Leishmania tropica experimental infection in the rat using luciferase-transfected parasites

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Veterinary Parasitology 187 (2012) 57– 62

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

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eishmania tropica experimental infection in the rat usinguciferase-transfected parasites

alit Talmi-Franka, Charles L. Jaffeb, Abedelmajeed Nasereddinb,c, Gad Banetha,∗

School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot 76100, IsraelDepartment of Microbiology and Molecular Genetics, Kuvin Centre for the Study of Infectious and Tropical Diseases, IMRIC, Hebrew University-Hadassahedical School, Jerusalem, IsraelAl-Quds Nutrition and Health Research Center, Faculty of Medicine, Al-Quds University, Abu-Deis, P.O. Box 20760, West Bank, Palestine

r t i c l e i n f o

rticle history:eceived 5 October 2011eceived in revised form5 December 2011ccepted 29 December 2011

eywords:eishmania tropicaat

a b s t r a c t

Leishmania tropica is the causative agent of zoonotic cutaneous leishmaniasis in differentparts of the Old World. Although it is a common cause of disease in some areas of the world,there is insufficient knowledge on the pathogenicity of this parasite in mammalian hostsand animal models. L. tropica luciferase-transfected metacyclic-stage promastigotes wereinoculated into the footpad or ear of Sprague Dawley (SD) rats. Parasite DNA was detectedby kDNA real time PCR in the blood at varying levels from 2 days to 5 weeks post infection(PI) in the absence of clinical signs. Parasite DNA was found in the spleen of all rats at the endof the study, and the parasitic load was up to 40 times higher in the spleen when compared

isceralizationuciferase

with inoculation sites. Parasites were cultured from the spleen, and skin inoculation sites5 weeks PI. Bioluminescent parasites were observed by in vivo imaging at one day PI, but thetechnique was not sufficiently sensitive to follow parasite spread after this time. This studyprovides new evidence for the viscerotropic spread of L. tropica in the rat and demonstratesthat the rat can serve as a model for persistent visceralizing infection with this parasite.

. Introduction

Leishmania tropica is a major cause of cutaneous leish-aniasis in the Middle East and in some areas of Africa. It

nflicts a disfiguring cutaneous disease in humans and maylso cause a visceral form of the disease in humans (Magillt al., 1994; Sacks et al., 1995) and dogs (Guessous-Idrissit al., 1997). The main natural animal reservoir for L. trop-ca in Israel is the rock hyrax, a wild life mammal abundantn areas where human L. tropica infection is found (Talmi-

rank et al., 2010a). Natural infection with L. tropica haslso been reported from other mammals including golden

∗ Corresponding author at: School of Veterinary Medicine, Hebrew Uni-ersity, P.O. Box 12, Rehovot 76100, Israel. Tel.: +972 3 9688557;ax: +972 3 9604079.

E-mail address: [email protected] (G. Baneth).

304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.12.035

© 2012 Elsevier B.V. All rights reserved.

jackals and red foxes (Talmi-Frank et al., 2010b), as well asrats (Massamba et al., 1998; Pratlong et al., 2009).

Experimental studies on the feeding preferences ofsand flies on different hosts have shown that Phleboto-mus halepensis females, vectors of L. tropica, feed readilyon rats (Sádlová et al., 2003), and that Phlebotomus sergentidevelop infection after feeding on asymptomatic L. trop-ica experimentally infected rats (Svobodová et al., 2003).Published information on animal experimental infectionswith L. tropica is lacking, in part because it is diffi-cult to establish infection in vivo with this Leishmaniasp. (Bastien and Killick-Kendrick, 1992). Sprague Dawley(SD) rats infected with Leishmania major or with Leish-mania donovani promastigotes showed no clinical signs(Giannini, 1985) and black rats (Rattus rattus) infected with

L. tropica did not develop clinical signs at the inoculationsite in another study (Svobodová et al., 2003). Further-more, an animal viscerotropic model of L. tropica has beenexplored only in a limited number of studies (Bastien and

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Killick-Kendrick, 1992; Lira et al., 1998; Svobodová et al.,2003; Mahmoudzadeh-Niknam et al., 2007; Anderson et al.,2008), thus a rat model might contribute to the study of thehost innate and adaptive immune responses to this para-site, considering the wide range of reagents and markers forinflammatory and cellular reactions available for researchin rats.

