Bioluminescent imaging of Trypanosoma cruzi - Feinberg Labs

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International Journal for Parasitology xxx (2008) xxx–xxx

Bioluminescent imaging of Trypanosoma cruzi infection

Kenneth V. Hyland, Sofya H. Asfaw, Cheryl L. Olson,Melvin D. Daniels, David M. Engman *

Department of Microbiology – Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

Department of Pathology, Northwestern University Feinberg School of Medicine, Ward Building 6-175, 303 East Chicago Avenue, Chicago, IL 60611, USA

Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL, USA

Received 22 January 2008; received in revised form 31 March 2008; accepted 3 April 2008

Abstract

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central andSouth America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. Therecent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases.We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes andamastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signalstrength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, wherea stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination ofparasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives pro-vided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skel-etal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection andunveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents,roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite–host straincombinations.� 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Trypanosoma cruzi; Luciferase; Bioluminescent imaging; Chagas disease

1. Introduction

Trypanosoma cruzi, the causative agent of Chagas dis-ease, is an intracellular, eukaryotic parasite of the familyTrypanosomatidae. Endemic to vast regions of Centraland South America, Chagas disease remains the leadingform of infectious heart disease worldwide (Kirchhoff etal., 2004). Whilst previous reports from the World HealthOrganization estimated that 16–18 million people areinfected with Chagas disease (Moncayo, 1999, 2003), amore recent analysis indicates that this number has been

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* Corresponding author. Tel.: +1 312 503 1288; fax: +1 312 503 1265.E-mail address: [email protected] (D.M. Engman).

Please cite this article in press as: Hyland, K.V. et al., Bioluminescentdoi:10.1016/j.ijpara.2008.04.002

reduced to nearly 8 million, due to progress in the controlof vectorial transmission in Latin American countries(World Health Organization, www.who.int/mediacentre/news/releases/2007/pr36/en/index.html). However, despitethe optimistic nature of this statistic, the report also statesthat cases are now being identified outside the typical ende-mic regions due to increasing incidences of blood transmis-sion (Kirchhoff et al., 2006) and organ transplantation(Nowicki et al., 2006). Chagas disease can occur in an acutephase, typically characterised by high parasitism, fever andlymphadenopathy, but more commonly progresses to achronic phase where cardiac alterations or gastrointestinaldisorders are observed.

Although the tissue tropism can vary amongst parasitestrains (Melo and Brener, 1978), it is generally thought

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imaging of Trypanosoma cruzi infection, Int. J. Parasitol. (2008),

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that, whilst capable of invading virtually any cell in thebody (Lenzi et al., 1996), T. cruzi preferentially targets neu-ronal and muscle cell-types (Lenzi et al., 1996) and its asso-ciated pathogenicities have typically been found tocorrespond to parasitosis of the myocardium (Ben Youn-es-Chennoufi et al., 1988; Jones et al., 1993; Bellotti etal., 1996; Anez et al., 1999; Zhang and Tarleton, 1999) ordigestive tract (Vago et al., 1996). These organs are oftenthe focus of Chagas disease research, since these are thewell-characterised disease manifestations. Whilst otherstudies have identified trypanosomes in liver, spleen andlung tissue (Melo and Brener, 1978) and, more recently,bone and cartilage (Morocoima et al., 2006), these distribu-tions generally follow the use of immunosuppressive ther-apy (Calabrese et al., 1992; Calabrese, 1999; Taniwaki etal., 2005) to facilitate robust parasite proliferation andexpansion. Regardless of the mode of infection or treat-ment regimen, the sacrifice of animals has typically beenrequired to obtain information on dissemination of para-sites and detection of parasites in specific tissues followinginfection. Furthermore, quantification of whole animal andorgan-specific parasite burden has been both cumbersomeand inconsistent, incorporating such techniques as PCRamplification or in situ hybridisation of parasite-specificgenes from tissue (Lane et al., 1997, 2003; Zhang and Tarl-eton, 1999; James et al., 2002), parasite antigen-specificimmunofluoresence (Ben Younes-Chennoufi et al., 1988;Chandler and Watts, 1988; Taniwaki et al., 2007) and thecounting of either nests of parasite amastigotes in tissuesections or free-swimming trypomastigotes in the blood(Nunes et al., 1990; Mortatti et al., 1992; Russo et al.,1996; Pinto et al., 1999). Whilst these approaches have cer-tainly been adequate for a variety of studies of experimen-tal Chagas disease pathogenesis, they are also accompaniedby significant limitations.

