Graduate PhD's Degree Program in Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.
Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Parasitol Res. 2019 Jun;118(6):1885-1897. doi: 10.1007/s00436-019-06311-z. Epub 2019 Apr 10.
Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 μg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 μg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.
东方利什曼原虫（Mundinia）是一种最近描述的新物种，它在泰国引起利什曼病。为了便于描述这个新物种，开发了一种体外培养系统来生成东方利什曼原虫的无共生性内阿米巴型。通过温度和 pH 值的操作，优化了无共生性培养衍生的内阿米巴型的体外培养条件。使用四种标准评估产生的东方利什曼原虫无共生性内阿米巴型，即形态、核酶的同工酶分析、循环转化和对人单核细胞系（THP-1）细胞的感染力。结果表明，东方利什曼原虫无共生性内阿米巴型的最佳培养条件是 Grace 的昆虫培养基，补充有 20%的 FCS、2%的人尿、1%的 BME 维生素和 25μg/ml 的硫酸庆大霉素，pH 值为 5.5，温度为 35°C。对于前鞭毛体，条件是 M199 培养基，补充有 10%的 FCS、2%的人尿、1%的 BME 维生素和 25μg/ml 的硫酸庆大霉素，pH 值为 6.8，温度为 26°C。形态学特征表明，寄生虫包括内阿米巴型、前鞭毛体、前环毛蚓型前鞭毛体、毛形前鞭毛体、幼期前鞭毛体和副体鞭毛体等六个主要阶段。此外，形态变化伴随着核酶同工酶图谱的变化。在两种培养系统中，东方利什曼原虫的体外发育周期在 12 天内完成。无共生性内阿米巴型对 THP-1 巨噬细胞的感染力和在细胞内的生长与 THP-1 衍生的细胞内内阿米巴型相似。这些结果证实了东方利什曼原虫无共生性内阿米巴型的成功培养。无共生性内阿米巴型和前鞭毛体可用于进一步研究在允许的载体和动物中的感染。