The aim of this study was to evaluate the spread andpersistence of L. tropica in a rodent model, and to evaluatethe magnitude of visceral involvement.

2. Materials and methods

2.1. Rat infection and follow up

Two months old female SD rats obtained from HarlanLaboratories (Harlan Laboratories, Rehovot, Israel) wereinfected by metacyclic promastigotes expressing the fire-fly luciferase gene Lt:pSSU-int/LUC (L. tropica/LUC) isolatedfrom stationary phase cultures (6 days old) by Ficoll (GEHealthcare) gradient as previously described (Späth andBeverley, 2001). Two rats were injected with 4 × 107 par-asites into the ear dermis (Ear 1 and Ear 2). Two otherrats were injected with 4 × 107 parasites into the dorsalside of foot (FP1-footpad 1 and FP2-footpad 2) and fournegative control rats were injected with saline into theear dermis and footpad, respectively. Rats were weighedand inspected for lesion development. In addition, bloodwas collected from the tail vein on days 1, 2, 4 and 8 postinfection (PI) and also 2, 3, and 6 weeks PI. This study wasapproved by the Animal Ethics Committee of the HebrewUniversity of Jerusalem (approval number AG 08-11090-03) and animals were handled adhering to the HebrewUniversity institutional guidelines for research animals.

2.2. Parasite load assessment using real-time PCR

Blood and tissue parasite loads were quantified usingkDNA real-time PCR of DNA extracted from whole blood,spleen, and skin. DNA from blood was extracted using theIllustra Extraction Kit (Illustra Blood GenomiPrep Mini Kit;GE, Healthcare, Little Chalfont, UK). DNA from the skin andspleen was extracted using the guanidine thiocyanate tech-nique (Höss and Pääbo, 1993). Samples were amplified bykDNA real-time PCR (Nicolas et al., 2002), using the Rotor-Gene 6000 real-time PCR cycler (Corbett Life Science). Astandard curve generated using rat blood spiked with L.tropica promastigotes was linear over a 5-log range of DNAconcentrations.

2.3. Plasmid construction for rat PGE2˛R

The rat prostaglandin E2 (PE2) receptor gene (PGE2R),a single copy gene was used to normalize the par-asite loads relative to the host DNA. This gene wasshown to be a single copy gene in dogs (Hibbset al., 1999), mice (Katsuyama et al., 1995) and

humans (Smock and Owen, unpublished observations).The primersPGE2RF (5′-TCTCGCTGTTCCACGTGCT-3′) andPGE2RR (5′-CCAGGCTGAAGAAGGTCATGG-3′) were used toamplify a 174 bp fragment of the rat PGE2�R by real-time

asitology 187 (2012) 57– 62

PCR. This fragment was cloned into the Topo TA plasmid(Invitrogen, Carlsbad, CA, USA), and used as a template forquantitative validation of assay sensitivity, and as posi-tive controls and standards. The plasmid was introducedinto competent Escherichia coli bacteria (RBC bioscienceCorp, USA) and purified using the Ultraclean MimiPrep Kit(MoBio Carlsbad, CA, 92010). Serial dilutions from 106 to1 molecules/�l in double distilled water were prepared,and DNA concentrations determined using a Nanodrop®

ND-1000 Spectrophotometer (Nanodrop Technologies, DE,USA).

2.4. Cultivation of parasites

Tissue samples from the inoculation site in the ears andfootpads of rats, as well as spleen samples of all infectedrats, were placed in semi-solid Novy–MacNeal–Nicollemedium (NNN) (Limoncu et al., 1997) and cultivated in 24well plates as described by Maurya et al. (2010).

2.5. L. tropica ELISA assay

ELISA plates were coated overnight at 4 ◦C with75 ng/well of L. tropica antigen LRC-L1239 isolated froma human patient in Israel. The coated plates werewashed with phosphate-buffered saline (PBS) contain-ing 0.05% Tween 20 (PBS-T) and blocked with PBSplus 2% milk for 1 h at room temperature (RT). Afterwashing, rodent sera diluted 1:50 in 2% milk wereadded to each well and incubated at 37 ◦C for 1 h,followed by washing and then incubation with biotincoupled protein G diluted 1:250 (Adar Biotech, Rehovot,Israel). Binding was detected following incubation withperoxidase-conjugated streptavidin (Jackson ImmunoRe-search, Laboratories, Inc., West Grove, PA, USA) diluted1:250 in 2% milk. The plates were developed using 2,2-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) andabsorbance read at 405 nm. The positive and negative-control sera were taken from experimentally infectedhyraxes and from non-infected rats, respectively.