During the past several years, bioluminescence imaging(BLI) techniques have overcome these limitations in theanalysis of many disease processes, including various mod-els of cancer tumourigenesis (Edinger et al., 2002; Vooijs etal., 2002; Shachaf et al., 2004; Lyons et al., 2006; Wendt etal., 2008) and infections caused by bacteria, viruses, fungiand parasites, nicely summarised in a recent review (Hut-chens and Luker, 2007). The incorporation of in vivoBLI has not only provided a means by which to evaluatethe spatiotemporal progression of disease in real-time,but has brought about the opportunity to observe poten-tially biologically relevant interactions of host and patho-gen, in the case of infectious disease, that may haveotherwise gone unnoticed. In general, BLI detects lightresulting from the reaction of luciferase enzymes with aspecific substrate. This is made possible by utilising eitherbacterial luciferase genes capable of encoding both enzymeand substrate that are typically transferred to other speciesof bacteria, or by using luciferase enzymes from higherorganisms such as the firefly or sea pansy (Hutchens andLuker, 2007). Whilst the use of bacterial genes precludesthe use of exogenous substrate to initiate the luciferase


enzymatic reaction, employing genes from the firefly andother such organisms requires that luciferin substrate beprovided to produce detectable light.

In this study, we sought to develop bioluminescentT. cruzi for in vivo BLI analysis of infection using ourwell-established model of experimental Chagas disease.To do this, we engineered the Brazil strain of T. cruzi toexpress firefly luciferase using standard transfection meth-ods. Following infection of animals with different numbersof luminescent T. cruzi trypomastigotes, we observed clearqualitative and quantitative differences in parasite burdenup to 2 weeks p.i., after which time a similar burden wasachieved and maintained throughout the remainder ofthe acute infection. In addition to in vivo imaging of para-site infection in A/J mice, we were able to detect lumines-cence in all three life cycle stages of T. cruzi by differentmethods. Finally, we illustrate the ability to detect lumines-cence in several harvested organs in a terminal, ex vivoanalysis.

2. Materials and methods

2.1. Parasites

The Brazil, heart-derived strain of T. cruzi (Hyland etal., 2007) was used for the experiments described here.Epimastigotes, used for transfection and specific in vitroassays, were maintained in supplemented, liver digest-neu-tralised, tryptose medium (LDNT) as described previously(Kirchhoff et al., 1984). Epimastigote transfectant cultures,consisting of differentiated metacyclic trypomastigotes,were used to infect monolayers of H9C2 rat myoblasts(American Type Culture Collection, Manassas, VA) fromwhich trypomastigotes could be continually passaged andisolated. These trypomastigotes were used for all animalinfections described.

2.2. Generation of bioluminescent Trypanosoma cruzi

For stable integration of the firefly luciferase gene into thetubulin locus, we used plasmid pBS:THT-x-T (the generousgift of Wesley Van Voorhis, University of Washington). Inthis pBluescript (Stratagene, La Jolla, CA)-based plasmid,the HygTK gene is flanked by b-tubulin untranslated/inter-genic regions (UTR/IR) and the gene of interest is flanked bya-tubulin intergenic regions (Weston et al., 1999). The MluIsite in the b–a UTR/IR was converted to a unique SalI sitefor plasmid linearisation prior to transfection. The fireflyluciferase gene was amplified by PCR from the pGL3 basicvector (Promega, Madison, WI) with forward primer 50-GGATCCATGGAAGACGCCAAAAACATAAAG-30 andreverse primer 50-TCTAGATTACACGGCGATCTTTCC-30 which included the BamHI and XbaI restriction sites(underlined), respectively. The resulting amplicon wasligated into the pCR-blunt vector (Invitrogen, Carlsbad,CA) and was selected for kanamycin resistance. The lucifer-ase coding sequence was then liberated by digestion with


