of Leishmania amazonensis in vitro. Received: 11 June 1996 / Accepted: 30 July 1996. Leishmaniasis is a significant cause of morbidity and mortality in tropical ...
Parasitol Res (1997) 83: 401–403
Springer-Verlag 1997
SHORT COMMUNICATION
Denis Sereno · Jean-Loup Lemesre
Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro
Received: 11 June 1996 / Accepted: 30 July 1996
Leishmaniasis is a significant cause of morbidity and mortality in tropical and subtropical areas. These protozoan parasites exist as a flagellate extracellular promastigote form in their sandfly vectors. In the mammalian hosts, only the nonflagellate amastigote form persists, surviving and dividing in the phagolysosome of macrophages. Leishmania promastigotes have often been quantified in vitro by an enzymatic method that involves the conversion of a tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into a colored, insoluble formazan product, the amount of which depends on the number of viable parasites present (Mossman 1983; Cole 1986; Rabinovitch et al. 1986; Berg et al. 1994). The use of amastigote forms has been hampered by difficulties in obtaining sufficient amounts of the purified mammalian stage. Toward that end, we and other investigators have recently developed culture systems for the in vitro growth of large amounts of extracellular amastigote forms that are free of host-cell contamination (Bates 1993; Lemesre, patent PCT/FR 94/00577). In the present paper we report the use of an MTT-based microassay to quantify the proliferation and the viability of axenically grown amastigote forms of L. amazonensis. The efficiency of the method was compared with that of another conventional method, cell counting. A general procedure was used in the current study to generate large quantities of each of the parasite stages. Active and dividing populations of axenically grown amastigote forms of L. amazonensis (MHOM/BR/76/ LTB-012) were maintained at 32 ± 1 °C by weekly subpassage in a cell-free medium called MAA (medium for axenically grown amastigotes) in 25-cm2 flasks D. Sereno (&) · J.-L. Lemesre ORSTOM, Laboratoire d’Epide´miologie des Maladies a` Vecteurs, Unite´ de Biologie Parasitaire, BP 5045, F-34032 Montpellier Ce´dex 1, France
(Lemesre et al. 1994; Lemesre, patent PCT/FR 94/00577). From a starting inoculum of 5 × 105 amastigote forms/ ml a cell density of about 5 × 107 parasites/ml was obtained on day 7. Promastigote cultures were maintained at 25 ± 1 °C in RPMI 1640 medium (Gibco BRL) buffered with 25 mM HEPES and 2 mM NaHCO3 (pH 7.2) and supplemented with 20% heat-inactivated fetal calf serum (FCS). Initial parasite concentrations were 5 × 105 flagellates/ml of medium. For verification of the correlation between the MTT-based assay and the number of parasites, increasing concentrations of latelog-phase parasites, ranging from 2.5 to 40 × 105 cells/ well, were distributed in triplicate in 100 ll of media in 96-well flat-bottom microtrays. Plates were then incubated with 10 ll of MTT (10 mg/ml) for 4 h at 25° or 32 ± 1 °C, depending upon the parasite stage. The enzyme-substrate reaction was stopped by the addition of 100 ll of a solution of 50% isopropanol and 10% sodium dodecyl sulfate (SDS; pH 5.4). Microplates were further incubated for 30 min at room temperature. The optical density (OD) was determined at 570 nm with a Titertech 96-well scanner. To evaluate the possibility of using the MTT-based assay to achieve growth kinetics curves as is possible with cell-counting experiments, microorganisms were seeded at two inocula (1 and 2 × 105 parasites/well) and the dehydrogenase activities were measured daily as described above. Cell concentrations were determined by daily counting of parasites in a Thoma chamber at 400x magnification after adequate dilution in 0.01 M phosphate-buffered saline (PBS, pH 7.2). Morphological aspects of parasites were determined daily by microscope examination to detect the possible occurrence of differentiation phenomena in 96well plates. As shown in Fig. 1, for densities higher than or equal to 2.5 × 105 parasites/well a linear correlation between the OD signal and the parasite number was revealed when both amastigote and promastigote forms were used. Amastigote forms of L. amazonensis showed lower levels of dehydrogenase activities than did their corresponding promastigote forms. This difference was
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Fig. 1 Relationship between the number of amastigote and promastigote forms of Leishmania amazonensis and the dehydrogenase activities, respectively. Results are given as mean values for three experiments; The SDs ranged between 2% and 8%
