Bioluminescence Imaging of Toxoplasma gondii Infection in Living Mice Reveals Dramatic Differences between Strains

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INFECTION AND IMMUNITY, Feb. 2005, p. 695–702
Vol. 73, No. 2
0019-9567/05/$08.00 0
doi:10.1128/IAI.73.2.695–702.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Bioluminescence Imaging of Toxoplasma gondii Infection in Living
Mice Reveals Dramatic Differences between Strains
Jeroen P. J. Saeij,1† Jon P. Boyle,1† Michael E. Grigg,1,2 Gustavo Arrizabalaga,1‡ and
John C. Boothroyd1*
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California,1
and Department of Medicine, University of British Columbia, Vancouver,
British Columbia, Canada2
Received 17 August 2004/Returned for modi?cation 22 September 2004/Accepted 11 October 2004
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We examined the in vivo growth, dissemination, and reactivation of strains of the protozoan parasite
Toxoplasma gondii using a bioluminescence-based imaging system. Two T. gondii strains, one with a highly
virulent disease phenotype in mice (S23) and the other with a 1,000-fold-lower virulence phenotype (S22), were
engineered to stably express the light-emitting protein luciferase. One clone of each wild-type strain was
isolated, and the two clones (S23-luc7 and S22-luc2) were found to express similar levels of luciferase. Mice
were infected intraperitoneally with S23-luc7 (50 or 5 parasites) or S22-luc2 (500, 50, or 5 parasites), and the

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progress of the infections was examined noninvasively following injection of the substrate for luciferase,
D-luciferin. In mice infected with 50 S23-luc7 parasites, the parasites grew exponentially within the peritoneal
cavity (as measured by light emitted from luciferase-expressing parasites) during days 1 to 10 p.i., and this
proliferation continued until there was severe disease. In mice infected with 500 S22-luc2 parasites, the

at SERIALS CONTROL Lane Medical Library on September 19, 2007
parasites proliferated in a fashion similar to the S23-luc7 proliferation during days 1 to 6, but this was followed
by a precipitous drop in the signal to levels below the limit of detection. Using this technique, we were also able
to observe the process of reactivation of T. gondii in chronically infected mice. After treatment with dexameth-
asone, we detected reactivation of toxoplasmosis in mice infected with S23-luc7 and S22-luc2. During reacti-
vation, growth of S23-luc7 was initially detected primarily in the head and neck area, while in S22-luc2-infected
mice the parasites were detected primarily in the abdomen. This method has great potential for identifying
important differences in the dissemination and growth of different T. gondii strains, especially strains with
dramatically different disease outcomes.

Toxoplasma gondii is an obligate intracellular parasite that is
strongly dependent on the infecting T. gondii strain. The ma-
able to infect virtually any nucleated cell from a wide range of
jority of T. gondii strains comprise three distinct clonal lineages
mammalian and avian species (12). In humans, Toxoplasma
(20) and differ genetically by 1% or less (24). Strains with the
infections are widespread and can lead to severe disease in
type I genotype are highly virulent in mice, and regardless of
individuals with immature or suppressed immune systems.
the genetic background of the mouse host, the lethal dose is a
Consequently, toxoplasmosis became one of the major oppor-
single viable parasite. In contrast, type II and III strains have
tunistic infections of the AIDS epidemic (14). Acute infection,
50% lethal doses (LD s) of
103 parasites, and the precise
50
which is associated with the rapidly dividing form or tachyzoite,
outcome of infection is dependent on the genotype of the host.
is normally controlled by innate and adaptive immune re-
The virulence of type I strains is in part a result of their
sponses. Sterile immunity is not achieved, however, and in-
enhanced migration and higher growth rates, which allow them
stead an asymptomatic, chronic phase of infection ensues. The
to disseminate more rapidly (2) and reach higher tissue bur-
parasite persists by differentiating into bradyzoites, a form that
dens, which ultimately leads to a cascade of proin?ammatory
expresses surface antigens that are distinct from those of
mediators that induce pathology (8, 17). Recently, we reported
tachyzoites. The bradyzoites exist within cysts that remain in
that a cross between the less virulent type II strain ME49 and
the host for long periods in less immunologically active tissues,
the type III strain CEP can give rise to progeny with enhanced
such as the central nervous system, apparently impervious to
virulence (9). One of the F1 progeny, designated S23, has an
the robust immune response induced by the tachyzoite stages
LD
that is at least 3 logs lower than that of either parent.