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BamH1 and Xba1 and the purified insert was directionallycloned into pBS:THT-x-T using these sites. The resultingplasmid, pBS:THT-Luc-T was linearised with SalI and10 lg of DNA was transfected into T. cruzi by electropora-tion with a Gene Pulser (Bio-Rad, Hercules, CA) using thefollowing conditions: 500 lF, 450 V, in a 0.2 cm cuvette.Briefly, mid-log stage (�2 � 107/ml) epimastigotes of T. cru-

zi Brazil heart strain (Hyland et al., 2007) were harvested bycentrifugation, washed twice in PBS supplemented with1 mg/L glucose and resuspended at a final concentration of1 � 108 cells in 0.4 mL of electroporation buffer (PBS with0.5 mM MgCl2 and 0.1 mM CaCl2). Following electropora-tion, the cells were placed on ice for 15 min and were trans-ferred to flasks containing 5 mL of LDNT. Forty-eighthours post-transfection, cells were selected for resistance tohygromycin (Boehringer–Mannheim, Mannheim, Ger-many) at 0.75 mg/ml. Drug-resistant parasites were analysedfor luciferase activity 6 weeks following transfection.

2.3. In vitro bioluminescent imaging

To confirm the expression of luciferase in antibiotic-resistant transfectants, T. cruzi epimastigotes were seriallydiluted from 1 � 106–500 parasites in Dulbecco’s PBS(Gibco-BRL, Grand Island, NY) into a black 96-well plate(Costar, Acton, MA). A 50-lL cell suspension was mixedwith 50 lL of Steady Glo reagent (Promega, Madison,WI), according to the manufacturer’s instructions. After5 min, the plate was read using a SpectraMax Gemini XS(Molecular Devices, Sunnyvale, CA) and analysed withSoftmaxPro 4.8 software (Molecular Devices). Wild-type(untransfected) Brazil heart T. cruzi was included as a neg-ative control. For further analysis of luminescence in tryp-omastigote and amastigotes life stages, active, in vitro ratmyoblast infections, using a variety of parasite-to-myoblastratios (as described in Fig. 1) were also analysed for biolu-minescence. Infections were given 5 days to become estab-lished in a clear 6-well plate (BD Biosciences, San Jose,CA), luciferin was added to 150 lg/ml, and plates wereimaged using the Xenogen IVIS system (see below for adescription of the IVIS imaging system and analysis soft-ware) after a 5-min incubation.

2.4. Experimental animals and T. cruzi infections

Four- to 6-week-old male A/J mice (Jackson Laborato-ries, Bar Harbor, ME) were housed under specific patho-gen-free conditions. Mice were infected by i.p. injectionof either 1 � 106, 1 � 105, or our standard quantity of1 � 104 Brazil heart strain, luminescent T. cruzi trypom-astigotes derived from infection of tissue culture H9C2rat myoblasts. Wild-type Brazil heart (untransfected)T. cruzi and uninfected controls that had received an i.p.injection of PBS were included for in vivo analysis. Theuse and care of mice were conducted in accordance withthe guidelines of the Center for Comparative Medicine atNorthwestern University.


2.5. In vivo and ex vivo bioluminescent imaging

Prior to bioluminescent imaging, mice were anaesthetisedwith 1.5% isofluorane. After anaesthesia was achieved,150 mg/kg body weight of substrate luciferin potassium saltdissolved in PBS and filtered through a 0.22 lm filter (Molec-ular Therapeutics, Ann Arbor, MI) was administered by asingle i.p. injection. Mice were placed into the camera cham-ber, where a controlled flow of 1.5% isofluorane in air wasadministered through a nose cone via a gas anaesthesia sys-tem designed to work in conjunction with the bioluminescentimaging system (IVIS 100; Xenogen, Alameda, CA). Thisimaging system consists of a cooled charge-coupled devicecamera mounted on a light-tight specimen chamber, a cam-era controller, and a Windows computer system. In order toallow adequate dissemination of the luciferin substrate(Contag et al., 1995), mice were maintained for 10 min afterinjection of the substrate. Mice were imaged in dorsal, ven-tral and left lateral positions by capturing a grayscale bodyimage overlaid by a pseudocolour image representing thespatial distribution of the detected photons. Images were col-lected with 0.5–2 min integration times depending on signalintensity. For the analysis of parasites in specific organs,mice were administered the luciferin substrate, as described,maintained for 5 min and sacrificed for organ harvest. Dataacquisition and analysis were performed by using the Living-Image software (Xenogen) where luminescence could bequantified as the sum of all detected photon counts per sec-ond within a chosen region of interest.

