Axenic amastigote cultivation and in vitro development of Leishmania orientalis

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. 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 d...

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Main Authors: Wetpisit Chanmol, Narissara Jariyapan, Pradya Somboon, Michelle D. Bates, Paul A. Bates
Format: Journal
Published: 2019
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http://cmuir.cmu.ac.th/jspui/handle/6653943832/65254
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Institution: Chiang Mai University
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spelling th-cmuir.6653943832-652542019-08-05T04:44:13Z Axenic amastigote cultivation and in vitro development of Leishmania orientalis Wetpisit Chanmol Narissara Jariyapan Pradya Somboon Michelle D. Bates Paul A. Bates Agricultural and Biological Sciences Immunology and Microbiology Medicine Veterinary © 2019, Springer-Verlag GmbH Germany, part of Springer Nature. 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. 2019-08-05T04:30:58Z 2019-08-05T04:30:58Z 2019-06-01 Journal 14321955 09320113 2-s2.0-85065840133 10.1007/s00436-019-06311-z https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85065840133&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/65254
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Agricultural and Biological Sciences
Immunology and Microbiology
Medicine
Veterinary
spellingShingle Agricultural and Biological Sciences
Immunology and Microbiology
Medicine
Veterinary
Wetpisit Chanmol
Narissara Jariyapan
Pradya Somboon
Michelle D. Bates
Paul A. Bates
Axenic amastigote cultivation and in vitro development of Leishmania orientalis
description © 2019, Springer-Verlag GmbH Germany, part of Springer Nature. 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.
format Journal
author Wetpisit Chanmol
Narissara Jariyapan
Pradya Somboon
Michelle D. Bates
Paul A. Bates
author_facet Wetpisit Chanmol
Narissara Jariyapan
Pradya Somboon
Michelle D. Bates
Paul A. Bates
author_sort Wetpisit Chanmol
title Axenic amastigote cultivation and in vitro development of Leishmania orientalis
title_short Axenic amastigote cultivation and in vitro development of Leishmania orientalis
title_full Axenic amastigote cultivation and in vitro development of Leishmania orientalis
title_fullStr Axenic amastigote cultivation and in vitro development of Leishmania orientalis
title_full_unstemmed Axenic amastigote cultivation and in vitro development of Leishmania orientalis
title_sort axenic amastigote cultivation and in vitro development of leishmania orientalis
publishDate 2019
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85065840133&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/65254
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