Theileria annulata: In vitro cultivation of schizont-infected bovine lymphocytes

• Published in: Veterinary parasitology Vol. 65; no. 1; pp. 11 - 20
• Main Authors: Shkap, Varda, Frank, M., Fish, L., Pipano, E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-10-1996
• Subjects: Animals; Animals, Newborn; BOVIN; CATTLE; Cell Division - drug effects; Cells, Cultured; CULTIVO IN VITRO; CULTURE IN VITRO; CULTURE MEDIA; Culture Media, Conditioned; Culture Techniques - instrumentation; Culture Techniques - methods; GANADO BOVINO; IN VITRO CULTURE; INFECCIONES POR PROTOZOOS; Insulin - pharmacology; LINFOCITOS; LYMPHOCYTE; LYMPHOCYTES; Lymphocytes - cytology; Lymphocytes - drug effects; Lymphocytes - parasitology; MEDIO DE CULTIVO; Mercaptoethanol - pharmacology; MILIEU DE CULTURE; Oxaloacetates - pharmacology; PROTOZOAL INFECTIONS; PROTOZOOSE; Pyruvic Acid - pharmacology; Schizont-infected cells; THEILERIA ANNULATA; Theileria annulata - pathogenicity; Theileria annulata - physiology; VACCIN; VACCINES; VACUNA; Schizont-infected cells; Theileria annulata; In vitro culture
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/0304-4017(96)00944-2

Experimental parameters for optimization of the in vitro growth conditions for
Theileria annulata schizont-infected bovine lymphoid cells are presented. Of the nine different media tested in the course of 14 successive passages, Leibovitz L-15 (L-15) and a combination of McCoy and Leibovitz L-15 (ML) were preferable to McCoy, Dulbecco (DMEM), RPMI 1640 or Eagle's minimum essential medium (MEM) based on either Hank's or Earle's salts. The lowest multiplication rate was obtained with M-199 medium based on Hank's or Earle's salts. The highest yield of cells was obtained when L-15 was supplemented with 20% newborn bovine serum, while lowering the serum concentration by half resulted in a 25% decrease in cell yield. There was no effect on multiplication rate observed during ten passages when 2-mercaptoethanol and a mixture of oxaloacetate, sodium pyruvate and insulin were added to the growth medium. On substitution of conditioned medium for 20 or 50% of the growth medium, the yield of cells decreased by 12 or 20%, respectively, a factor which might be considered in calculations of the cost of anti-
T. annulata vaccine production. When cells were grown in stationary cultures in Roux bottles for 7 days without change of medium, the highest yield resulted from seeding at 10
7 cells per vessel (100 ml) in L-15 supplemented with 20% serum. In Roller bottles, with the same type and volume of medium and cultivation for 7 days without medium change, best yield resulted from seeding with the highest inoculum size of 4 × 10
7 cells per vessel.