Cultivation of Babesia divergens in vitro

This article presents the results of a study on the optimal conditions for in vitro cultivation of Babesia divergens. Cultivating species of the genus Babesia is essential for studying their biology, pathogenesis, and for developing diagnostic and therapeutic strategies. Different Babesia species require specific cultivation conditions, including optimal medium composition, serum and erythrocyte concentrations, oxygen levels, and additional nutritional components. Research on B. divergens in vitro is particularly relevant, as this species is a causative agent of human babesiosis and has limited therapeutic options available. In this study, we evaluated the effectiveness of three different media for cultivation: RPMI 1640, DMEM, and M-199. The culture was maintained by regularly replacing the medium and performing passages every two days. This process involved centrifuging the culture to remove the supernatant, transferring the infected erythrocytes into fresh medium, and adding fresh erythrocytes to achieve an optimal hematocrit concentration of 5 %. Parasitemia levels were routinely monitored by preparing blood smears from infected samples. The RPMI 1640 medium supplemented with bovine erythrocytes and 10% fetal calf serum yielded the highest parasitemia level and stable replication. The results indicated that the absence of serum adversely affected the viability of B. divergens, with parasites dying by the fourth day of culture without serum components. The optimal erythrocyte ratio was 5 %, providing a stable environment for the prolonged replication of the parasite. Regular monitoring of parasitemia showed sustained growth of B. divergens over six days, with parasitemia levels increasing from 2 % to 20 %. Confocal microscopy with fluorescent staining of genetic material confirmed active parasite growth, allowing visualization of intracellular forms. The data obtained for B. divergens emphasize the necessity of serum factors in the medium, distinguishing this species from others. This study enhances cultivation methods and may support future research aimed at understanding the biology, pathogenesis, and potential therapeutic strategies for controlling babesiosis caused by B. divergens.