50
(6, 11, 22).
To investigate the biological basis for this enhanced viru-
The outcome of toxoplasmosis in the mouse model is
lence, we compared the dissemination of S23 in mice with the
dissemination of one of its nonvirulent siblings, S22 (9). To do
this, we engineered the two strains to express ?re?y luciferase.
The resulting bioluminescent strains were then used to deter-
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Fairchild Building D305, Stanford University
mine the spatiotemporal distribution of infection of each strain
School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5124. Phone:
in mice. Using this method, we observed both qualitative and
(650) 723-7984. Fax: (650) 723-6853. E-mail: [email protected]
quantitative differences in the dissemination patterns of the
.edu.
two strains. We were also able to use this technique to observe
† J.P.J.S. and J.P.B. contributed equally to this article.
the time course of reactivation induced by immunosuppres-
‡ Present address: Department of Microbiology, Molecular Biology,
and Biochemistry, University of Idaho, Moscow, Idaho.
sants.
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FIG. 1. (A) Construct used to generate luciferase-expressing T. gondii clones S23-luc7 and S22-luc2. pDHFR-Luc (obtained from David Roos,
University of Pennsylvania), a plasmid with a pBluescript KS( ) backbone and the ?re?y luciferase gene driven by the T. gondii DHFR promoter,
was modi?ed by addition of a GFP expression cassette driven by the GRA1 promoter. The plasmid was linearized with NotI prior to transfection.
Enzyme abbreviations: N, NotI; S, SalI; B, BglII; P, PstI. (B) Dilution of S23-luc7 and S22-luc2 tachyzoites, demonstrating the similar levels of light
production in the presence of D-luciferin. Freshly harvested tachyzoites were quanti?ed and serially diluted in twofold increments in 96-well plates
with black walls in quadruplicate. D-Luciferin was added, and each plate was imaged 10 min later. The topmost wells contained 5
105 tachyzoites.
In the pseudocolor images the luciferase photon intensity ranges from the lowest intensity (blue) to the highest intensity (red). (C) S22-luc2
tachyzoites were serially diluted in 10-fold increments, and different amounts were injected intraperitoneally into BALB/c mice. Luciferin was
injected immediately after this, and 10 min later mice were imaged as described in Materials and Methods. The minimum number of tachyzoites
necessary to detect a bioluminescent signal greater than the background signal in these experiments was between 10,000 and 100,000.
MATERIALS AND METHODS
GFP-expressing clones, intracellular parasites of each clone were isolated by
syringe lysis and quanti?ed with a hemocytometer, and equal numbers of the
Parasites. Strains were maintained in vitro by serial passage on monolayers of
parasites were serially diluted in 96-well plates with black walls (Costar, Corning,
human foreskin ?broblasts (HFFs) at 37°C in the presence of 5% CO2 as
N.Y.).
previously described (19). HFFs were grown in Dulbecco modi?ed Eagle me-
D-Luciferin potassium salt (Xenogen, Alameda, Calif.) was added to a
concentration of 0.25 mg/ml, and the cultures were incubated for 10 min, after
dium (GIBCO BRL) supplemented with 10% NuSerum (Collaborative Biomed-
ical Products), 2 mM glutamine, 50 g of penicillin per ml, 50 g of streptomycin
which each plate was directly imaged for 5 min with the Xenogen IVIS system
per ml, and 20
g of gentamicin per ml. S23 is the virulent offspring of two
(see below for a description of the IVIS imaging system and the image analysis
relatively avirulent parents, ME49 and CEP. S22 is the genetically most similar
software). The background settings were adjusted to 6,000 photons/s/cm2/sr.
(21) but less virulent sibling of S23 (9).
These settings were the same settings used for all mouse imaging experiments
Generation of bioluminescent T. gondii. Plasmid pDHFR-Luc was provided by
(see below).
