Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Chanmol, Wetpisit and Jariyapan, Narissara and Somboon, Pradya and Bates, Michelle D. and Bates, Paul A. (2019) Axenic amastigote cultivation and in vitro development of Leishmania orientalis. Parasitology Research, 118 (6). pp. 1885-1897. ISSN 0044-3255

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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.

Item Type: Journal Article
Journal or Publication Title: Parasitology Research
Additional Information: The final publication is available at Springer via http://dx.doi.org/10.1007/s00436-019-06311-z
Subjects:
Departments: Faculty of Health and Medicine > Biomedical & Life Sciences
ID Code: 133312
Deposited By: ep_importer_pure
Deposited On: 03 May 2019 08:40
Refereed?: Yes
Published?: Published
Last Modified: 15 Sep 2019 03:34
URI: https://eprints.lancs.ac.uk/id/eprint/133312

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