 January 1968. Supported in part by research grant A1-07488 from the NIAID, U. S. Public Health Service, and by a traineeship from the National Science Foundation. Published as Journal Paper no. 744, Utah Agricultural Experiment Station. ture Collection Cell Repository in Rockville, Maryland. The fourth type, L cell (mouse fibroblast) cultures, was obtained from Dr. Rex Spendlove, Department of Bacteriology, Utah State University. Between serial passages, cultures were maintained in Brockway prescription bottles in a 37 C incubator. Madin-Darby bovine kidney (MDBK) cells and embryonic bovine trachea (EBTr) cells were cultured in Eagle's minimal essential medium (MEM) with Earle's salts, 1% sodium pyruvate, 1% nonessential amino acids, and 10% fetal bovine serum. Embryonic human intestinal (Int 407) cells (Henle and Deinhardt, 1957) were cultured in Eagle's basal medium (BME) with Hank's salts and 15% fetal bovine serum. L cells (Sanford, Earle, and Likely, 1948) were cultured in MEM and 10% fetal bovine serum. The cells to be used in each experiment were transferred, at the rate of 150,000 cells/ml, to Leighton tubes containing 9by 35-mm or 10.5by 50-mm cover slips. These tubes were incubated at 37 C until the cells attached to the cover slip, at which time sporozoites were added. The terminology used for cell cultures is that recently proposed by the Tissue Culture Association (Fedoroff, 1967). By the use of methods previously described (Fayer and Hammond, 1967), oocysts were collected, cleaned, sterilized, and excysted. The total number of sporozoites in suspension was determined with the aid of a hemocytometer. Concentrations of 150,000 to 350,000 sporozoites per 1.7 ml aliquot were obtained by diluting the suspension with serum-free tissue culture medium appropriate for the cell type being investigated. A sterile Cornwall syringe was used to pipette 1.7 ml of suspended sporozoites into each of six to 20 Leighton tubes containing cell cultures for each experiment. In each experiment up to 20