Hostname: page-component-586b7cd67f-gb8f7 Total loading time: 0 Render date: 2024-11-26T02:16:22.720Z Has data issue: false hasContentIssue false
  • English
  • Français

The impact of distinct culture media in Leishmania infantum biology and infectivity

Published online by Cambridge University Press:  05 September 2013

NUNO SANTARÉM ,
JOANA CUNHA ,
RICARDO SILVESTRE ,
CÁTIA SILVA ,
DIANA MOREIRA ,
MARC OUELLETTE  and
ANABELA CORDEIRO-DA-SILVA
NUNO SANTARÉM
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal Centre de Recherche en Infectiologie du Centre de Recherche du CHUL, Québec, Canada
JOANA CUNHA
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar e Faculdade de Medicina, Universidade do Porto, Portugal
RICARDO SILVESTRE
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
CÁTIA SILVA
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
DIANA MOREIRA
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
MARC OUELLETTE
Affiliation:
Centre de Recherche en Infectiologie du Centre de Recherche du CHUL, Québec, Canada
ANABELA CORDEIRO-DA-SILVA*
Affiliation:
Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal
*
*Corresponding author: Parasite Disease Group, Unit of Infection and Immunity, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

An ideal culture medium for Leishmania promastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinct in vitro and in vivo infectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for the in vitro maintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

