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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Akhoundi M, Downing T, Votýpka J, Kuhls K, Lukeš J, Cannet A, Ravel C, Marty P, Delaunay P, Kasbari M, Granouillac B, Gradoni L, Sereno D (2017) Leishmania infections: molecular targets and diagnosis. Mol Asp Med 57:1–29
Barratt J, Kaufer A, Peters B, Craig D, Lawrence A, Roberts T, Lee R, McAuliffe G, Stark D, Ellis J (2017) Isolation of novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida:Trypanosomatidae) provides support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae. PLoS Negl Trop Dis 11:e0005215
al-Bashir NT, Rassam MB, al-Rawi BM (1992) Axenic cultivation of amastigotes of Leishmania donovani and Leishmania major and their infectivity. Ann Trop Med Parasitol 86:487–502
Bates PA (1994) Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology 108:1–9
Bates PA (2007) Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol 37:1097–1106
Bates PA (2018) Revising Leishmania’s life cycle. Nat Microbiol 3:529–530
Bates PA, Robertson CD, Tetley L, Coombs GH (1992) Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology 105:193–202
Chiewchanvit S, Tovanabutra N, Jariyapan N, Bates MD, Mahanupab P, Chuamanochan M, Tantiworawit A, Bates PA (2015) Chronic generalized fibrotic skin lesions from disseminated leishmaniasis caused by Leishmania martiniquensis in two patients from northern Thailand infected with HIV. Br J Dermatol 173:663–670
Debrabant A, Joshi MB, Pimenta PF et al (2004) Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Int J Parasitol 34:205–217
Espinosa OA, Serrano MG, Camargo EP et al (2018) An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum. Parasitology 145:430–442
Ghosh S, Goswami S, Adhya S (2003) Role of superoxide dismutase in survival of Leishmania within the macrophage. Biochem J 369:447–452
Gupta N, Goyal N, Rastogi AK (2001) In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 17:150–153
Hodgkinson VH, Soong L, Duboise SM, McMahon-Pratt D (1996) Leishmania amazonensis: cultivation and characterization of axenic amastigote-like organisms. Exp Parasitol 83:94–105
Jain SK, Sahu R, Walker LA et al (2012) A parasite rescue and transformation assay for antileishmanial screening against intracellular Leishmania donovani amastigotes in THP1 human acute monocytic leukemia cell line. J Vis Exp 70:4054
Jariyapan N, Daroontum T, Jaiwong K, Chanmol W, Intakhan N, Sor-suwan S, Siriyasatien P, Somboon P, Bates MD, Bates PA (2018) Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localized cutaneous leishmaniasis. Parasit Vectors 11:351
Joshi MB, Hernandez Y, Owings JP, Dwyer DM (2012) Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections. Mol Cell Biochem 361:169–179
Li J, Zheng ZW, Natarajan G et al (2017) The first successful report of the in vitro life cycle of Chinese Leishmania: the in vitro conversion of Leishmania amastigotes has been raised to 94% by testing 216 culture medium compound. Acta Parasitol 62:154–163
Moradin N, Descoteaux A (2012) Leishmania promastigotes: building a safe niche within macrophages. Front Cell Infect Microbiol 2:121
Paladi CS, Pimentel IA, Katz S et al (2012) In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis. PLoS Negl Trop Dis 6:e1626
Pan AA (1984) Leishmania mexicana: serial cultivation of intracellular stages in a cell-free medium. Exp Parasitol 58:72–80
Pothirat T, Tantiworawit A, Chaiwarith R, Jariyapan N, Wannasan A, Siriyasatien P, Supparatpinyo K, Bates MD, Kwakye-Nuako G, Bates PA (2014) First isolation of Leishmania from Northern Thailand: case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex. PLoS Negl Trop Dis 8:e3339
Rainey PM, Spithill TW, McMahon-Pratt D, Pan AA (1991) Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture. Mol Biochem Parasitol 49:111–118
Rogers ME, Chance ML, Bates PA (2002) The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitology 124:495–507
Saar Y, Ransford A, Waldman E et al (1998) Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani. Mol Biochem Parasitol 95:9–20
Schuster FL, Sullivan JJ (2002) Cultivation of clinically significant hemoflagellates. Clin Microbiol Rev 15:374–389
Seblova V, Sadlova J, Vojtkova B, Votypka J, Carpenter S, Bates PA, Volf P (2015) The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is capable of developing late stage infections of Leishmania enriettii. PLoS Negl Trop Dis 9:e0004060
Siripattanapipong S, Leelayoova S, Ninsaeng U et al (2018) Detection of DNA of Leishmania siamensis in Sergentomyia (Neophlebotomus) iyengari (Diptera: Psychodidae) and molecular identification of blood meals of sand flies in an affected area, Southern Thailand. J Med Entomol 55:1277–1283
Späth GF, Beverley SM (2001) A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol 99:97–103
Sunter J, Gull K (2017) Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding. Open Biol 7:170165
Teixeira MC, de Jesus Santos R, Sampaio RB et al (2002) A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species. Parasitol Res 88:963–968
Yao C, Chen Y, Sudan B, Donelson JE, Wilson ME (2008) Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model. Exp Parasitol 118:129–133
We thank Assoc. Prof. Dr. Sirida Yangshim of the Department of Microbiology, Faculty of Medicine, Chiang Mai University for the human monocytic cell line.
This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant number: PHD/0065/2556 to NJ for WC), the Faculty of Medicine Endowment Fund (grant number: 024/2558 to NJ), and the Diamond Research Grant (grant number: PAR-2560-04663 to NJ), Faculty of Medicine, Chiang Mai University. In addition, the Chiang Mai University provided the budget for our Excellence Center in Insect Vector Study (grant number: 2562 to NJ).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Section Editor: Nawal Hijjawi
About this article
Cite this article
Chanmol, W., Jariyapan, N., Somboon, P. et al. Axenic amastigote cultivation and in vitro development of Leishmania orientalis. Parasitol Res 118, 1885–1897 (2019). https://doi.org/10.1007/s00436-019-06311-z
- Axenic amastigote