Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Abstract

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.

LGAAP: Leishmaniinae Genome Assembly and Annotation Pipeline

“…1 ). Genomic DNA was extracted from a previously developed culture system for L. orientalis axenic amastigotes ( 3 ) and sequenced using two standard technologies, i.e., short read (Illumina) and long read (Oxford Nanopore Technologies [ONT]).…”

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) orientalis, Isolate LSCM4, Strain LV768

“…Parasites were grown using an in vitro culture system previously developed for L. ( M. ) orientalis axenic amastigotes ( 11 ), in Schneider’s insect medium at 26°C as promastigotes, then in M199 medium supplemented with 10% fetal calf serum (FCS), 2% stable human urine, 1% basal medium Eagle vitamins, and 25 μg/ml gentamicin sulfate, with subpassage to fresh medium every 4 days to sustain the parasite growth and viability. DNA was extracted and purified using a Qiagen DNeasy blood and tissue kit using the spin column protocol, according to the manufacturer’s instructions.…”

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) enriettii, Isolate CUR178, Strain LV763

“…We obtained intracellular amastigote parasites from six naturally infected guinea pigs from the rural area of Mandirituba (state of Paraná, Brazil). The parasites were grown using an in vitro culture system previously developed for Leishmania ( Mundinia) orientalis axenic amastigotes ( 6 ) in Schneider’s insect medium at 26°C as promastigotes, then in M199 medium supplemented with 10% fetal calf serum (FCS), 2% stable human urine, 1% basal medium Eagle vitamins, and 25 μg/ml gentamicin sulfate, with subpassage to fresh medium every 4 days to sustain the parasite growth and viability. DNA was extracted and purified using a Qiagen DNeasy blood and tissue kit using the spin column protocol, according to the manufacturer’s instructions.…”