David Roos (University of Pennsylvania). This plasmid has the ?re?y luciferase
One clone of strain S23 (S23-luc7) and one clone of strain S22 (S22-luc2) had
coding sequence (GenBank accession no. P08659) (except for the last three
comparable luciferase expression levels (see below), and these clones were used
codons) cloned behind the promoter and the 5 untranslated region (UTR) of
in all subsequent experiments. Plasmid rescue was used to determine the site(s)
the T. gondii gene for dihydrofolate reductase (DHFR) (positions 933 to 1383 of
of insertion of pDHFR-Luc-GFP in both strains as described previously (1).
the gene; GenBank accession no. L08489). The luciferase coding sequence was
Brie?y, genomic DNA was harvested from S23-luc7 and S22-luc2 by the TELT
followed by the 3 UTR from DHFR (positions 7792 to 8175) (Fig. 1A). This
method (1), and 2
g was digested in a 100- l mixture with 20 U of SalI. Thirty
construct was modi?ed by insertion of a green ?uorescent protein (GFP) expres-
nanograms of SalI-digested DNA was diluted to a concentration of 3 ng/ l and
sion cassette into the SalI restriction site. The GFP cassette contained the
incubated overnight at 16°C with 2 U of T4 DNA ligase. One-half of the ligation
promoter and 5 UTR sequence from the T. gondii GRA1 gene (positions 8 to
reaction mixture was used to transform chemically competent Escherichia coli,
612 of the gene; GenBank accession no. M26007) and the 3 region from the T.
and bacterial colonies carrying the plasmid were selected by growth on Luria-
gondii GRA2 gene (positions 1179 to 1296 of the mRNA; GenBank accession no.
Bertani agar plates containing 100
g of ampicillin per ml. For each strain, at
J04018). The resulting plasmid, pDHFR-Luc-GFP (Fig. 1A), was linearized with
least three colonies were examined by restriction digestion and sequencing. The
NotI, and 50 g of DNA was introduced into parasites by standard methods (23).
insertion sites were identi?ed by BLAST analysis of the rescued genomic se-
After parasites were passaged sequentially for 2 weeks, tachyzoites that stably
quence against the T. gondii genome (available at http://www.toxodb.org).
expressed GFP were isolated by ?uorescence-activated cell sorting and were
Growth competition assay. To determine if luciferase-expressing strains were
subsequently cloned by limiting dilution.
impaired in their in vitro growth capacities, equal numbers of each luciferase-
To determine the relative expression levels of luciferase in the T. gondii
expressing strain and its wild-type counterpart were mixed and inoculated into

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T25 ?asks with HFFs. At the same time 100 parasites from the mixture were
marker) and luciferase were isolated. One clone of each strain
inoculated in triplicate into a 24-well plate with HFFs for a plaque (viability)
was chosen based on the production of comparable levels of
assay. After lysis of a monolayer by the parasites, one-twentieth of the monolayer
light when the organisms were incubated with D-luciferin (S23-
was transferred to another T25 ?ask. After 12 passages another plaque assay was
performed, and the ratio of the different parasite strains (as determined by the
luc7 and S22-luc2) (Fig. 1B). We estimated the sensitivity of
presence or absence of GFP ?uorescence) was calculated.
the imaging system for the detection of bioluminescent Toxo-
Infection of mice. Female BALB/c mice that were 5 to 10 weeks old (Jackson
plasma strains in terms of the number of parasites necessary to
Laboratories, Bar Harbor, Maine) were used in all experiments. For intraperi-
detect a signal greater than the background signal in vivo. We
toneal (i.p.) infection, S23-luc7 or S22-luc2 tachyzoites were grown in vitro and
injected mice intraperitoneally with 1
105, 1
104, or 1
extracted from host cells by passage through a 27-gauge needle, and they were
quanti?ed with a hemocytometer. Parasites were diluted in phosphate-buffered
103 S22-luc2 tachyzoites and imaged them 10 min later. Signals
saline, and mice were inoculated intraperitoneally with either 50 or 1,000
that were greater than the background signal in the abdomen
tachyzoites of each strain (in 100
l) by using a 27-gauge needle. Since the
were consistently detected only for mice injected with 1
105
viability of parasites isolated in this manner varied from batch to batch, plaque
tachyzoites (Fig. 1C).
assays were performed with these preparations to determine the number of
viable tachyzoites inoculated (4). Due to the comparatively high virulence of T.
