Visceral leishmaniasis: Experimental models for drug discovery

Axenic amastigotes

A direct comparison of the drug susceptibility towards standard antileishmnial drugs, between amastigotes and axenic amastigotes, demonstrates that the latter express specific susceptibility to many if, not all the drug tested8. Screening against axenic amastigotes (Fig. 2) presents several advantages; (i) the test is directed against the relevant stage of parasite, (ii) this stage is as easy to manipulate as the promastigote model, and (iii) quantification of drug activity is simple and often inexpensive.

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Fig. 2

Axenic amastigotes (100 X under oil immersion lens).

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Axenic amastigotes system for drug screening has been used earlier8–10. Several investigators used different methods for evaluating activity of compound against axenic amastigotes such as viability of cell population with a 3-(4-5 dimethylthiazol–2– yl) – 2, 5 diphenyl tetrazolium bromide, Thiazole blue (MTT) based method1112, determining ornithine decarboxylase activity8 or using a fluorescent dye like propidium iodide (PI) and fluorescence-activated-cell-sorter (FACS)1314. Several Leishmania parasites expressing reporter genes have been selected and the capacity of some of them to be used in axenic amastigote drug screening protocol has been assessed (Table I). Sereno et al15 assessed luciferase expressing DNA transformed axenically grown L. infantum amastigotes and showed its use in high-throughput screening for new antileishmanial drugs.

Rapid fluorescent assay using Alamar Blue for screening drugs on axenic amastigotes of L. donovani and L. tropica was done recently by Shimony and Jaffe16. But the assay is semi – predictive, it neither tests for penetration of the compound into the host cell nor for activity in the peculiar environment of the macrophage phagolysosome. In addition, axenic amastigotes may have different metabolic processes than intracellular amastigotes. Also screening with axenic amastigotes from clinical isolates is not possible because they require time to get adapted in the cultures.

Intracellular amastigotes

The most widely used models for testing drugs against Leishmania species have involved either murine peritoneal macrophages or human-monocyte transformed macrophages (THP-1, U937, and HL-60) as host cells (Fig. 3). These models show species/strain variation in drug sensitivity1718. In these differentiated non-dividing macrophages, the rate of amastigote division in host cells and drug activity can be clearly assessed. The activity of test drug is measured by either microscopical counting of percentage of infected cells or number of amastigotes/macrophage19 or by colorimetric or fluorometric methods. The slow rate of division of L. donovani and L. infantum amastigotes in this model is a limitation. Assays that use dividing host cells must ensure that the confounding effects of drug activity on both parasite and host cell number are considered6.

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Fig. 3

Stained (with giemsa stain) infected macrophages bearing amastigotes (100 X under oil immersion lens).

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GFP fluorescent assay:

GFP is an autofluorescent and stable protein, which originates from the jellyfish Aequorea victoria103132. GFP based assays offer several advantages over other non-reporter or reporter gene-based assays including greater simplicity, easier kinetic monitoring, low cost and enhanced biosafety33. Expression of GFP in several parasite species has been achieved and applied for drug evaluating studies10. GFP Leishmanial fusion proteins have been synthesized for localization and trafficking analysis34.

GFP expression in Leishmania was first achieved by Ha et al35. Since then its expression by episomal vector has been carried out in several species of Leishmania3637 wherein the fluorescence intensity in parasites decreased with time in the absence of geneticin sulphate (antibiotic G 418), thereby necessitating its regular addition38 (Table II). Okuno et al39 used the recombinant L. amazonensis expressing egfp or the beta-galactosidase gene in drug screening. Dube et al40 have also described the advantages of GFP based intramacrophagic amastigote screening assay using transfected field isolates of L. donovani strains.

Generally transfectants do not express sufficient levels of fluorescence for spectrofluorometric measurement on micro plate. To overcome this problem Chan et al41 have developed a spectrofluorometric assay wherein multimeric form of the GFP was engineered and expressed in L. amazonensis promastigotes. As expected, parasites expressing the multimeric GFP form bear fluorescence quantifiable in 96 wells with spectrofluorometric analysis. The integration of the GFP gene downstream of the 18 S rRNA gene promoters was done by Singh et al42 at the ribosomal locus within the genome of the parasite, which also represents a valuable tool for drug screening in macrophages.

Generally, methods that use catalytic reporter genes technology like luciferase, β-galactosidase, and β-lactamase are more sensitive than methods based on fluorescent proteins4344.