2.6. Production of L. tropica promastigotes expressing thefirefly luciferase gene (Lt:pSSU-int/LUC)

The Lt:pSSU-int/LUC plasmid (Lt:LUC) was prepared asfollows. The luciferase gene (LUC) was excised from thepCEP4-LUC (Invitrogen, San Diego, CA) using the restrictionenzymes Hind III and BamH I, and ligated into pBlue-script II KS(+) (Fermentas, MA, USA) predigested with thesame enzymes. pBluescript:LUC was excised with ClaI andSpeI, and the LUC gene cloned into the polylinker of thepredigested plasmid pSSU-int/�-GAL (Misslitz et al., 2000)which was a gift from T. Aebischer, Robert Koch Institute,Germany.

The destination vector was used to transform DH5�competent bacteria cells, and plasmid DNA purified frommini-preparations of positive colonies (QIAprep Spin

Miniprep Kit, Qiagen).

For transfection, the plasmid pSSU-int/LUC (5 �g) waslinearized by digestion with PmeI and PacI accord-ing to the manufacturers instructions (New England

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ig. 1. Parasite loads in blood of the infected and control SD rats. Parasiteingle copy gene. Footpad inoculation (FP1, FP2), and ear pinna inoculatio

iolabs, MA, USA), and purified with Wizard SV Gelnd PCR Clean-Up (Promega, Madison, WI, USA). Theurified linearized vector used to transfect L. tropicaMHOM/IL/1990/P283, LRC-L590 or MHOM/IL/2002/LRC-863) promastigotes essentially as previously describedBeverley and Clayton, 1993). Linearized DNA (5 �g) wasdded to 400 �l parasites (108 cells) diluted in cytomixuffer in a EC Gene Pulser cuvette (2 mm cuvette, Bio-ad, Hercules, CA, USA), and pulsed twice with 1600 V, 200EM, 25 �F (ECM 630, BTX, Holliston, MA). Parasites were

ncubated on ice for 10 min, and cultured for 24 h afterhich stably transfected Lt:LUC (mutant) promastigotesere selected with hygromycin (25 �g/ml).

.7. Measurement of in vitro and in vivo bioluminescence

In vitro luciferase activity of the different Leishma-ia/LUC parasites (MHOM/IL/2006/LRC-L590; MA 11/8nd MA 3/8, and MHOM/IL/2002/LRC-L863) was evalu-ted using a luminometer (Berthold®, GmbH) and theightOwl® imaging system (Berthold technologies). Ten-

old parasite dilutions (7 × 107 to 70 cells/50 �l well)ere made in a 96 well black microtiter plate (NUNCTM,enmark) and evaluated 5, 10 and 15 min after the luciferin

ubstrate addition.The in vivo activity and spread of the L. tropica/LUC par-

sites was followed in the experimentally infected rats.ats were anaesthetized using ketamine hydrochloride 10%

nd xylazine 2%. A working stock containing 2:1 ratio ofetamine and xylazine, respectively, was prepared, andats were given a dose of 0.1 ml/100 g using the intra-uscular route at different time points after infection and

s were quantified using kDNA real-time PCR and adjusted to the PGE2�R, Ear 2).

were then injected intra-peritoneally with 1.5 ml luciferin(Caliper Life Sciences) per rat (15 mg/ml final concentrationdissolved in sterile water for injection). Measurements ofluminescence using the NightOwl® imaging system using acharge couple device (CCD) camera began 5 min after sub-strate injection to allow the spread of the luciferin in therat’s body. Exposure time was one minute with high reso-lution pixel binning of 5 × 5 mode at 5, 7, 10, 13 and 15 minpost substrate injection. Images were taken at dorsal aswell as ventral positions creating a grey-scale result andwere overlaid with pseudo-colored luminescence show-ing the number of photons emitted from chosen region ofinterest (ROI).