3. Results

3.1. Evaluation of T. cruzi bioluminescence

To examine the live, in vivo dissemination of T. cruzi ina non-invasive manner, we engineered bioluminescentepimastigotes by integrating the firefly luciferase codingsequence into the tubulin locus. The luciferase codingsequence from the pGL3 basic vector was directionallysubcloned into the pBS:THT-x-T tubulin integration vec-tor (Weston et al., 1999), and integrated into the intergenicregions of parasite a- and b-tubulin after electroporation(Fig. 1A). Integration of the luciferase gene into the tubulinlocus was confirmed by luciferase Southern blot hybridisa-tion of genomic DNA digested with SacII (Weston et al.,1999) as well as chromosomes prepared by pulsed-field gra-dient gel electrophoresis (data not shown). Eight weekspost-transfection, an antibiotic-resistant, epimastigotepolyclonal population displayed notable luminescencemeasured by a standard plate-reading apparatus, indicat-ing successful integration and expression of the luciferasegene (Fig. 1B). Further analysis of this luminescent popu-lation was conducted by allowing the epimastigote popula-tion to differentiate into metacyclic trypomastigotes. Oncemetacyclogenesis occurred, trypomastigotes were used toinfect myoblasts at variable ratios from which both infec-tious trypomastigotes and intracellular, replicating


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Fig. 1. Generation and analysis of luminescent Trypanosoma cruzi. (A) Construct and cloning strategy used to generate luminescent T. cruzi. pBS:THT-x-T (obtained from Wesley Van Voorhis, University of Washington), a plasmid with a pBluescript backbone and a hygromycin-resistance gene, wasmodified by insertion of the firefly luciferase gene. After linearisation with SalI, the plasmid was transfected into T. cruzi epimastigotes and the Hyg andLuc genes integrated into the tubulin locus by homologous recombination. (B) Serial dilutions of antibiotic-resistant, T. cruzi epimastigote transfectantsand wild-type epimastigotes were examined for luciferase activity. A black, 96-well plate containing 50 lL suspensions of parasites, mixed with 50 lL ofSteady Glo reagent was read by a SpectraMax Gemini XS Microplate Spectrofluorometer and analysed with SoftmaxPro 4.8 software. (C) Luminescenttrypomastigotes and amastigotes were examined for luciferase activity in active, in vitro rat myoblast infections using parasite:myoblast ratios of 40:1,20:1, 10:1, 5:1, 2:1 or uninfected (NI). Five days p.i., D-luciferin was added to each well and the 6-well plate was imaged with the Xenogen IVIS systemafter a 5-min incubation (see Section 2 for detailed description). In the pseudocolour image the luciferase activity or photon intensity ranges from thelowest intensity (blue) to highest intensity (red). Abbreviations: RLU, relative light units; min, minimum.


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amastigotes could be imaged for luminescence. The imag-ing of this in vitro infection revealed significant biolumines-cence with an expected correlation of signal strength toparasite number (Fig. 1C).

