Cultivation of Besnoitia jellisoni in Bovine Cell Cultures

Abstract

Besnoitia jellisoni organisms obtained from cysts in white mice or kangaroo rats (Dipodomys ordii) multiplied in monolayer cell cultures of bovine embryonic spleen (BES), embryonic bovine trachea (EBTr), and adult bovine kidney (MDBK) in Leighton tubes. BES and EBTr cell lines provided more favorable conditions for development than did MDBK established cell lines. Peak numbers of intracellular organisms were found 3 and 4 days after inoculation. In two experiments an additional peak occurred on days 8 and 9, suggesting a second cycle of multiplication. Multiplication occurred by endodyogeny and stages possibly representing binary fission and schizogony were also seen. Flexing, gliding, and pivoting movements as well as penetration and leaving of host cells were observed. One hundred live intracellular organisms averaged 7.2 by 2.4 /t and 75 live extracellular organisms averaged 8.0 by 2.4 . Cytopathic effects were observed in cell cultures beginning 6 days after inoculation. No detailed reports have yet appeared concerning in vitro cultivation of Besnoitia jellisoni. Frenkel (1965) reported infection of many unspecified tissue culture cell lines. Suggs (1967) and Suggs and Walls (1967) obtained infection of human fetal lung cell cultures with proliferative organisms. Bigalke (1962) reported cultivation of proliferative and cyst organisms of B. besnoiti in lamb and calf kidney monolayer cell cultures. We have obtained growth of B. jellisoni in cultured bovine cells inoculated with cyst organisms from kangaroo rats (Ernst et al., 1968) and white mice. These results are reported herein. MATERIALS AND METHODS Three cell types including cell line cultures of embryonic bovine trachea (EBTr) and embryonic bovine spleen (BES) as well as established cell line cultures of Madin-Darby adult bovine kidney (MDBK) were used in this study. EBTr and MDBK cells were obtained from the American Type Culture Collection Cell Repository in Rockville, Maryland. BES cells were obtained from a 4to 6-month calf fetus and begun as primary cells in the tissue culture laboratory at Utah State University. All 3 cell types were grown in 32-oz Brockway prescription bottles using Eagle's minimal essential medium (MEM) with Earle's salts, Received for publication 20 February 1968. * Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service, and by a traineeship from the NSF. Published as Journal Paper No. 860, Utah Agricultural Experiment Station. t Present address: Beltsville Parasitological Laboratory, Animal Disease and Parasite Research Division, ARS, USDA, Beltsville, Maryland 20705. t Present address: Department of Biology, Andrews University, Berrien Springs, Michigan 49104. 1% sodium pyruvate, 1% nonessential amino acids, and 5 to 10% fetal calf serum. The cells to be used in each experiment were transferred at the rate of 100,000 cells/ml to Leighton tubes containing 9by 35-mm cover slips. These tubes were incubated at 37 C for approximately 24 hr, at which time the cyst organisms were added. Cysts containing Besnoitia organisms were removed from the fascia beneath the skin on the back of naturally infected kangaroo rats (Dipodomys ordii) collected in the Curlew Valley near Snowville, Utah. Other cysts were removed from the peritoneum of artificially infected white mice. Cysts obtained from either host were suspended in MEM and crushed in a sterile glass tissue grinder, releasing motile organisms. In each experiment these organisms were counted with the aid of a hemocytometer, diluted with MEM to concentrations ranging from 110,000 to 270,000 organisms/ ml, and inoculated in 1-ml aliquots into 10 to 12 Leighton tubes containing cell monolayers. Nine experiments were conducted in which each of the 3 cell types was used 3 times (Table I). In each of 4 experiments, EBTr or MDBK cell cultures were inoculated with cyst organisms from white mice; in 2 other experiments, each of the 2 cell types was inoculated with cyst organisms from kangaroo rats. In 2 experiments BES cell cultures were inoculated with cyst organisms from kangaroo rats and in one experiment with cyst organisms from a white mouse. In an EBTr and an MDBK experiment (Exps. 1, 4) with white mouse cyst organisms, double cover slip preparations were examined with phase-contrast microscopy 1 hr after inoculation and at daily intervals thereafter for 10 days. Measurements were made of 100 organisms occurring singly in vacuoles in the host cell and 75 extracellular organisms in such EBTr cell cultures beginning 5 days after inoculation. After observation of the living specimens, the cover slips were fixed in Schaudinn's fluid and stained with iron hematoxylin. Cover slips in the other experiments were removed daily from Leighton tubes, fixed and stained as described above, and then examined. Quantitative data regarding intracellular organisms present in the cultures 1 through 10 days after inoculation were obtained for each of the 9