3. Results

3.1. Parasite quantification and tissue distribution

Parasites were isolated and cultured from the spleen aswell as from the inoculation sites of all rats at the end of thestudy, 7 weeks PI. Quantification of the Leishmania parasiteload in the spleen and skin was done using kDNA real-timePCR normalized to the rat PGE2R gene as described above.Each sample was run in duplicates and used for amplifica-tion of both kDNA and PGE2R. Parasites were detected inthe blood at 4 days PI among all rats (Fig. 1). There was apeak in the parasite loads in the ear of infected rats at day4 PI and at day 2 or 8 in the foot of infected rats, followed

by a drop and disappearance of parasites from the bloodat 15 days PI. Two rats infected in the dorsal foot expe-rienced a rise in parasite loads in blood between 22 and43 days PI, while the ear infected rats continued to have low

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Fig. 2. Parasite loads in spleen samples taken post mortem, adjusted to

Fig. 3. Parasite load in SD rats inoculated intra-dermally with luciferase-expressing metacyclic L. tropica parasites one day post inoculation. Similar

the single copy gene PGE2� and related to the parasite numbers at theinoculation site. Footpad inoculation (FP1, FP2); and ear pinna inoculation(Ear 1, Ear 2).

blood parasite loads throughout the whole study period.Parasite loads at two sites were also examined post mortemat 7 weeks PI. The spleen had 2–40 times higher parasiteloads when compared to the inoculation sites (Fig. 2). Theparasite numbers in the spleen tissue ranged between 10and 100/per 1000 tissue cells following 7 weeks of infec-tion, regardless of the inoculation site chosen.

3.2. L. tropica ELISA assay

Sera samples were collected at 7 weeks PI from the tailvein of four infected rats and two non-infected rats, andexamined for antibodies to crude L. tropica antigen. No sig-nificant difference was observed between the infected andnon-infected negative control animals (data not shown).

3.3. In vitro bioluminescence, uptake of substrate andemission of L. tropica/LUC cultured parasites

Luciferase activity of the three L. tropica/LUC para-site isolates was examined. MA11/8 (LRC-L590) originallyisolated from a cutaneous leishmaniasis patient in the vil-lage of Kfar Adumim, showed the highest activity of thethree mutant isolates. This mutant isolate showed a higherreading, and the lowest threshold of detection, 5 × 104

promastigotes, was reached by using 10-fold dilutions ofL. tropica/LUC cultured promastigotes, and the NightOwl®

system as described above. Both live and lysed parasitesfrom the MA11/8 LRC-L590 parasites showed the highestRLU activity when compared to the two others (data notshown), and it was selected for the experimental rat infec-tion.

3.4. In vivo imaging of infected rats

LUC-transfected Leishmania parasites were visible one-day PI at the inoculation site (Fig. 3). The rats were scannedalso at 2, 4, and 8 days PI and 2, 3 and 6 weeks PI, but nobioluminescent parasites were detectable.

4. Discussion

Despite the widespread endemicity of L. tropica and theability of this parasite to cause a visceralizing disease, there

results were obtained for all rats (n = 4). Parasite burdens were visualizedby in vivo imaging on a NightOwl® imaging system. Luminescence wasexpressed in (Photons/sec/ROI). Use the same abbreviations throughout.

is currently insufficient information on animal models forviscertropic leishmaniasis caused by L. tropica. Further-more, information is lacking on the host responses to thisdisease and the dynamics of the infection in the animalhost.

By infecting rats with metacyclic-stage promastigotes,we have demonstrated the growth and persistence in theblood, as well as splenic visceralization, of L. tropica para-sites despite the absence of apparent clinical disease. Theabsence of clinical signs and persistence of infection arein agreement with the study by Svobodová et al. (2003),that found that rats infected with L. tropica had varyingamounts of parasites (5 × 103–106) at the inoculation siteand remained asymptomatic for two years, but did notdemonstrate visceralization of the parasite. In a separatestudy, L. tropica infected C57BL/6 mice (Anderson et al.,2008) were shown to develop very small (1 mm) lesions8 weeks PI. Another study compared the clinical outcome ofBALB/c mice infection with hamster infection using severalL. tropica isolates, and showed differences in the progres-sion and manifestations of infection depending on theparasite strain used for infection (Lira et al., 1998). Lesionsin mice inoculated into footpad were non-progressive andnon-ulcerative, but did not heal. Parasites were retrievedfrom the inoculation site and draining lymph nodes upto 9 months PI, without evident dissemination to the liverand spleen by any of the strains examined. When hamsterswere inoculated in the footpad, it was possible to cultureor detect parasites by microscopy in tissues 12 monthspost-inoculation in the absence of any gross pathologyof the foot. However, parasites, especially from isolatesoriginating in patients with visceral L. tropica infection,did visceralize to the spleen and liver. These experimen-tal studies show that L. tropica strains may have differenttissue tropism and virulence in different animal hosts.

