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

Experimental parameters of 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 Eagles'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 x 10(7) cells per vessel.