Keywords

InfectivityLeishmania infantumculture mediapromastigote cultivation
Type
Research Article
Information
Parasitology , Volume 141 , Issue 2 , February 2014 , pp. 192 - 205
Copyright
Copyright © Cambridge University Press 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Allahverdiyev, A. M., Bagirova, M., Elcicek, S., Koc, R. C. and Oztel, O. N. (2011). Effect of human urine on cell cycle and infectivity of Leishmania species promastigotes in vitro . American Journal of Tropical Medicine 85, 639–643. doi: 85/4/639 [pii] 10.4269/ajtmh.2011.10-0207.Google Scholar
Alvarez-Rueda, N., Biron, M. and Le Pape, P. (2009). Infectivity of Leishmania mexicana is associated with differential expression of protein kinase C-like triggered during a cell-cell contact. PLoS ONE 4, e7581. doi: 10.1371/journal.pone.0007581.Google Scholar
Armstrong, T. C. and Patterson, J. L. (1994). Cultivation of Leishmania braziliensis in an economical serum-free medium containing human urine. Journal of Parasitology 80, 1030–1032.Google Scholar
Bates, P. A. (2007). Transmission of Leishmania metacyclic promastigotes by phlebotomine sandflies. International Journal for Parasitology 37, 1097–1106. doi: S0020-7519(07)00126-9 [pii] 10.1016/j.ijpara.2007.04.003.Google Scholar
Bates, P. A. (2008). Leishmania sandfly interaction: progress and challenges. Current Opinion in Microbiology 11, 340–344. doi: S1369-5274(08)00083-0 [pii] 10.1016/j.mib.2008.06.003.Google Scholar
Bates, P. A. and Tetley, L. (1993). Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH. Experimental Parasitology 76, 412–423. doi: S0014-4894(83)71050-7 [pii] 10.1006/expr.1993.1050.CrossRefGoogle ScholarPubMed
Bello, A. R., Nare, B., Freedman, D., Hardy, L. and Beverley, S. M. (1994). PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major . Proceedings of the National Academy of Sciences USA 91, 11442–11446.Google Scholar
Brun, R. and Schonenberger, M. (1979). Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Tropica 36, 289–292.Google Scholar
Carvalho, S., Cruz, T., Santarem, N., Castro, H., Costa, V. and Tomas, A. M. (2009). Heme as a source of iron to Leishmania infantum amastigotes. Acta Tropica 109, 131–135. doi: S0001-706X(08)00277-5 [pii] 10.1016/j.actatropica.2008.10.007.Google Scholar
Chang, C. S. and Chang, K. P. (1985). Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis. Molecular and Biochemical Parasitology 16, 267–276.Google Scholar
Cunha, J., Carrillo, E., Sánchez, C., Cruz, I., Moreno, J. and Cordeiro-da-Silva, A. (2013). Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV− patients in the murine model of visceral leishmaniasis. Parasites and Vectors 6, 122. doi: 10.1186/1756-3305-6-122.Google Scholar
Freitas, V. C., Parreiras, K. P., Duarte, A. P., Secundino, N. F. and Pimenta, P. F. (2012). Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis . American Journal of Tropical Medicine and Hygiene 86, 606–612. doi: 86/4/606 [pii] 10.4269/ajtmh.2012.11-0386.Google Scholar
Fritsche, C., Sitz, M., Weiland, N., Breitling, R. and Pohl, H. D. (2007). Characterization of the growth behavior of Leishmania tarentolae: a new expression system for recombinant proteins. Journal of Basic Microbiology 47, 384–393. doi: 10.1002/jobm.200710111.Google Scholar
Gholamhosseinian, A. and Vassef, A. (1988). Superiority of hemoglobin to hemin for cultivation of Leishmania tropica promastigotes in serum-free media. Journal of Protozoology 35, 446–449.Google Scholar
Gossage, S. M., Rogers, M. E. and Bates, P. A. (2003). Two separate growth phases during the development of Leishmania in sandflies: implications for understanding the life cycle. International Journal for Parasitology 33, 1027–1034. doi: S0020751903001425 [pii].Google Scholar
Grekov, I., Svobodova, M., Nohynkova, E. and Lipoldova, M. (2011). Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium. Journal of Microbiological Methods 87, 273–277. doi: S0167-7012(11)00299-5 [pii] 10.1016/j.mimet.2011.08.012.Google Scholar
Hendricks, L. D., Wood, D. E. and Hajduk, M. E. (1978). Haemoflagellates: commercially available liquid media for rapid cultivation. Parasitology 76, 309–316.Google Scholar
Holzer, T. R., McMaster, W. R. and Forney, J. D. (2006). Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana . Molecular and Biochemical Parasitology 146, 198–218. doi: S0166-6851(05)00366-X [pii] 10.1016/j.molbiopara.2005.12.009.Google Scholar