3.2. In vivo characterisation of luciferase T. cruzi

Once the myoblast infections were established and prop-agated by serial passage, it was not possible to determinewith accuracy the number of parasites responsible for gen-


erating the luminescent signals shown in Fig. 1C. However,based on a comparison of the number of trypomastigotesused to initiate the in vitro infection and the numbers ofepimastigotes used for the experiment of Fig. 1B, itappeared that the IVIS instrument was far superior tothe standard plate-reading apparatus in luminescence sensi-tivity. For this reason, and to determine the minimal inoc-ulum required to follow the course of infection in ourmouse model of Chagas disease, we infected mice with106, 105 or 104 (our normal inoculum) luminescent trypom-



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astigotes by i.p. injection. Imaging of these mice as soon as1 h p.i. revealed the capability of the IVIS system to detectall amounts used, with clear differences in signal strength(Fig. 2A). The magnitude of light emission was noticeablyhigher in the in vivo infection than in the in vitro infectionwhen comparing the maximum values indicated on thescales. Interestingly, whilst infection with 104 luminescenttrypomastigotes produced a signal clearly above that ofwild-type parasites in vivo (not shown), nearly2 � 105 epimastigotes were required to generate a lumines-cent signal above that of wild-type cells (Fig. 1B). The dis-semination of parasites was monitored in infectionsinitiated with the different inocula over 25 days, showingthe highest parasite burden at 10 days p.i. when the highestnumber of trypomastigotes was used, but then a reducedand more dispersed burden was similar amongst the threeanimals by 3 weeks p.i. (Fig. 2A). When quantified, the sig-nal intensity in all infections indicates a peak of parasiteburden at 10 days p.i. when using either 1 � 106 or1 � 105 parasites, with a slight lag in peak signal (14 daysp.i.) when using 1 � 104 parasites (Fig. 2B). Additionally,at 2 weeks p.i., the original number of parasites used forinfection became irrelevant as photon emission, or lumines-cence, was indistinguishable amongst the groups (Fig. 2B)and parasitosis appears to become more organ-specific atlater time-points (Fig. 2A). It is noteworthy that the 10-fold differences of parasite quantity used for the initialinfection correlated with 10-fold differences in lumines-cence signal intensity observed 1 h p.i. (Fig. 2B).

3.3. Course of intraperitoneal infection observed in the A/J–Brazil strain–strain model of experimental chagas disease

Since our experimental model of acute Chagas disease isnormally initiated with the i.p. injection of 104 Brazil straintrypomastigotes, we conducted a thorough analysis of par-asite dissemination from three perspectives. As expected,the majority of luminescence observed early in the infectionwas in the lower left quadrant, where the parasites wereadministered. This was consistently observed from ventral,dorsal and left lateral viewpoints (Fig. 3). As the infectionprogressed, the overall intensity of luminescence decreasedand specific regions retained parasites, indicative of possi-ble occupancy in specific organs. For instance, at both 21and 25 days p.i., it appeared that parasites were persistentin regions corresponding to lung or heart, as might beexpected (Fig. 3, ventral). The persistence of luminescenceemitted from the lower portion of the mouse abdomen sug-gested a general maintenance of parasite proliferation inthis i.p. region or specific habitation of parasites in the gas-trointestinal tract. To further examine the parasite burdenin an organ-specific manner, several organs were harvestedfrom animals 25 days p.i. and imaged for luminescence.These animals were provided D-luciferin substrate, allowedtime for adequate systemic dissemination and sacrificed fororgan harvest. As shown in Fig. 4, the majority of T. cruzi

was found in the gastrointestinal system, particularly the


large intestine (LI), with a notable presence in the smallintestine (SI), lungs (L) and kidneys (K). Smaller amountsof luminescence were also observed in the heart (H) andskeletal muscle (Sk) whilst either a completely absent orbarely detectable luminescent signal was seen in spleen(Sp) and liver (Lv). Whole blood (B) revealed a low-levelsignal.

4. Discussion

To make bioluminescent imaging of T. cruzi possible, amethod in which the firefly luciferase enzyme could be sta-bly expressed within the parasite was required. ThepBS:THT-x-T plasmid, having been previously employedfor the expression of an FL-160-GFP fusion in T. cruzi(Weston et al., 1999), was chosen for its ease of modifica-tion, possession of an antibiotic resistance gene for positiveselection, and design for integration into an essential regionof the trypanosome genome (tubulin locus) to ensure itsmaintained expression. This transfection strategy producedtransgenic parasites that could be used for animal infec-tions without the need for continuous drug selection,unlike those using episomal vectors. The administrationof hygromycin following transfection produced a popula-tion of epimastigotes that readily display luminescent activ-ity using a standard plate-reading luminometer (Fig. 1B).Although the modest sensitivity of this instrument pre-cludes assessment of luciferase activity at the single-celllevel, there is potential for such a tool in the screening ofpotential parasiticidal compounds. This high-throughput,efficient method has been effective for the investigation ofanti-Leishmania compounds (Ashutosh et al., 2005; Langet al., 2005) and could similarly be applied to luminescentT. cruzi.