Genomic sequences ?anking the insertion site of the lucif-
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gondii clone S23-luc7, a subset of the mice infected intraperitoneally with S23-
erase construct were determined for S23-luc7 and S22-luc2 by
luc7 was given either 100 or 400 mg of sulfadiazine per liter in the drinking water
plasmid rescue, and the sequences were subjected to a BLAST
during days 3 to 10 postinfection (p.i.) to facilitate survival of the mice beyond
analysis against the 9X version of the T. gondii genome (avail-
the acute stage of the infection (13).
able at http://www.toxodb.org). For S23-luc7 the insertion was
Reactivation. Dexamethasone (DXM) was used to reactivate T. gondii accord-
ing to the model developed by Djurkovic-Djakovic and Milenkovic (7). Dexa-
at position 2792490 of genomic scaffold TGG_995364, while in
methasone (dexamethasone 21-phosphate disodium salt; Sigma) was dissolved at
S22-luc2 the vector was inserted at position 448636 of genomic
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a concentration of 5 mg/liter in the drinking water. Treatment was started 80 days
scaffold TGG_995285. For each strain at least three indepen-
after infection. When no reactivation was seen after 25 days of treatment, the
dent bacterial clones were found to contain the same genomic
DXM concentration in the drinking water was doubled to 10 mg/liter.
In vivo imaging. The in vivo imaging system (IVIS; Xenogen, Alameda, Calif.),
sequence, suggesting that only one copy of pDHFR-Luc-GFP
consisting of a cooled charge-coupled device camera mounted on a light-tight
was present in S23-luc7 and S22-luc2 and therefore that the
at SERIALS CONTROL Lane Medical Library on September 19, 2007
specimen chamber, a camera controller, a camera cooling system, and a Win-
insertions were at only one locus. Genomic sequences ?anking
dows computer system, was used for data acquisition and analysis. D-Luciferin
the insertion sites were subjected to BLAST searches against
potassium salt (Xenogen), the substrate for ?re?y luciferase, was dissolved in
the T. gondii expressed sequence tag (EST) database to deter-
phosphate-buffered saline at a concentration of 15.4 mg/ml and ?ltered through
a 0.22- m-pore-size ?lter before use. Mice were injected with 200 l of luciferin
mine if the insertion had disrupted any transcribed genes. The
(3 mg) and immediately anesthetized in an oxygen-rich induction chamber with
insertion in S23-luc7 was found to be in the 3 UTR of the gene
2% iso?uorane. The mice were maintained for at least 10 min so that there was
for ROP9 (after position 1711 of the gene; GenBank accession
adequate dissemination of the injected substrate (5) and so that the animals were
no. AJ401616), 386 bp downstream of the stop codon. The
fully anesthetized. Mice were imaged in dorsal, ventral, and sometimes lateral
positions by collecting two images, a grayscale reference image obtained under
sequences near the insertion site for S22-luc2 were found to
low-level illumination and an image of light emission from luciferase expressed
contain two expressed sequence tags, one ending 339 bp up-
in the parasites within the animals and transmitted through the tissue. Anesthe-
stream of the insertion site (GenBank accession no. W06251)
sia was maintained during the entire imaging process by using a nose cone
and the other starting 555 bp downstream of the insertion site
iso?uorane-oxygen delivery device in the specimen chamber. Images were col-
(GenBank accession no. CN658789). When the insertion site
lected with 1- to 5-min integration times depending on the intensity of the
bioluminescent signal, and pseudocolor representations of light intensity (red
was compared to a set of predicted polypeptides encoded by
was the most intense, and blue was the least intense) were superimposed over the
the T. gondii genome, it was found to be after the ?rst base of
grayscale reference image. In certain cases mice were sacri?ced after imaging,
predicted
codon
101
of
Twinscan-predicted
protein
and individual organs were excised from the mice and imaged ex vivo. Data
995285.056.1 (Twinscan2 predictions are available at http:
acquisition and analysis were performed by using the LivingImage (Xenogen)
software with the IgorPro image analysis package (WaveMetrics, Seattle, Wash.).
//www.toxodb.org). However, neither the EST sequences nor
To allow comparisons between images from different days and different exper-
the Twinscan predicted protein was found to have signi?cant
iments, a background setting that could be applied to all images was determined
BLAST homology (Expect
10) to any characterized protein
empirically. Mice were injected with luciferin and imaged as described above,
sequences in the GenBank database.
and the background cutoff of the images was adjusted so that no signal was
Comparisons of bioluminescent and wild-type strains. It
detected in any of these control mice. The value obtained, 6,000 photons/s/cm2/
sr, was used as the background cutoff value for all images unless otherwise noted.
