Effect of Inoculum Size on Development of Eimeria tenella in Cell Cultures

Abstract

To study the in vitro propagation of Eimeria tenella, primary 2-ml cultures of chick embryo kidney cells, grown in Eagle's medium supplemented with calf serum, were inoculated with doses of 5,000, 25,000, 50,000, 100,000, and 500,000 sporozoites per ml. The time and incidence of invasion of the host cell were established by determining the number of sporozoites that were washed from the cultures 1, 3, 12, and 24 hr after inoculation, and by finding the number of sporozoites which, when inoculated onto monolayers, resulted in maximum cell infection and asexual development. The decrease in the number of sporozoites removed by washing, consistent with the number of hours the parasites were on the cultures, ranged from an average of 62% removal 1 hr after inoculation to 22% at 24 hr. The rate of infection 1 hr after inoculation was approximately 2%, increasing to about 4% at 24 hr and to approximately 5% at 96 hr. The highest rate of asexual development occurred in those cultures inoculated with 5,000 sporozoites per ml, while the lowest rate occurred in the unrinsed cultures inoculated with 500,000 sporozoites per ml. Considering the extent of infection, the total number of developmental stages on a slide, and the condition of the cell monolayer, we obtained the best results with an inoculum of 500,000 sporozoites per ml when the cultures were washed 1 hr after inoculation, or with 100,000 sporozoites per ml with no washing or replacement of the culture medium. In vitro cultivation of the asexual stages of coccidia has been reported by Patton (1965), Strout et al. (1965), Fayer and Hammond (1967), Doran and Vetterling (1967a, b), and Hammond and Fayer (1968). Although these papers have shown the successful asexual development of avian and bovine coccidia in a variety of cells cultured in vitro, to date there has been no report of a detailed study on the relationship of the size of inoculum to infection of the host cell and development of the parasite. Fayer and Hammond (1967) reported using a concentration of sporozoites calculated from the number of oocysts. In a later paper Hammond and Fayer (1968) used 150,000 to 350,000 sporozoites per culture, but they did not study the rates of infection and development. Doran and Vetterling Received for publication 15 March 1968. * Supported in part by Research Grant AI 0574504 from the NIAID, U. S. Public Health Service, and by grants-in-aid from Eli Lilly and Company, Greenfield, Indiana, and Sandoz Pharmaceuticals, a division of Sandoz, Inc., Hanover, New Jersey. Published with the approval of the Director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 433. (1967a, b), using several million sporozoites per culture, found the number of parasites that invaded cells and became schizonts was extremely small. Our experience has also indicated that of the many sporozoites that infect cells, only a very limited number develop into schizonts. The work reported in this paper is concerned with finding the number of sporozoites which, when inoculated onto 48-hr chick embryo kidney monolayers, will result in maximum infection and subsequent maturation of the asexual stages. We also attempted to establish the time and incidence of invasion by determining the number of sporozoites that we were able to wash from the cultures at various time intervals. MATERIALS AND METHODS