β - galactosidase

β-galactosidase presents the advantage that colorimetric detection can be performed. Okuno et al39 selected Leishmania Promastigotes expressing β-galactosidase and evaluated their use in drug screening procedures. Expression of lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (CPRG). Fluorescence signal and beta galactosidase activity measured by a colorimetric reaction with chlorophenol red beta D galactopyranoside. The inhibitory concentration (IC₅₀) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assays. Further, the fluorescence signal and absorbance of CPRG correlated well with the numbers of Lz/lacZ amastigotes in macrophages, respectively, suggesting La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in the macrophages in vivo. However, some commonly cited drawbacks of β-galactosidase include its large size (the monomer is 116 kDa), sensibility and the endogenous expression of β-galactosidase by some mammalian cell types including macrophages (Table II).

β–lactamase

To circumvent above shortcomings, a catalytic reporter system based on β-lactamase was developed. Two species of Leishmania: L. major and L. amazonensis expressing β-lactamase were engineered and overall, the antileishmanial results obtained demonstrate that this methodology could be valuable for drug screening procedures4546. A simple colorimetric β-lactamase assay for quantifying Leishmania amastigotes grown in microtiter plates has also been reported47. The β-lactamase gene was integrated into rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis and post- kala-azar dermal leishmaniasis isolates were transfected with β-lactamase gene. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leismanial drugs in the clinical isolates4647.

Luciferase assay

The luciferase reporter gene technology is being widely used to monitor cell growth and proliferation under in vitro culture systems and to monitor the cellular events associated with gene expression48 and signal transduction. The use of firefly luciferase reporter genes in a number of intracellular microorganisms including Mycobacterium tuberculosis49 has facilitated antimicrobial drug testing and discovery. The firefly luciferase50 represents one of the most efficient biological reporter molecules, which allows monitoring host-microbe interactions51, rapid testing of cellular viability, and thus is most suitable for biological screening. The method is rapid, very sensitive, and highly reproducible and does not require expensive specialized instrument or training. Main drawback of this system is the use of expensive substrate and lyses buffer (Table II).

Various species of Leishmania parasites expressing luciferase were recently developed and their susceptibility towards classical antileishmanial agents investigated153852. L. donovani cell lines expressing firefly luciferase reporter gene (luc.) have been developed as a part of episomal vector and suitability of these cell lines for in vitro screening of antileishmanial agents has been establised52. This system has been adapted to evaluate compounds in a 96 well micro plate format and is being employed53–55 for primary screening of novel synthetic compounds and marine extracts.

For assessing the activity of compounds against amastigote stage of the parasite, mouse macrophage cell line (J-774A.1) infected with promastigotes expressing luciferase firefly reporter gene is used5253. The main advantages of this technology include high sensitivity of the test and absence of background activity in the host cell.

Recently, a refined work performed by Lang and co-workers56 demonstrated that L. amazonensis parasites expressing firefly luciferase could be used to monitor Leishmania infection in real time, through imaging analysis. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracellular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. This proves that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing monitoring parasite-load before any clinical signs of Leishmaniasis are detectable. In short, the luciferase imaging study is useful to monitor the efficacy of antiLeishmanial drugs on live cell culture and to trace Leishmanial infection in animal models.

Limitations

Reporter genes present several limitations (Table II). Cross-resistance conferred by the presence of the antibiotic resistance is one of those. Neomycin confers resistance toward paromomycin57. Development of a method to create defined mutants lacking selectable markers could help to overcome this problem58.

The way by which the reporter gene is introduced could also have an impact on the throughput of the screen. When reporters are part of plasmids, the relative output of reporter may depend on the copy number of the transfected plasmid (which vary from cell to cell) rather than on the activity of the drug. Secondly, transforming parasites could have biological consequences either by disrupting the genomic architecture or just by the presence of the foreign reporter gene product. Thirdly, for the β-galactosidase technology, the reporter could have by itself some limitations (i.e. sensibility, background activity from host macrophages) that make it inaccurate for an in vitro determination of drug activity against intracellular parasites10.

Mouse model

Murine models of leishmaniasis have been extensively used to study the pathogenesis of the disease and to test novel therapeutic and immunoprophylactic agents65, where a relatively low amount of compound is required. These are available as SPF and inbred strains enabling reproducible results with five animals per group. Mice are susceptible to most strains and species of Leishmania in both non-cure and self cure models6667.

Outbred mice are generally resistant to infection with L. donovani (visceral Leishmaniasis) but inbred strains of mice are widely used with susceptible, resistant and intermediate strains that share some similarities with human visceral Leishmaniasis. There is a generic basis for susceptibility to infection with L. donovani, based on the presence of Sc11 1a1 locus: mice with a wild type locus (CBA) have an earlier (more vigorous) parasite growth than those with the mutated Scl 1 1a1 locus (BALB/c and C57BL/6)68–70. The infection in each mouse strain needs to be characterized for each parasite strain used to ensure that drugs are tested appropriately. Athymic and SCID mice provide a model for treatment of VL in immunosuppressed cases6.