Kar, K. (1997). Folic acid: the essential supplement to brain heart infusion broth for cultivation and cloning of Leishmania donovani promastigotes. Parasitology 115, 231–235.Google Scholar
Kedzierski, L. (2010). Leishmaniasis vaccine: where are we today? Journal of Global Infectious Diseases 2, 177–185. doi: 10.4103/0974-777X.62881.Google Scholar
Krishnamurthy, G., Vikram, R., Singh, S. B., Patel, N., Agarwal, S., Mukhopadhyay, G., Basu, S. K. and Mukhopadhyay, A. (2005). Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket. Journal of Biological Chemistry 280, 5884–5891. doi: M411845200 [pii] 10.1074/jbc.M411845200.Google Scholar
Limoncu, M. E., Balcioglu, I. C., Yereli, K., Ozbel, Y. and Ozbilgin, A. (1997). A new experimental in vitro culture medium for cultivation of Leishmania species. Journal of Clinical Microbiology 35, 2430–2431.Google Scholar
McCarthy-Burke, C., Bates, P. A. and Dwyer, D. M. (1991). Leishmania donovani: use of two different, commercially available, chemically defined media for the continuous in vitro cultivation of promastigotes. Experimental Parasitology 73, 385–387. doi: 0014-4894(91)90112-A [pii].Google Scholar
McNicoll, F., Drummelsmith, J., Muller, M., Madore, E., Boilard, N., Ouellette, M. and Papadopoulou, B. (2006). A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum . Proteomics 6, 3567–3581. doi: 10.1002/pmic.200500853.Google Scholar
Mendez, S., Fernandez-Perez, F. J., Santin, M., De La Fuente, C., Cuquerella, M., Gomez-Munoz, M. T. and Alunda, J. M. (2001). Correlation between in vitro and in vivo infectivity of Leishmania infantum clones. Journal of Eukaryotic Microbiology 48, 616–621.Google Scholar
Merlen, T., Sereno, D., Brajon, N., Rostand, F. and Lemesre, J. L. (1999). Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms. American Journal of Tropical Medicine and Hygiene 60, 41–50.Google Scholar
Mitchell, G. F., Handman, E. and Spithill, T. W. (1984). Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection. Australian Journal of Experimental Biology and Medical Science 62, 145–153.Google Scholar
Moreira, D., Santarem, N., Loureiro, I., Tavares, J., Silva, A. M., Amorim, A. M., Ouaissi, A., Cordeiro-da-Silva, A. and Silvestre, R. (2012). Impact of continuous axenic cultivation in Leishmania infantum virulence. PLOS Neglected Tropical Diseases 6, e1469. doi: 10.1371/journal.pntd.0001469. PNTD-D-11-00375 [pii].Google Scholar
Mougneau, E., Bihl, F. and Glaichenhaus, N. (2011). Cell biology and immunology of Leishmania . Immunological Reviews 240, 286–296. doi: 10.1111/j.1600-065X.2010.00983.x.CrossRefGoogle ScholarPubMed
Muniaraj, M., Lal, C. S., Kumar, S., Sinha, P. K. and Das, P. (2007). Milk of cow (Bos taurus), buffalo (Bubalus bubalis), and goat (Capra hircus): a better alternative than fetal bovine serum in media for primary isolation, in vitro cultivation, and maintenance of Leishmania donovani promastigotes. Journal of Clinical Microbiology 45, 1353–1356. doi: JCM.01761-06 [pii] 10.1128/JCM.01761-06.Google Scholar
Nicolle, C. (1908). Culture du parasite du bouton d'Orient. Comptes Rendus de l'Académie des Sciences 146, 842–843.Google Scholar
Nolan, T. J. and Herman, R. (1985). Effects of long-term in vitro cultivation on Leishmania donovani promastigotes. Journal of Protozoology 32, 70–75.Google Scholar
Ouakad, M., Bahi-Jaber, N., Chenik, M., Dellagi, K. and Louzir, H. (2007). Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages. Parasitology Research 101, 473–477. doi: 10.1007/s00436-007-0491-1.Google Scholar
Palomino, J. C. (1982). Peptone-yeast autolysate-fetal bovine serum 10, a simple, inexpensive liquid medium for cultivation of Leishmania spp. Journal of Clinical Microbiology 15, 949–950.CrossRefGoogle ScholarPubMed
Pescher, P., Blisnick, T., Bastin, P. and Spath, G. F. (2011). Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation. Cellular Microbiology 13, 978–991. doi: 10.1111/j.1462-5822.2011.01593.x.CrossRefGoogle ScholarPubMed
Rochette, A., Raymond, F., Corbeil, J., Ouellette, M. and Papadopoulou, B. (2009). Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum . Molecular and Biochemical Parasitology 165, 32–47. doi: S0166-6851(09)00005-X [pii] 10.1016/j.molbiopara.2008.12.012.Google Scholar
