Abstract:
The establishment of in vitro culture methods has greatly facilitated the research of Babesia. However, the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum, which intensively limits the culture and is unable to satisfy the demands of long-term studies. In this study, AlbuMAX I (2 mg/mL) and 2.5% dog serum (vol/vol) were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD (2.5%), and the effectiveness of this medium was assessed by the growth of B. gibsoni. The results showed that VP-SFMAD (2.5%) could support the continuous growth of the parasite, and the parasitemia has no difference with the cultivation in RPMI 1640 with 20% dog serum. In contrast, either a low concentration of dog serum or absence of AlbuMAX I will significantly lower the parasite growth or fail to maintain B. gibsoni growth in the long term. The strategy of reducing the hematocrit was also evaluated, and VP-SFMAD (2.5%) improved the parasitemia to over 50% within 5 days. The high parasitemia is helpful for larger numbers of parasite collection, which is valuable for studying the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites. In addition, VP-SFMAD (2.5%) medium was successfully used for monoclonal parasite screening, which obtained monoclonal strains with parasitized erythrocytes about 3%, which is similar to RPMI-1640D (20%) medium that obtains monoclonal strains on the 18th day. Those results showed that VP-SFMAD can be applied to B. gibsoni continuous long-term, expansion culture, and subclone culture. IMPORTANCE The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia. Importantly, several technical problems impeding such studies have been overcome.
摘要:
体外培养方法的建立极大地促进了巴贝虫的研究。然而,目前的gibsoni巴贝斯的体外培养基需要高浓度的犬血清,这严重限制了文化,无法满足长期研究的需求。在这项研究中,将AlbumMAXI(2mg/mL)和2.5%狗血清(vol/vol)添加到VP-SFM培养基中,以开发名为VP-SFMAD(2.5%)的低浓度血清培养基,并且通过B.gibsoni的生长来评估该培养基的有效性。结果表明,VP-SFMAD(2.5%)可以支持寄生虫的持续生长,寄生虫血症与20%犬血清的RPMI1640培养没有差异。相比之下,低浓度的狗血清或不存在AlbMAXI将显著降低寄生虫生长或不能长期维持双歧杆菌生长。还评估了降低血细胞比容的策略,VP-SFMAD(2.5%)在5天内将寄生虫血症改善至50%以上。高寄生虫血症有助于收集更多的寄生虫,这对研究生物学很有价值,发病机制,以及巴贝斯虫和其他红细胞内寄生虫的毒力。此外,VP-SFMAD(2.5%)培养基成功用于单克隆寄生虫筛选,获得了约3%被寄生红细胞的单克隆菌株,这类似于在第18天获得单克隆菌株的RPMI-1640D(20%)培养基。这些结果表明,VP-SFMAD可以连续长期应用于B.gibsoni,扩张文化,和亚克隆文化。重要性VP-SFM作为补充有AlbuMAXI和低浓度犬血清(2.5%)的基础培养基,可以在小体积和大体积下连续体外培养gibsoni巴贝斯虫,这是为了满足不同的实验需求,如长期培养和获得高寄生虫血症和亚克隆培养。体外培养系统的建立使研究人员能够更好地了解巴贝虫的代谢和生长模式。重要的是,已经克服了阻碍此类研究的几个技术问题。