Continuous Cultivation of Trypanosoma theileri at 37 C in Bovine Cell Culture*

Abstract

Trypanosoma theileri was cultivated at 37 C in bovine bone marrow cell culture through 50 consecutive subcultures. Medium 199, supplemented with Bacto-peptone, vitamin B12, and fetal bovine serum, was utilized both for primary and continuous cultivation. The number of trypanosomes produced in culture averaged 8 x 10(6) (1-26 x 10(6)) trypanosomes/ml. In each subculture the organisms divided as epimastigotes and transformed into trypomastigotes; a round from was observed during the stationary and declining phase of growth. Gradual changes such as increased generation time, size reduction, and decreased trypomastigote production were observed as subculturing progressed. Cultured trypanosomes were infective for the bovine through the 48th serial transfer and could be cultivated at 26 degrees C.

Energy Metabolism of Bloodstream Form Trypanosoma theileri

“…Therefore, isolated bloodstream form T. theileri trypanosomes were cultured at 37°C in the presence of 5% CO 2 in a humidified incubator in RPMI 1640 medium contain-ing 50 IU/ml penicillin, 50 g/ml streptomycin, 2 mM L-glutamine (all from Invitrogen-Gibco), 10% (vol/vol) fetal calf serum (Bodinco, The Netherlands), 5 ϫ 10 Ϫ5 M ␤-mercaptoethanol (Flow Laboratories, Ivume, United Kingdom), and 2 ϫ 10 6 (irradiated) peripheral blood mononuclear cells (PBMCs) per ml. The necessity of blood cells or cell products in the culture medium for T. theileri species is in agreement with earlier reports (8,18,19).…”

“…Glucose was degraded by T. theileri for 47 Ϯ 3% to acetate and for 45 Ϯ 4% to succinate (calculated from the data in Table 1). Only a very limited amount of the degraded [6][7][8][9][10][11][12][13][14] C]glucose (less than 1%) was broken down by T. theileri to 14 C-labeled CO 2 , the end product resulting from full Krebs cycle activity.…”

Isolation of Trypanosoma (Megatrypanum) theileri from Dairy Cattle in Taiwan

“…As has been previously described, bovine bone marrow [16], human or monkey cells [4,31,32], HeLa, human epidermoid (Hep), human embryonic lung (Hel), monkey stable (MS) cell [32], and LLCMK2 (monkey) cells (a common cell line used to culture intracellular protozon such as Toxoplasma) [19] can be used to propagate T. theileri. Hatama et al [11] isolated a Megatrypanum trypanosome which is closely related to T. theileri from a sika deer shortly after death using kidney primary trypsinizating culture, however, subsequent attempts to propagate it in a bovine kidney cell line (Madin-Darby bovine kidney cell) and chicken cell primary culture (chicken embryo fibroblast) were not successful.…”

The electrokaryotype and molecular characterization of Trypanosoma cervi isolates using recombinant DNA techniques

“…Continuous culcivation of the T. cervi isolates was generally conducted according to the methods of McHolland-Raymond et al, (1978) at 37°C in a media overlay of a feeder cell monolayer. In some cases, Keratinocyte Growth Medium (Clonetics Corporation) was used to reduce clumping (agglutination).…”

“…Most fungicides reportedly prove lethal to trypanosome culturas but antibiotics were routinely used and are listed below (McHolland-Raymond et al, 1978). The trypanosomes were harvested from culture by centrifugation (1500x g, 10 min), washed in TDB and pelleted in a microfuge (1300 RPM, 1 min Viable cells could be recovered from a fetus stored at 4°c or on ice as long as one week after collection from the mother.…”