Leishmania donovani s.l.: Evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry


Leishmania infantum causes visceral leishmaniasis in all countries in the Mediterranean basin. It uses Phlebotomine sandflies as vectors where the promastigote stage develops, reproduces and becomes infective. Therefore the reproductive power of the promastigotes determines the inoculum size of the iso- late. Ten Leishmania strains from Cyprus: two Leishmania donovani and eight L. infantum were used to study the proliferation capacity of the promastigotes. Population increase during a 6-day culture period was assessed quantitatively, by haematocytometer enumeration, and qualitatively by following the divi- sion history of each population during the same period by CFSE staining and flow cytometry. The strains exhibited different proliferation rates with L. infantum showing higher multiplication rates than L. donovani. These differences may represent their fitness capabilities and their ability to synchronize the multiplication activity of individual members in the population for the production of a sizeable inoculum in time for the vector’s blood meal.

1. Introduction

Leishmania, a protozoan parasite belonging to the family Try- panosomatidae, causes leishmaniases, a group of diseases with clin- ical manifestations that range from self-healing cutaneous to fatal visceral disease (VL) if untreated. Over 10–15 million people are estimated to be infected worldwide, with 400,000 new cases each year (WHO, 1981). Leishmania infantum is the causative agent of human and canine visceral leishmaniasis in the Mediterranean ba- sin, where it is endemic (Gramiccia et al., 1992).

Leishmania presents a complex life cycle that involves Phlebot- omine sandfly vectors and more than 100 species of mammalian hosts (Lima et al., 2008). The parasite leads a digenetic life cycle; the amastigote that resides within the phagolysosome of macro- phages and the promastigote in the midgut of the sandfly insect vector (Descoteaux and Turco, 1999). The sandfly takes amastig- otes with infected macrophages when feeding on the mammal res- ervoir host. Condition changes (decrease in temperature, increase in pH) triggers the development of the parasite in the vector (Kam- hawi, 2000; Bates and Rogers, 2004). The amastigotes, released from the macrophages into the midgut live extracellularly (Brittingham et al., 1995) and transform into non-infective procyclic promastigotes (Sacks and Perkins, 1984) multiplying by binary fis- sion. In less than a week the parasites enter a stationary phase and differentiate into nectomonad promastigotes that move from the posterior to the interior midgut where they transform into lepto- monads resuming replication at the anterior midgut (Gossage et al., 2003). After a final transformation into highly infective meta- cyclic promastigotes (Sacks and Perkins, 1984; Rogers et al., 2002), they move to the oesophagus and the salivary glands; the key step in the transmission of the parasite to the mammalian host (Antoine et al., 1998; Rittig and Bogdan, 2000; Burchmore and Barrett, 2001; Kima, 2007; McConville and Handman, 2007).

Quantifying Leishmania population growth is involved in a number of investigations for comparing strains in competition studies associated with co-infections, in assays for testing drug efficacy, etc. (Choisy et al., 2004). In such studies the promastigote stage is most often used. Since the promastigote is the infective form of the parasite, the proliferation capacity of a strain plays a key role in its infectivity potential. However, no qualitative studies on the multiplication rate of the promastigote have been done. The aim of this work was to assess and compare the growth kinetics of 10 strains of Leishmania promastigotes in vitro. For this, two L. donovani, MON-37 isolated from human patients and eight L. infantum, MON-1 isolated from dogs in Cyprus (Antoniou et al., 2008), were used. The vital fluorescent stain CFSE (carboxyfluorescein succinimidyl ester) was used to assess cell divisions in each population by measuring the relative fluorescence intensity of the culture daily during a 6-day period using flow cytometry. CFSE stain attaches to the cytoplasm and thereby stains the whole cell, and its fluorescence is halved during each cell division thus allow- ing the monitoring of cell proliferation while it does not adversely affect cellular function (Lyons, 2000). The flow cytometric data were compared to daily counts of population growth obtained by haematocytometer enumeration.

2. Materials and methods

2.1. Parasites and culture conditions

Ten Leishmania strains isolated in Cyprus from different areas of the island; seven from Pafos and Limassol (coastal areas) and three from Nicosia (inland) (Antoniou et al., 2008), were used. The para- sites were isolated from the blood of eight dogs (L. infantum MON- 1, D1–D8) and two patients (L. donovani MON-37, H1 and H2) (Ta- ble 1). Two media were used for the isolation of the parasites at 26–27 °C: the NNN (Novy – MacNeaL – Nicolle) medium consisting of 14 g/l agar (plain, non-nutrient), 6 g/l NaCl and 150 ml/l defi- brinated rabbit or sheep blood (Ozbilgin et al., 1995) and the RPMI 1640 liquid culture medium (Gibco–BRL, UK) supplemented with 2 mM L-glutamine, 20 mM Hepes buffer, 10% FBS, 100 IU/ml peni- cillin and 100 lg/ml streptomycin (Lightner et al., 1983; Lemesre et al., 1988). Following successful culture, the parasites were stored in liquid nitrogen with 8% DMSO. Freshly defrosted cultures in supplemented RPMI 1640 culture medium were used for the study using parasites after the 3rd or 4th passage.