was possible that insertion of the luciferase expression con-
The units of this value re?ect the fact that the data were normalized with respect
struct resulted in decreased ?tness of the bioluminescent
to imaging time, area imaged, and the distance between the light source (i.e., the
clones compared with their wild-type counterparts. Moreover,
mouse) and the charge-coupled device camera (5). For quantitation of the
the different insertion sites of pDHFR-Luc-GFP could have
detected light over the course of infection, regions of interest were drawn by
using IgorPro, and the light emitted from each region was recorded by recording
resulted in phenotypic differences between S23-luc7 and S22-
the total number of photons per second (total ?ux).
luc2. Therefore, we assessed both the in vitro growth capacities
and the in vivo virulence phenotypes of these two strains com-
pared to their nontransgenic parents. When cultures were ini-
RESULTS
tiated with equal numbers of S23-luc7 and S23 or with equal
Characterization of S23-luc7 and S22-luc2. To visualize T.
numbers of S22-luc2 and S22, the ratio of GFP-expressing
gondii infection by bioluminescence imaging, it was necessary
parasites to wild-type parasites was approximately 1:3 after 12
to engineer the parasite to express a luciferase gene. To do
in vitro passages. While this ?nding indicates that there was an
this, we used ?re?y luciferase, which Matrajt and colleagues
approximately 10% difference in the amount of growth per
have described previously as a heterologous reporter for this
passage between the bioluminescent and wild-type strains
parasite (15). Several clones of each wild-type strain (S23 and
(0.331/12
0.9), this is a relatively minor disparity compared
S22) with stable expression of both GFP (as the selectable
to the disparities for well-described fast-growing strains. For

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FIG. 2. Luminescent T. gondii, imaged on the days postinfection indicated with an IVIS imaging system (Xenogen). Tachyzoites (tz) were
isolated from HFFs by syringe lysis, and BALB/c mice were infected with S23-luc7 (S23) (50 tachyzoites) or S22-luc2 (S22) (500 tachyzoites). Mice
were imaged ventrally starting 1 day after infection, and the data are representative of two experiments (three mice per group). For all images
shown, the color scale ranges from blue (just greater than the background noise; set to 6,000 photons/s/cm2/sr) to red (at least 1
105
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photons/s/cm2/sr). (A) Typical course of i.p. infection of mice infected with T. gondii strain S23-luc7 (50 parasites). (B) Typical course of i.p.
infection of mice infected with T. gondii strain S22-luc2 (500 parasites).
example, in similar cultures starting with a 50:1 ratio of either
cent signal in mice that allowed the progress of infection to be
S23-luc7 or S22-luc2 to the rapidly growing type I strain RH,
monitored noninvasively (no bioluminescence was observed in
we could not detect any GFP-expressing parasites after 12
mice infected with either wild-type strain or in uninfected mice
passages, as expected; i.e., RH fully outcompeted the lucif-
[data not shown]). In both the S23-luc7 and S22-luc2 infec-
erase-expressing strains. We also determined whether S23-luc7
tions, a bioluminescent signal was detectable by day 4 p.i., and
retained the virulent phenotype of the wild-type strain and
it came exclusively from the abdomen. The mice infected with
whether S22-luc2 retained the nonvirulent phenotype of the
S23-luc7 shown in Fig. 2A illustrate the typical disease pro-
wild-type strain (9). In infections initiated in mice, injection of
gression that we observed in infections with this virulent strain.
50 S23-luc7 tachyzoites was uniformly lethal (three of three
The amount of signal detected in the abdomens of S23-luc7-
mice died from the infection; data not shown), while all ?ve
infected mice increased over the course of the infection (Fig.
mice survived infection with 1,000 tachyzoites of S22-luc2.
2A), and quantitative analysis showed that the increase was
These results are highly consistent with the reported LD s for
exponential during days 1 to 10 (Fig. 3). In addition, signals
50
the wild-type S22 and S23 strains (9). Overall, these data sug-
were detected in the thorax and neck on days 10 and 12. All
gest that the genetic manipulation necessary to express lucif-
three mice in this group succumbed to the infection on day 13
erase in S23 and S22 did not result in any signi?cant alterations
or 14.
in the behavior of the organisms either in vivo or in vitro and
The course of the infection with nonvirulent S22-luc2 was
therefore that S23-luc7 and S22-luc2 could be used as tools to
dramatically different from the course observed for virulent
infer differences between the wild-type strains during mouse
S23-luc7 (Fig. 2B). For mice infected with 5 or 50 S22-luc2
infections.
tachyzoites there was a signal only at the site of injection (data
Bioluminescence imaging of S23-luc7 and S22-luc2 reveals
not shown). For the mice infected with 500 S22-luc2
differences in dissemination. Our overall goal is to determine
tachyzoites a bioluminescent signal greater than the back-
whether there are differences in dissemination between the
ground signal was observed in the peritoneal cavity at day
virulent Toxoplasma strain S23 and the less virulent strain S22.
