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In vitro maintenance of Eperythrozoon suis
N Nonaka1, B J Thacker, T W Schillhorn van Veen
1Department of Large Animal Clinical Sciences, Michigan State University, East Lansing 48824, USA.
Abstract:
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.
Insights
Researchers developed a novel in vitro system to maintain Eperythrozoon suis (E. suis) in pig erythrocyte cultures. This optimized system allows for metabolic studies and radiolabeling of E. suis for antigen analysis.
Area of Science:
- Veterinary Microbiology
- Parasitology
- Cell Culture Technology
Background:
- Eperythrozoon suis (E. suis) is an important swine pathogen.
- In vitro culture systems are crucial for studying E. suis metabolism and developing diagnostic tools.
Purpose of the Study:
- To establish an optimal in vitro culture system for maintaining Eperythrozoon suis (E. suis) attached to erythrocytes.
- To evaluate the metabolic activity of E. suis in culture.
- To enable metabolic radiolabeling of E. suis for further analysis.
Main Methods:
- Erythrocyte culture system optimization using varying anticoagulants, CO2 concentrations, media, and serum types.
- Metabolic activity assessment via glucose consumption, lactate, and pyruvate production.
- E. suis viability and attachment evaluated using EDTA and medium refreshment protocols.
Main Results:
- Optimal conditions identified: heparin anticoagulant, 5% CO2, 37°C, and reduced Eagle's minimum essential medium (rEM).
- E. suis demonstrated glucose consumption and lactate production, confirming its glycolytic activity.
- Fetal calf serum and frequent medium refreshment significantly improved E. suis maintenance and metabolic activity.
Conclusions:
- A robust in vitro system for E. suis culture was successfully developed.
- The system facilitates metabolic studies and enables metabolic radiolabeling for protein/antigen analysis.
- This advancement supports further research into E. suis pathogenesis and diagnostics.