We employed the IVIS instrument, typically used forwhole animal imaging, to assess bioluminescence of try-pomastigotes and amastigotes from a live, in vitro infectionof rat cardiomyocytes. This method provides a more com-plete picture of luminescence by providing a visualisationof light and the ability to quantify emitted photons in aspecific region of interest. In addition to the utility of lumi-nescent trypanosomes for drug screening, studies of hostand parasite factors involved in susceptibility and resis-tance to infection could be accomplished using this technol-ogy. For instance, specific deletion or knockdown ofparasite or host cell components thought to be requiredfor invasion could be quickly analysed by determiningwhether infections can be established and maintained inculture before moving to an animal model.

The ability to image T. cruzi with IVIS instrumentationon a whole animal level will also enable investigators toconduct important studies of virulence from both hostand parasite perspectives. We initially had to determinethe threshold of detection as it relates to our standardmouse infection regimen. We previously established anexperimental model of Chagas disease in which A/J miceare infected with 104 trypomastigotes and analysed in the


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Fig. 2. Luminescent T. cruzi imaged at various times p.i. with an IVIS imaging system. (A) Trypomastigotes were isolated from in vitro myoblastinfections and different amounts, as indicated, were injected i.p. into A/J mice. Mice were imaged ventrally starting 1 h after infection and monitored thedays p.i. as shown. For all images shown, the colour scale ranges from blue (just above background with a minimum set to 15,000 photons/s/cm2/sr) to red(maximum of 1 � 106 photons/s/cm2/sr). (B) The course of whole-body parasite burden expressed in terms of the photonic signal resulting from infectionof A/J mice with either 106, 105 or 104 luminescent or 106 wild-type T. cruzi trypomastigotes. The total light emission from the entire mouse body wasmeasured and data points were generated from the analysis of at least two mice per infection condition. Day 0 p.i. corresponds to measurements acquired1 h p.i. (A). The signal shown for wild-type infection corresponds to background noise of the IVIS instrument. Abbreviation: min, minimum.


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Please cite this article in press as: Hyland, K.V. et al., Bioluminescent imaging of Trypanosoma cruzi infection, Int. J. Parasitol. (2008),doi:10.1016/j.ijpara.2008.04.002

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Fig. 3. Course of parasite dissemination in a mouse model of experimental Chagas heart disease. A/J mice were injected i.p. with 1 � 104 luminescentTrypanosoma cruzi trypomastigotes and imaged either ventrally, dorsally or laterally over the course of infection prior to death typically observed by30 days p.i.. For all images shown, the colour scale ranges from blue (just above background with a minimum set to 50,000 photons/s/cm2/sr) to red(maximum of 1 � 106 photons/s/cm2/sr). The minimum for this scale was adjusted to avoid signal saturation during the peak of signal intensity. Theventral, dorsal and lateral perspectives for each timepoint were taken from the same animal and all images are representative of at least two animals.Abbreviation: min, minimum.


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acute phase (21 days p.i.) at which time severe inflamma-tion, fibrosis and parasitosis of the heart is typicallyobserved. To assess the sensitivity of the IVIS instrumentfor imaging our luciferase parasites we found that, whilstluminescence detection was minimal 1 h p.i., the prolifera-tion and dissemination over a 25-day period was sufficientto produce a maintained signal throughout the course ofinfection (Fig. 2A). Interestingly, when infecting with either10- or 100-fold more trypomastigotes, an initial correlationof luminescence to parasite number was observed (Fig. 2B,1 h p.i.), followed by a gradual equilibration of parasiteburden by 14 days p.i.. One possible explanation for thiscould be a combination of enhanced immunity againstT. cruzi coupled with an impaired capacity for immuneevasion typically associated with the parasite. A numberof reports have described different ways in which T. cruzi