3 p.i., and this signal increased by day 6 (Fig. 2B and 3). By day
To do this, we injected mice with bioluminescent T. gondii
10, however, a signal was no longer detectable. In these mice
strains S23-luc7 and S22-luc2 and monitored the course of
we never observed signal in any area except the abdomen, and
infection using an IVIS imaging system.
after day 8 we never observed a signal greater than the back-
Figure 2 shows biophotonic images of three representative
ground signal anywhere in these mice. None of the mice in-
mice infected with either bioluminescent S23-luc7 (Fig. 2A) or
fected with S22-luc2 died. Thus, using bioluminescence imag-
bioluminescent S22-luc2 (Fig. 2B) over a 13-day i.p. infection.
ing, we could detect dramatic differences in growth and
To obtain the data shown, mice were infected with approxi-
dissemination between the virulent S23-luc7 strain and the less
mately 50 S23-luc7 plaque-forming tachyzoites (Fig. 2A) or 500
virulent S22-luc2 strain.
S22-luc2 plaque-forming tachyzoites (Fig. 2B) based on a
Mice were also infected with a lower dose of S23-luc7, and
plaque assay. Both strains produced a signi?cant biolumines-
by using the plaque assay it was determined that these mice

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699
were
injected
with
approximately
?ve
plaque-forming
tachyzoites. One of the mice in this group died 14 days p.i., and
the course of infection was very similar to that of the mice
infected with 50 S23-luc7 tachyzoites (data not shown). Two
mice in this experiment survived the acute phase of the infec-
tion with S23-luc7, and the time course of the infection is
shown in Fig. 4A. In one S23-luc7-infected mouse, the biolu-
minescent signal in the abdomen peaked at day 10 and then
progressively decreased during days 12 to 14 (Fig. 4A). When
this mouse was imaged again on day 18, a signal was no longer
detected in the peritoneal cavity, but there was a signal from
the thorax, head, and what seemed to be the eyes. By dissection
of a similar mouse in another experiment, the thorax signal was
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determined to be due to extensive infection of the lungs, as
expected (18). The lung signal was not detected on day 25 p.i.
FIG. 3. Course of infection with luciferase-expressing T. gondii
The head signal was still present on day 25 p.i., but it was not
strains expressed in terms of the photonic signal. Mice were infected
detected on day 32 p.i. or during later imaging sessions. An-
intraperitoneally with either 50 S23-luc7 tachyzoites or 500 S22-luc2
tachyzoites and imaged three times per week. The images were ana-
other mouse died at day 29 p.i., presumably from encephalitis.
lyzed by measuring the total light ?ux (number of photons per second)
In mice infected with S23-luc7, a characteristic signal in a
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coming from the abdominal cavity, and the data points are the aver-
region around the neck was visible within 24 h before death
ages for three mice per strain. The emitted signal from the S23-luc7-
(Fig. 2A, day 12). After dissection of one such mouse, this
infected mice increased exponentially during days 4 to 10. In contrast,
the detected light from S22-luc2-infected mice peaked at day 6 and
signal was found to come from the super?cial cervical lymph
began to decrease soon after this, and the level was below the back-
nodes (Fig. 4C). In addition, after dissection, bioluminescent
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ground level by day 10 p.i.
FIG. 4. (A) Course of i.p. infection of a mouse that survived infection with T. gondii strain S23-luc7 (?ve parasites). One ventral image is shown
for days 4 to 14, while for days 18 and 25 ventral (V), lateral (L; day 18 only), and dorsal (D) images (of the same mouse) are shown. The color
scale for the images is the same as that in Fig. 2. (B) Course of i.p. infection in a mouse infected with T. gondii strain S23-luc7 (50 parasites) and
treated from day 3 to 10 with 100 mg of sulfadiazine per liter. Ventral images taken on days 4 to 18 are shown. The color scale for the images is
the same as that in Fig. 2. (C) Organs dissected from a mouse infected with 50 S23-luc7 parasites on day 13 p.i. (without sulfadiazine treatment).