2.2. Quantitative determination of the multiplication rate of the 10 isolates

Log phase promastigotes of each of the 10 isolates were inocu- lated into 5 ml of supplemented RPMI 1640 culture medium, in
triplicates, at 26 °C ± 1 °C at a starting concentration of blue stains dead cells and was used in order to count only live parasites.

2.3. Qualitative determination of the multiplication rate of the 10 isolates

Log phase promastigotes of each of the 10 isolates were inocu- lated into 5 ml supplemented RPMI 1640 culture medium, in trip- licates, at 26 °C ± 1 °C at a starting concentration of 1 106/ml in a six-well plate. Subsequently, the parasites were labelled with 1 ml of 10 lM CFSE solution (Molecular Probes, Eugene, OR) in PBS, for 5 min at 26 °C. Labelling was quenched with an equal volume (6 ml) of supplemented RPMI 1640 culture medium (Smirlis et al., 2006). Parasites were then washed twice in PBS and cultured in 5 ml supplemented RPMI 1640. The parasites were maintained at 26 °C±1 °C and the culture medium was changed every 2 days, after centrifugation of the plates at 700g, for 10 min, at ambient temperature. The rate of multiplication was determined daily, for 6 days, as follows: 0.5 ml of the culture, at ambient temperature, with 10 lg/ml propidium iodide (PI) to differentiate dead cells (Ka- mau et al., 2000) was analysed per sample using flow cytometry to read the CFSE signal (Epics Elite Coulter, Florida, USA). This was performed in triplicate, and the average was calculated. The fluo- rescent intensity is reduced by half after each cell division, thus providing readout of the mitotic activity within each population. The distribution of the fluorescent intensity was divided into five channels. The initial population had the strongest signal (undi- vided cells; channel 1) and subsequent channels (2–5) indicated the number of cell divisions at a given time frame. WinMDi 2.8 software was used to generate the 3D histogram overlays.

3. Results

Quantitative determination of the multiplication rate of the promastigotes measured using the haemocytometer was different for each of the 10 isolates. With all isolates’ starting population of 1 × 106 parasites/ml, the population size after a 6-day culture period reached 4 × 106 (minimum) to 80 × 106 (maximum) parasites/ ml. The cultures were maintained in six-well plates (Costar,×Corning Inc., NY) and the culture medium was replaced every 2 days with fresh, after centrifugation of the plates at 700g for 10 min, at ambient temperature.
The rate of multiplication was determined under the light microscope by counting the number of parasites per ml every day for 6 days, using a haemocytometer. For this, 0.1 ml of the par- asite culture was diluted 1/10 in 10% formaldehyde solution (Scharlau Chemie S.A., Spain) in PBS, pH 7.2, to slow down promas- tigote movement; this was further diluted with Trypan blue solu- tion (Sigma–Aldrich, USA) so as to contain 0.4 mg dye/ml. Trypan ml. The two L. donovani isolates had the lowest final population size of 4 106 and 8 106 parasites/ml (Fig. 1) compared to the eight L. infantum isolates (10.1–80 106 parasites/ml). Over the 6-day culture period, the number of promastigotes increased be- tween 300% and 7900% with L. donovani isolates having the lowest increase, 300% and 700% (Fig. 1). The average daily multiplication rate over the 6-day culture period ranged between an average of 32.35% and 168.08%. Again the two L. donovani isolates presented the lowest averages (32.35% and 52.56%) (Table 2).

3.1. Qualitative determination of the multiplication power of the promastigotes

The number of live and non-apoptotic parasites dividing each day, measured using flow cytometry and the vital stain CFSE, was different for each isolate. The average percentage of parasites dividing five times (five generations) by day 6 ranged between 0.3% and 53.5% (Table 3 and Figs. 2 and 3). During the 6 day cultur- ing period, 0.1% and 4.1% of the parasites of the two L. donovani iso- lates did not divide at all whilst all members of the populations of L. infantum divided at least once during the first 2 days (day 3 of the experiment) (Table 3 and Figs. 2 and 3).