is capable of evading host immune responses during theacute phase of infection, allowing the persistence to gradu-ally contribute to chronic pathology (Kierszenbaum, 1981;Garcia et al., 1997; Brodskyn et al., 2002; Kotner and Tarl-eton, 2007). With the extreme nature of infecting with 105

or 106 trypomastigotes, an inoculum far surpassing anyused for a variety of T. cruzi-based experimental animalsystems, the ability of the parasite to avoid the robustadaptive immune response mounted could be reduced. By10 days p.i., we observed a peak in parasite burden in theseanimals followed by a decrease which approaches that seenin animals infected with our normal quantity of parasites.The timing of this event likely corresponds to the peak ofthe adaptive immune response, which appears to controlthe infection once the parasite burden reaches a certainpoint. Interestingly, animals infected with the lowest num-


Fig. 4. Detection of luminescent T. cruzi in the internal organs of infectedA/J mice. Mice were infected with 104 trypomastigotes and injected withD-luciferin substrate (as described in Section 2) prior to sacrifice and organdissection. Twenty-five days p.i. luminescence was analysed in heart (H),spleen (Sp), skeletal muscle (Sk), lung (L), kidney (K), large intestine (LI),liver (Lv), small intestine (SI) and whole blood (B). For all images shown,the colour scale ranges from blue (just above background with a minimumset to 7500 photons/s/cm2/sr) to red (maximum of 2.5 � 105 photons/s/cm2/sr). The minimum and maximum for this scale was adjusted toenhance signal detection whilst avoiding saturation and is consistent forall organs imaged. Abbreviation: min, minimum.


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ber of parasites show increased parasite burden until 14days p.i., at which point the animals appear to have thesame burden as those receiving higher inocula. In all cases,once the adaptive immune response presumably initiatedfull control of parasitism, we observed a sharp decline inburden by 21 days p.i., at which point parasites appearedto have taken up residence in specific organs, rather thanbeing dispersed throughout the animal. As the animals pro-gress to 25 days p.i., the luminescent signal plateaued, sug-gestive of a scenario in which host immunity has controlledthe infection and parasite persistence has been achieved. Inthis particular experimental disease model, using the Brazilparasite strain with A/J mice, animals succumb to diseaseby 30 days p.i., prohibiting the examination of lumines-cence into the chronic phase of disease. The developmentof luminescent T. cruzi in other parasite strains and per-forming infections in different strains of mice will facilitatethe investigation of parasite dissemination in other, long-term chronic animal models of Chagas disease.

Further analysis of our standard experimental model,using 104 luminescent trypanosomes, provided a clearerpicture of parasite dissemination by imaging from three


perspectives (Fig. 3; ventral, dorsal and lateral). The siteof injection, in the lower left quadrant, displayed the mostprominent photon emission early after infection, suggestingthat parasites immediately invade and initiate replication insurrounding tissue. The infection spread over the next2 weeks, at which point the peak of parasite load wasobserved, followed by the sharp decrease in luminescentsignal. By 3 weeks p.i., the intensity in the lower abdominalregion was decreased but maintained, and luminescencewas observed in areas of the thoracic region from the ven-tral perspective, suggestive of parasitosis of heart or lung.In addition, the dorsal view suggested potential parasitismof either the spleen or kidney at both 21 and 25 days p.i..Whilst the signal observed in the abdominal region dimin-ished over time, it remained strong enough to potentiallymask the parasite burden associated with nearby tissue(e.g. skeletal muscle and liver).

