Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro

Abstract

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1×10 9 red blood cell (RBC)/ml, 1–2.5×10 5 infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [ 3H]hypoxanthine incorporation. An excellent correlation was demonstrated between A 405 of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A 405 and [ 3H]hypoxanthine incorporation for [ 3H]hypoxanthine concentrations lower than 4 μCi/ml. Our assays also highlighted the inhibitory effect of [ 3H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 μCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A 405 and parasitemia counting. In conclusion, A 405 measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.