4. Discussion

The results obtained using two complementary methods to study population growth, flow cytometry and the haemocytometer, were consistent for the 10 Leishmania isolates studied. Quanti- tatively and qualitatively analysis showed similarities and differences amongst strains’ multiplication potential. In a closed experimental system, the accumulation of toxins tends to decrease the pH of the medium and the non-availability of nutrients causes reduction in population growth. At the same time, the decrease in pH causes the transformation of procyclic into metacyclic prom- astigotes, as it happens in the midgut of the sandfly. In order to avoid this stress factor, the culture medium was changed every 2 days and so cell proliferation continued for 6 days. The number of dead parasites in the 10 Leishmania cultures, during the experi- ments, was similar in all conditions used and varied between 0.3% and 2%. The parasites used were cultures after 3–4 passages only to make sure that the isolates had not adapted to the in vitro conditions.

The results of the daily counts of the 10 populations were compared with the results from the flow cytometry data indicating the proliferation activity of individual parasites in the population. Since each daughter cell inherits approximately half of the CFSE fluorescence, quantification of cell divisions is possible (Kamau et al., 2001) and so the division history of each population can be followed. Population growth during the 6-day period resulted be- tween 4 106 and 80 106 parasites/ml with all, nearly all or just a proportion of the population members undergoing divisions. However, the isolates with the lowest population growth rate were the two L. donovani strains isolated from patients (Figs. 1 and 4) and these were the only isolates in which a proportion 0.1% and 4.1% of the parasites underwent no divisions at all. Only 0.3% and 1.9% of the two L. donovani populations achieved five divisions by day 6 of culture, whilst L. infantum isolates showed five divi- sions in 1.4–53.5% of the population members (Table 3 and Figs. 2, 3). The two human isolates achieved a lower number of parasites/ ml each day due to the low proliferation activity of the members of the population. As Table 3 and Figs. 2 and 3 show the CFSE fluores- cence of the two L. donovani strains migrated between the five channels at lower rates than those of the L. infantum isolates. The average daily multiplication rate of the 10 isolates during the 6 days of culture ranged from 32.35% to 168.08% with L. donovani having the lowest rate (32.35–52.56%) in relation to L. infantum (76.60–168.08%) (Table 2).

Fig. 2. The fluorescence intensity of promastigotes as they migrated from channel 1–5 during a 6-day culture period using CFSE and read by flow cytometry. The results of three representative isolates H1, D3 and D8 are shown.

Fig. 3. Single parameter overlaid histograms of CFSE fluorescence intensity of promastigotes as they migrated from channels 1–5 during the 6-day culture period, indicating the on-going cell divisions. On the above 3D histograms isolates H1, D3 and D8 are shown.

The results of growth kinetics show that promastigotes of different species and strains have different division potential with L. donovani, isolated from patients, lower than L. infantum, isolated from dogs. These differences may be due to the different species of the parasite, the different host origin or the different Phleboto- mine sandfly vector (Diptera-Psychodidae). Phlebotomus tobbi is incriminated as the vector of both Leishmania spp. in Cyprus (Anto- niou et al., 2008, 2009; Leger and Depaquit, 2008). No correlation was found between the isolate’s multiplication capacity and the clinical symptoms of the patients or dogs. Within a Leishmania population it is possible to have a mixture of two or more different parasite strains; aneuploids or even hybrids (Ravel et al., 2006). In case of mixed populations, Leishmania can undergo genetic ex- changes during growth and development in the sandfly vector and can transmit the hybrid progeny to a mammalian host. The diversity of Leishmania species is thought to have arisen by gradual accumulation of divergent mutations rather than by sexual recom- bination (Akopyants et al., 2009). The greater the percentage of fast dividing members in a population the greater the chance of a hy- brid, that has arisen, to be passed on. The new characteristics could be related to parasite virulence or resistance to drugs, which would affect the epidemiology of the disease and public health.

Fig. 4. Population growth of the 10 isolates after a 6-day culture period in supplemented RPMI 1640 medium, counted using a haematocytometer.

It would be interesting to study strain susceptibility to drugs in relation to its proliferation capacity. Are non-dividing members of the population more/less susceptible to drugs? What is the role of these non-dividing or slow dividing parasites in the dispersal and success of the population? The in vitro multiplication of the prom- astigotes represents the parasite growth in the anterior midgut of the insect vector and is related to the parasite load to be injected to a mammalian host during the insects’ blood meal. The greater the number of promastigotes produced, the bigger the inoculum and so the greater the number of macrophages likely to be infected. An- other important factor is the population’s ability to synchronize its members’ multiplication rate for the production of a sizeable inoc- ulum in time for the vector’s next blood meal on a mammalian host, at the same time not harming the vector on the fitness of which lay its survival and dispersion.

The multiplication capacity of the isolates however may represent their adaptation potential to culture conditions, in which case, their differences indicate their fitness capabilities. The use of CFSE stain and flow cytometry for the determination of the proliferation capacity of isolates is rapid, precise, sensitive and non-costly and can be used for in the vitro screening of antileishmanial com- pounds as well as the comparison of strain characteristics.