Summary: | Human babesiosis is an emerging tick-borne disease, caused by haemoprotozoa genus of <i>Babesia</i>. Cases of transfusion-transmitted and naturally acquired <i>Babesia</i> infection have been reported worldwide in recent years and causing a serious public health problem. <i>Babesia duncani</i> is one of the important pathogens of human babesiosis, which seriously endangers human health. The in vitro culture systems of <i>B. duncani</i> have been previously established, and it requires fetal bovine serum (FBS) to support long-term proliferation. However, there are no studies on serum-free in vitro culture of <i>B. duncani</i>. In this study, we reported that <i>B. duncani</i> achieved long-term serum-free culture in VP-SFM AGT<sup>TM</sup> (VP-SFM) supplemented with AlbuMax<sup>TM</sup> I. The effect of adding different dilutions of AlbuMax<sup>TM</sup> I to VP-SFM showed that 2 mg/mL AlbuMax<sup>TM</sup> I had the best <i>B. duncani</i> growth curve with a maximum percentage of parasitized erythrocytes (PPE) of over 40%, and it can be used for long-term in vitro culture of <i>B. duncani</i>. However, the commonly used 20% serum-supplemented medium only achieves 20% PPE. Clearly, VP-SFM with 2 mg/mL AlbuMax<sup>TM</sup> I (VP-SFMA) is more suitable for the in vitro proliferation of <i>B. duncani.</i> VP-SFM supplemented with CD lipid mixture was also tested, and the results showed it could support the parasite growth at 1:100 dilution with the highest PPE of 40%, which is similar to that of 2 mg/mL AlbuMax<sup>TM</sup> I. However, the CD lipid mixture was only able to support the in vitro culture of <i>B. duncani</i> for 8 generations, while VP-SFMA could be used for long-term culture. To test the pathogenicity, the VP-SFMA cultured <i>B. duncani</i> was also subjected to hamster infection. Results showed that the hamster developed dyspnea and chills on day 7 with 30% PPE before treatment, which is similar to the symptoms with un-cultured <i>B. duncani</i>. This study develops a unique and reliable basis for further understanding of the physiological mechanisms, growth characteristics, and pathogenesis of babesiosis, and provides good laboratory material for the development of drugs or vaccines for human babesiosis and possibly other parasitic diseases.
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