In vitro maintenance of Eperythrozoon suis.

In vitro maintenance of Eperythrozoon suis was attempted using a Petri dish erythrocyte culture system. In preliminary experiments, the optimal conditions for maintaining E. suis attachment to erythrocytes during incubation were anticoagulation with heparin or citrate solution, incubation with 5 or 10% CO2 at 37 degrees C, and incubation with reduced or non-reduced Eagle's minimum essential medium. Using heparin, a CO2 incubator and reduced Eagle's medium (rEM), E. suis metabolic activity was evaluated by measuring glucose consumption, and lactate and pyruvate production. Glucose consumption and lactate production were measurable while pyruvate production was not detected. Erythrocyte integrity was improved by the addition of inosine although no effect was observed on maintenance of E. suis attachment to erythrocytes or the rate of glucose consumption. To determine whether the glucose consumption observed in culture was due to E. suis glycolytic activity or enhanced erythrocyte glycolytic activity, the effect of E. suis killing by EDTA addition to medium was evaluated using rEM containing inosine (rEMI). Glucose consumption decreased proportionally with the decline in the percentage of parasitized erythrocytes induced by EDTA, indicating that glucose consumption was due to E. suis. In a subsequent experiment, the effect of different types of serum (pig or fetal calf serum) and different gaseous environments (5% CO2 incubator or candle jar) were evaluated using rEMI. Glucose consumption by E. suis was significantly increased by the addition of fetal calf serum; however, no difference in the maintenance of E. suis attachment to erythrocytes and in E. suis glycolytic activity was observed between a 5% CO2 incubator and a candle jar. Finally, the effect of medium refreshment (rEMI containing fetal calf serum) was evaluated. Maintenance of E. suis parasitism on erythrocytes and E. suis glycolytic activity were significantly improved by frequent medium refreshment. The maintenance system developed enabled successful metabolic radiolabeling of E. suis for protein/antigen analysis.