In the image of the small intestine (INT), much of the signal comes from discrete patches. Signals were also observed in images of the cervical
lymph nodes (CLN) and in ventral (V) and dorsal (D) views of the brain (CNS). Given the increased sensitivity of imaging of infected organs ex
vivo, background settings were adjusted for these images to allow the most intense signal sources to be visualized in the context of the entire organ.
Abbreviations: S23, S23-luc7; tz, tachyzoites; sulfa, sulfadiazine.

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FIG. 5. Reactivation of Toxoplasma in mice infected with either S23-luc7 (A and B) or S22-luc2 (C). Immunosuppressive therapy was started
80 days after infection by adding dexamethasone sulfate (5 mg/liter) to the drinking water. At day 105 p.i. the dose was doubled to 10 mg/liter. In
all images, the number of days postinfection is indicated in the upper right corner, the mouse number is indicated in the lower left corner, and
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the view (dorsal [D] or ventral [V]) is indicated in the lower right corner. (A) Dorsal and ventral views of reactivation 111 days p.i. in a mouse
infected with ?ve S23-luc7 tachyzoites (not treated with sulfadiazine). The signal was observed ?rst in the head, abdomen, and cervical lymph
nodes. The two images are images of the same mouse. (B) Reactivation in three different mice (mice 2, 3, and 4) infected with S23-luc7 and treated
with sulfadiazine (100, 400, and 400 mg/liter, respectively) during the acute phase of infection. After reactivation, the signal was ?rst observed in
all three mice in the cervical lymph nodes, and signal was also detected in the lungs in mice 3 and 4. (C) Examples of reactivation in mice infected
with S22-luc2. Mouse 5 was imaged ventrally, and mouse 6 was imaged dorsally. Reactivation occurred primarily in the peritoneal cavity in mouse
5, while it was restricted to the right thigh in mouse 6.
iai.asm.org
signals were detected in many other organs, including, in de-
Immunosuppression reveals distinct sites of reactivation of
at SERIALS CONTROL Lane Medical Library on September 19, 2007
scending order of total light ?ux, the intestine (6
107 pho-
S23-luc7 and S22-luc2. Mice that survived a previous infection
tons/s, mainly from discrete patches) (Fig. 4C), the lungs (1
were immunosuppressed with DXM to assess if either drug
107 photons/s), the liver (1
107 photons/s), peritoneal cells (7
treatment or the immune system had cleared all parasites and,
106 photons/s from 2.6
106 cells), the spleen (4
106
if all parasites had not been cleared, to monitor the course of
photons/s), the kidneys (4
105 photons/s), the brain (4
105
reactivation. Immunosuppression was begun 80 days after in-
photons/s), and the heart (3
105 photons/s).
fection by adding a water-soluble form of DXM to the drinking
Real-time monitoring of anti-Toxoplasma therapy. Sulfadia-
water. Reactivation was monitored by imaging treated mice
zine, an inhibitor of folic acid synthesis, is effective in treating
regularly. No bioluminescence was detected after 25 days of
Toxoplasma infections in mouse models and human patients.