consistent with the relatively poor development of the mitochondria in amastigote forms as compared with promastigotes (Marr 1980; Mukkada et al. 1985). The relationship between increasing concentrations of amastigote forms and dehydrogenase activities showed that an MTT-based assay can be applied to this parasite stage. When amastigote forms were cultured in 96-microwell plates, typical parasite-growth curves were obtained, showing an initial latent phase of growth followed by logarithmic and stationary phases and cell death, leading to a decrease in density (Fig. 2A). Cell yields at the culture confluence were about 5–6 × 106 parasites/well, depending on the initial inoculum, as has previously been shown for promastigote forms of different leishmanial species (Lemesre et al. 1988). The time at which the latent phase finished and the point at which the logarithmic phase merged into the stationary phase Fig. 2 A, B Growth curves generated for axenically grown amastigote forms of L. amazonensis in 96-well plates as determined by A counting and B MTT test. Parasites were seeded at two cell concentrations. The results are given as mean values for three experiments
were also dependent on the original density. Under our experimental conditions, analysis of the morphological parameters did not show the occurrence of differentiation phenomena of amastigote into promastigote forms during parasite growth. A comparison of the results given in Figs. 2A and 2B clearly demonstrated that whatever the initial density, the latent periods were not monitored by MTT, which showed an increase in the OD signal. After the 1st or 2nd day of culture an increase in the OD value was always correlated with an increase in the number of parasites until the 5th day of culture. However, after the 3rd and 4th days of culture, depending on the initial cell concentration, a slight increase in the OD signal was observed even if a large increase in parasite number was monitored (Fig. 2A, B). Cell death, which occurred after 8 days of culture when parasites were seeded at 2 × 105 cells/well, was poorly monitored by MTT, which showed only a slight decrease in the OD signal. The variations observed between the counting method and the MTTbased assay could be explained by the observation that dividing cells release more formazan than do resting cells (Mosmann 1983). These data showed that cultures initiated with 2 × 105 amastigote forms/well in 96-well flatbottom microtrays followed by 72 h of incubation were the most suitable conditions for performance of the MTT-based assay to quantify the proliferation of extracellular amastigote forms of L. amazonensis. As shown in Fig. 3, the leishmanicidal activity of pentamidine was tested successfully. Pentamidine depressed both the OD570 signal and the parasite number in a dose-dependent manner as compared with the respective controls. It is noteworthy that measurement via the MTT-based micromethod reflects cellular survival. Pentamidine had only poor activity, if any, at concentrations ranging from 1 to 2 lM. A maximal effect was seen at concentrations higher than 5 lM. These results demonstrate that pentamidine was directly toxic to dividing amastigote forms. Finally, dimethylsulfoxide (DMSO) and ethanol could be used as drug solvents
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screening of new potential agents, and a system in which the sensitivity of new isolates of Leishmania could be ascertained. It represents a general and useful tool in the field of parasitology. Acknowledgements This work was supported by grants from INSERM-CNAM (921303) and from the ORSTOM institute. We thank J. L. Chevrolier for revising the language in the manuscript.
References
Fig. 3 Leishmanicidal activity of increasing concentrations of pentamidine on L. amazonensis amastigote forms. Cells were seeded at 2 × 105 parasites/well; after an incubation period of 72 h, dehydrogenase activities were measured and parasites were counted by microscope examination. The results are expressed as the percentage of viable cells as compared with the control without drug and represent mean values for three experiments
with minor cytotoxic effects at concentrations lower than 1% and 2%, respectively (data not shown). This report demonstrates that it is now possible to perform a rapid, simple, and reliable colorimetric microassay for evaluation of the proliferation, viability, and dehydrogenase activities of axenically cultured amastigote forms. The currently available methods for in vitro drug screening on the amastigote forms are based on the use of infected mouse or human macrophages (Berman and Wyler 1980). To our knowledge, we describe for the first time the use of axenically cultured amastigote forms for the determination of drug sensitivity. This in vitro model may therefore provide a tool to investigate the pharmacological effect of antileishmanial agents at a cellular level, a method for early
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