Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi.
OBJECTIVE
To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi.


METHODS
Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi.


METHODS
Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled.


RESULTS
The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%.


CONCLUSIONS
Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.
Miranda
Joana P G
JP
Experimental and Technological Biology Institute-Chemical and Biological Technology Institute, Universidade Nova de Lisboa, 2780-901 Oeiras, Portugal.
Nascimento
Elisabete M
EM
Cruz
Helder J
HJ
Yin
Hong
H
Zweygarth
Erich
E
Oliva
Abel G
AG
D000818:Animals; D018929:Cell Culture Techniques; D015925:Cryopreservation / Q000662:veterinary; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D004912:Erythrocytes / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012756:Sheep; D012757:Sheep Diseases / Q000097:blood / Q000469:parasitology*; D016794:Theileria / Q000254:growth & development*; D013801:Theileriasis / Q000097:blood / Q000469:parasitology*
D016895:Culture Media, Serum-Free; D017931:DNA Primers