The BALB/c mouse is a commonly used strain, at 18-20 g, with highly reproducible levels of infection when an amastigote inoculum is administered intervenously. An assay in week one/two after infection examines the activity of the drug against the liver infection but not the spleen infection2571. Briefly, BALB/c mice (both sexes) are infected intravenously with 2 × 10⁷ L. donovani amastigotes and randomly sorted into groups of five. For routine in vivo screening of newly synthesised compound/extracted plant materials are diluted three-fold to obtain three different dose levels. Mice are dosed (by ip or oral route) 7 days post-infection for five consecutive days and sacrificed 3 days after the completion of treatment (day 14 post-infection). Groups of mice are weighed before and after treatment, and the per cent weight change is recorded. Impression smears are prepared from weighed livers; methanol fixed, and stained with 10 per cent Giemsa stain in water. The number of amastigotes per 500 liver cell nuclei is determined and multiplied by the liver weight in milligrams to obtain Leishman-Donovan (LD) units. The per cent inhibition was calculated for all drug-treated groups in relation to a non treated group, and ED₅₀ values are calculated by sigmoidal regression analysis using MicroSoftxlfit (ID Business Solution, Guildford, United Kingdom). For routine in vivo screening of newly synthesised/extracted synthetic compound, plant extract are diluted three-fold to obtain three different dose levels.

Rat model

The cotton rat (Sigmodon hispidus) represents one of the most susceptible animal hosts for L. donovani72. The infection remains 3-4 months and after the appearance of initial clinical signs, the disease progresses rapidly leading to death of the host. Earlier investigetons7273 infected the African white tailed rat (Mastomys albicandatus) which proved to be an excellent host for in vivo maintenance and long term experiments with L. donovani and L. braziliensis. Nolan & Farrell75 used M. natalensis, a multi-mammate rat as an experimental model for L. donovani and L. Major, and Dwivedi et al76 successfully used this model for L. donovani.

Hamster model

Although many hamster species are susceptible to L. donovani infection77, the Syrian golden hamster (Mesocricetus auratus) establishes a good model for VL and provides a more synchronous infection in the liver and spleen that can develop into a chronic non-cure infection more similar to human VL637879.

For in vivo evaluation of chemotherapeutic agents generally the hamsters are infected intra-cardiacally. Many workers have chosen different days (day 1, 3 and 15) for initiation of drug testing. Duration of treatment (ranges from 5 to10 days) and autopsy days after treatment also differ80–82. A critical appraisal of the screening techniques by Gupta et al83 shows that none of the above is able to provide comprehensive information about the total efficacy of the potential drug. This is because the total effect of a drug depends on the parasites, and host immune system. Many drugs are known to act through the immune machinery of the host.

Methods described by Beveridge84 are more logical as the pre-treatment parasitic burden is assessed by spleen biopsy to select experimental animals carrying similar parasitic load. However, the animals are sacrificed on day 7 post-treatment. Major drawback of these methods is the inability to assess the delayed action of drugs.

Bhatnagar et al2 modified the technique where the delayed action of drugs can also be assessed conducting repeated spleen biopsies on the same animal at different intervals of day 7, 14, and 28, thus making it suitable for studying the sequential effects of drug in the model. This is more rational as it gives all information regarding cure and survival time of treated animals and allowed sufficient time to the host immunity to play, if any, a role2.

Gupta and Tiwari85 reported the suitability and susceptibility of inbred hamsters in terms of parasite establishment and longer survival period as compared to outbred hamsters. Dea-Ayuela et al86 studied its suitability and established suitable immunobiological parameters for in vivo testing of new antileishmanial compounds in the golden hamster model of visceral leishmaniasis. The clinico-pathological features of the hamster model of VL closely mimic active human disease. Systemic infection of the hamster with L. donovani results in a relentless increase in visceral parasite burden, progressive cachexia, hepatosplenomegaly, pancytopenia, hypergamma-globulinaemia and ultimately death78. The advantage is that biopsy is possible to monitor pre- and post treatment infection status and all antileishmanials are active against liver as well as spleen parasites. De-Oliveira et al87 demonstrated that the golden hamster is the best experimental model to study VL, because it reproduces the clinical and pathogenesis of the disease, as seen in humans and dogs. Unfortunately, the wide use of hamsters is still limited due to lack of available reagents such as antibodies to cell markers and cytokines.