In our study, bioluminescent parasites were demon-strated in the ear pinna, as well as the foot, one-dayPI verifying that live parasites existed at the inoculation

site. However, 48 h PI, the majority of the parasites wereremoved from tissues, possibly by the innate immune sys-tem, resulting in the loss of bioluminescent signal. The limitof detection in vitro using the Nightowl® system was 104

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arasites, therefore we could not determine by this methodf parasites below this level remained at the site of injection.

Several other studies have used bioluminescent detec-ion systems to follow experimental infection of miceith Leishmania species other than L. tropica with dif-

erent detection limits (Roy et al., 2000; Lang et al.,005; Thalhofer et al., 2010). The detection limit of04 parasites per region of interest at the inoculationite was reached also in a study using mice infectedith Leishmania mexicana or L. chagasi and followedsing the in vivo imaging system Xenogen IVIS 200Caliper Life Sciences) (Thalhofer et al., 2010), whereas,nother study using L. amazonensis infection in BALB/cice, reported clinical signs in the ear at the site of

nfection and the minimum number of amastigotesxpressing luciferase was 103 (Lang et al., 2005).

Although limited number of rats was used in our study,he different inoculation routes did not seem to influencehe clinical outcome or ability to detect parasites. Despitehe lack of systematic or local signs of disease in the infectedats, parasites reached the animals viscera in all cases, andive parasites could be retrieved from the spleen post-

ortem and grown in culture indicating that infectionersisted in the rats.

Rats may serve as natural hosts of L. tropica. Wildodents including rats have been suspected as reser-oir hosts of cutaneous leishmaniasis (Giannini, 1985;aliba Disi et al., 1994). Natural infection with L. trop-ca has been described in rats from Iraq and KenyaMassamba et al., 1998; Pratlong et al., 2009), and Phle-otomus halepensis sand flies responsible for L. tropicaistribution fed readily on rats under laboratory set-ings (Sádlová et al., 2003). In addition to these reports,ild caught rats from Turkey experimentally infectedith L. tropica promastigotes showed long term parasiteersistence while remaining asymptomatic and infec-ious to sand flies (Svobodová et al., 2003). However,t was not demonstrated that these rats had visceralrgan infection. Furthermore, in a study performed inyprus by Psaroulaki et al. (2010), 7.3% of the rats thatere caught were serologically positive for Leishmania

pp., but no characterization of the infecting speciesas made. However, under our experimental settings,

era tested 8 weeks PI revealed very low titers of anti-. tropica antibodies indicating that serological tests mayot serve as a good marker for L. tropica infection inats.

The fact that the rats did not develop clinical signs due tonfection in this study, and in a previous study (Svobodovát al., 2003), indicates that they may serve as an experimen-al animal model for L. tropica behavior in reservoir wildlifeuch as hyraxes, in which L. tropica infection shows little oro cutaneous signs (Talmi-Frank et al., 2010a), yet it mightot mimic symptomatic human infection with cutaneousanifestations.In conclusion, experimental L. tropica infection was

nduced in the rat. It caused an asymptomatic, visceral-sing and persistent infection indicating that this model

ay be useful in further research on leishmaniasis caused

y this pathogen and the role of reservoir animals in theisease.

asitology 187 (2012) 57– 62 61

Acknowledgements

This study was supported by the Deutsche Forschungs-gemeinschaft (DFG) Grant no. SCHO 448/8-1 “Emergenceof Cutaneous Leishmaniasis in the Middle East: An inves-tigation of Leishmania tropica in the Palestinian Authorityand Israel”, and the Israel Science Foundation (ISF) Grantno. 135/08 “Cutaneous Leishmaniasis caused by Leishmaniatropica is an emerging zoonosis in Israel”.

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