In order to overcome this issue, we analysed specificorgans from infected mice 25 days p.i.. After allowing ade-quate dissemination of the injected luciferin substrate, micewere sacrificed and organs (heart, skeletal muscle, spleen,lungs, kidneys, liver, blood, large and small intestine) wereimaged using IVIS (Fig. 4). As anticipated, due to the typ-ical anatomical sites associated with Chagas disease, weobserved luminescent signals from heart and skeletal mus-cle as well as gastrointestinal organs. Substantial photonemission was observed in both lung and kidney tissuewhilst nearly a complete absence of signal was observedin spleen and liver. This was surprising, given the signifi-cant roles of these organs in the reticuloendothelial system.Whilst the blood appears to have high parasitaemia basedon visual appearance, the overall signal is actually quitelow with respect to the scale of intensity (Fig. 4; B). Thislow-level parasitaemia is in agreement with previous find-ings at the acute timepoint used for analysis. Whilst othershave reported parasite distribution to lung, spleen and liverin cases when animals have been immunosuppressed (Cala-brese et al., 1992) and even parasitism of bone and cartilage(Morocoima et al., 2006), it has become increasingly clearfrom a number of studies that the genetics of the parasiteand host play a defining role in the tissue distribution ofT. cruzi (Melo and Brener, 1978; Ben Younes-Chennoufiet al., 1988; McCabe et al., 1989; Andrade et al., 2002;Franco et al., 2003; Marinho et al., 2004). Although thepathogenesis of Chagas disease is multivariate, the persis-tence of T. cruzi has been suggested as playing a fundamen-tal role in disease progression (Anez et al., 1999; Zhang andTarleton, 1999; Tarleton, 2001). Despite the continueddebate and variability of the mechanism of pathogenesis,the development of luminescent T. cruzi provides the abil-ity to quickly screen organs and tissue samples for the pres-ence of parasites. Further correlation of whole body andharvested organ luminescence should permit the analysisof tissue-specific parasitisation in a non-invasive mannerusing this powerful technology. In infections in which smallnumbers of microorganisms persist at extremely low levels,methods of detection proven to be far more sensitive (e.g.,



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PCR) will continue to be essential for specific analyses. Theobservation of low-level parasitaemia indicated by ex vivoluminescence of blood, in conjunction with the apparentabsence of parasites in blood-filtering organs, illustratesthe limitations of luminescence detection. This limitationdoes not come unexpectedly, since the detection of a mini-mal presence of trypanosomes has also been challengingwith a number of other methods (see Section 1). Althoughluminescent trypanosomes do not overcome all the obsta-cles encountered in this field of research, they do providea powerful solution to a number of experimental challengesthat previously required invasive, more costly, analyses.

During the past several years several groups haveemployed IVIS technology to a variety of parasitic infec-tions (Heussler and Doerig, 2006; Hutchens and Luker,2007). Recent applications to Toxoplasma gondii virulencehave determined the importance of IFN-c and Toll-likereceptor signalling to parasite dissemination to the CNSfollowing immunosuppression (Dellacasa-Lindberg et al.,2007) or to overall host resistance (Hitziger et al., 2005),respectively. These studies were made possible using trans-genic, knockout mice coupled with multiple strains of lumi-nescent Toxoplasma. Others have used multiple strains todetermine differences in replication capacity over timeand to examine the reactivation of parasites during achronic infection (Saeij et al., 2005). Dissemination pat-terns resulting from different routes of Toxoplasma infec-tion have also been analysed (Boyle et al., 2007). Inaddition to in vitro drug screening, bioluminescent Leish-

mania amazonensis has also been used in vivo and ex vivoto examine the response to various therapeutics on bothliving mice and extracted, parasitised organs (Lang et al.,2005). Results of our study confirm the applicability ofIVIS technology for the study of Chagas disease pathogen-esis. In addition to the experiments conducted in other par-asitic disease models, we will now able to address questionspertaining to the relevance of parasite burden to the mag-nitude of organ-specific autoimmunity (Leon et al., 2001;Hyland et al., 2007), conduct rapid screening of new poten-tial parasiticidal drugs and test strategies for vaccine devel-opment against this parasite.

Acknowledgements

We are grateful to Dr. Wesley Van Voorhis for the gen-erous gift of the pBS-THT-x-T plasmid which was easilymodified for use in transfection, and to Dr. Dixon Kauf-man and Courtney Larson for the use of and expert tech-nical assistance on the IVIS 100 BLI instrument andsoftware. This work was supported by NIH GrantHL075822 (to D.M.E.) and pre-doctoral fellowship fromthe American Heart Association (to K.V.H.).

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