Rodrigues Ide, A., da Silva, B. A., dos Santos, A. L., Vermelho, A. B., Alviano, C. S., Dutra, P. M. and Rosa Mdo, S. (2010). A new experimental culture medium for cultivation of Leishmania amazonensis: its efficacy for the continuous in vitro growth and differentiation of infective promastigote forms. Parasitology Research 106, 1249–1252. doi: 10.1007/s00436-010-1775-4.CrossRefGoogle ScholarPubMed
Rogers, M. E., Chance, M. L. and Bates, P. A. (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.Google Scholar
Santos, M. G., Silva, M. F., Zampieri, R. A., Lafraia, R. M. and Floeter-Winter, L. M. (2011). Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes. Memorias do Instituto Oswaldo Cruz 106, 190–193. doi: S0074-02762011000200012 [pii].Google Scholar
Schuster, F. L. and Sullivan, J. J. (2002). Cultivation of clinically significant hemoflagellates. Clinical Microbiology Reviews 15, 374–389.Google Scholar
Sereno, D. and Lemesre, J. L. (1997). Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrobial Agents and Chemotherapy 41, 972–976.Google Scholar
Shamsuzzaman, S. M., Furuya, M., Korenaga, M., Imamura, K. and Hashiguchi, Y. (1999). Use of urine samples from healthy humans, nephritis patients or other animals as an alternative to foetal calf serum in the culture of Leishmania (L.) donovani in vitro . Annals of Tropical Medicine and Parasitology 93, 613–620.CrossRefGoogle ScholarPubMed
Sharief, A. H., Khalil, E. A., Omer, S. A. and Abdalla, H. S. (2008). Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species. Parasitology International 57, 138–142. doi: S1383-5769(07)00131-6 [pii] 10.1016/j.parint.2007.10.003.Google Scholar
Silvestre, R., Cordeiro-Da-Silva, A., Santarem, N., Vergnes, B., Sereno, D. and Ouaissi, A. (2007). SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection. Journal of Immunology 179, 3161–3170. doi: 179/5/3161 [pii].Google Scholar
Singh, S., Mohapatra, D. P. and Sivakumar, R. (2000). Successful replacement of fetal calf serum with human urine for in vitro culture of Leishmania donovani . Journal of Communicable Diseases 32, 289–294.Google Scholar
Soto, M., Quijada, L., Alonso, C. and Requena, J. M. (1997). Molecular cloning and analysis of expression of the Leishmania infantum histone H4 genes. Molecular and Biochemical Parasitology 90, 439–447. doi: S0166-6851(97)00178-3 [pii].Google Scholar
Srivastava, P., Sharma, G. D., Kamboj, K. K., Rastogi, A. K. and Pandey, V. C. (1997). Heme metabolism in promastigotes of Leishmania donovani . Molecular and Cellular Biochemistry 171, 65–68.Google Scholar
Steiger, R. F. and Black, C. D. (1980). Simplified defined media for cultivating Leishmania donovani promastigotes. Acta Tropica 37, 195–198.Google Scholar
Tasew, G., Kebede, A., Wolday, D., Gadisa, E., Britton, S., Eidsmo, L. and Akuffo, H. (2009). Low-cost liquid medium for in vitro cultivation of Leishmania parasites in low-income countries. Global Health Action 2. doi: 10.3402/gha.v2i0.2046.Google Scholar
Van Hellemond, J. J., Opperdoes, F. R. and Tielens, A. G. (1998). Trypanosomatidae produce acetate via a mitochondrial acetate : succinate CoA transferase. Proceedings of the National Academy of Sciences USA 95, 3036–3041.Google Scholar
Volf, P. and Volfova, V. (2011). Establishment and maintenance of sandfly colonies. Journal of Vector Ecology 36 (Suppl. 1), S1–S9. doi: 10.1111/j.1948-7134.2011.00106.x.Google Scholar
Wanderley, J. L., Pinto da Silva, L. H., Deolindo, P., Soong, L., Borges, V. M., Prates, D. B., de Souza, A. P., Barral, A., Balanco, J. M., do Nascimento, M. T., Saraiva, E. M. and Barcinski, M. A. (2009). Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis . PLoS ONE 4, e5733. doi: 10.1371/journal.pone.0005733.Google Scholar
Wheeler, R. J., Gluenz, E. and Gull, K. (2011). The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology. Molecular Microbiology 79, 647–662. doi: 10.1111/j.1365-2958.2010.07479.x.Google Scholar
Yao, C., Chen, Y., Sudan, B., Donelson, J. E. and Wilson, M. E. (2008). Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model. Experimental Parasitology 118, 129–133. doi: S0014-4894(07)00177-4 [pii] 10.1016/j.exppara.2007.06.012.Google Scholar
Zangger, H., Mottram, J. C. and Fasel, N. (2002). Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis? Cell Death and Differentiation 9, 1126–1139. doi: 10.1038/sj.cdd.4401071.CrossRefGoogle ScholarPubMed
Supplementary material: File

Santarem Supplementary Material

Supplementary Data

Download Santarem Supplementary Material(File)
File 28 MB