treatment, and so the dose of DXM was doubled. Six days after
To determine whether bioluminescence imaging can be used to
this, the ?rst signs of reactivation were seen in mice that had
monitor the ef?cacy of anti-Toxoplasma therapy in real time,
been infected with S23-luc7 (day 111) (Fig. 5A and B). Inter-
we monitored the effect of early sulfadiazine treatment of mice
estingly, while a weak signal was observed in the head region
infected with the virulent S23-luc7 strain. Mice were injected
(Fig. 5A) in some mice, reactivation in S23-luc7-infected mice
with a high, normally lethal inoculum (50 parasites) or a low
started mainly in the lungs (con?rmed after dissection) and
inoculum (5 parasites) and treated with 100 or 400 mg of
cervical lymph nodes (Fig. 5B). However, after dissection, par-
sulfadiazine per liter in the drinking water. Treatment started
asites were also detected in the brain (as expected) and the
on day 3 and continued through day 10. With both the high and
intestine in these mice. Interestingly, in the two cases in which
low doses of the drug and with either inoculum, all mice sur-
reactivation was seen with S22-luc2, the signal appeared in an
vived the acute phase of infection up to day 18. Figure 4B
area that was different from the area in which the S23-luc7
shows the typical course of infection of a mouse infected with
signal was seen. In one mouse (mouse 5) (Fig. 5C), the most
50 S23-luc7 tachyzoites and treated with 100 mg of sulfadiazine
intense signal was seen in the peritoneal cavity, although the
per liter. Bioluminescence was detected only in the abdomen
fact that a signal came from nearly every part of this mouse
during days 4 to 12, and from day 14 to day 18 a strong signal
suggests that it had an extremely high parasite burden. In
was also seen in the thorax and head; based on dissection of a
another mouse infected with S22-luc2 that reactivated (mouse
similar mouse in another experiment, these signals came from
6) (Fig. 5C), the most intense signal was detected in an area
the lungs and the brain, respectively. Two mice infected with 50
near the thigh. Thus, bioluminescence allowed the anatomical
S23-luc7 tachyzoites died at 20 and 25 days p.i., while one
sites of Toxoplasma reactivation induced by oral administration
mouse infected with 5 S23-luc7 tachyzoites died at 24 days p.i.
of DXM to be observed. Moreover, the results revealed dif-
With the high dose of sulfadiazine, no signal was detected
ferences that appear to be strain speci?c, although more mice
from day 8 onward in mice infected with ?ve S23-luc7
need to be examined before this conclusion can be drawn with
tachyzoites (data not shown). In mice infected with 50 S23-luc7
con?dence.
tachyzoites, the signal disappeared from the abdomen after day
8, and the only signal detected after this was a signal in the head
DISCUSSION
(brain) during days 14 to 25 (data not shown). None of the mice
treated with the high dose of sulfadiazine died. These results show
In this study, we demonstrated the use of photonic imaging
that bioluminescence is an effective way to monitor the ef?cacy of
to visualize bioluminescent Toxoplasma in live animals. We
a drug over and above simple survival curves.
found that differences due to strain and/or treatment can be

VOL. 73, 2005
BIOLUMINESCENCE IMAGING OF T. GONDII INFECTION
701
effectively observed with fewer animals and with much greater
dures. Third, the sensitivity varies from organ to organ, since
precision and subtlety than when previously described methods
different tissues absorb the light with different ef?ciencies and
are used. While head-to-head comparisons of the dissemina-
it is estimated that each centimeter of tissue lowers the detect-
tion patterns of different T. gondii strains and of the pharma-
able bioluminescent signal 10-fold (5). This limitation is also
codynamics of anti-Toxoplasma drugs have been made by other
partly overcome by opening the animal up (5).
workers, these workers have routinely relied on plaque assays
A good example of the added value of bioluminescent im-
and quantitative PCR with tissue homogenates to determine
aging was apparent when mice were injected with a low dose
parasite burdens in organs of interest (10, 17). While useful,
(?ve parasites) of S23-luc7. Because each mouse could be
these methods have several disadvantages. First, the animal
monitored individually over time, we were able to see the
analyzed must be sacri?ced, and, therefore, it is not possible to
individual differences between mice surviving the infection and
monitor the kinetics and extent of disease progression in the
mice that were about to die (Fig. 2A and B and 4A and B).
same animal over time. This obscures the true pattern of in-
Interestingly, all S23-luc7-infected mice that died in the acute
fection and also means that signi?cant insights from animal-
phase developed a signal around the ventral side of the neck,
Downloaded from
to-animal variations may be missed. It also means that many
and after dissection this signal was shown to be coming from
more animals must be used to obtain convincing data if mul-
the cervical lymph nodes (Fig. 4C). This provides important
tiple times are to be examined. Second, the traditional meth-
clues concerning the fatal pathogenesis in this model. This is
ods are laborious and depend on knowing which tissue to
similar to the situation in humans, where lymphadenopathy,
examine; dissemination to unexpected anatomical sites or even
primarily of the cervical type, is one of the most common signs
to particular subcompartments of the organ being examined
of symptomatic, acquired toxoplasmosis (16).
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may easily be missed. Third, they are dependent on obtaining
The advantages and disadvantages of the technique are also
reproducible, quantitative recovery of the parasites and on
apparent in the analysis of brain infection in animals infected
there being no variation in the ef?ciency of ampli?cation or
with S23-luc7. Although no head signal could be seen in living
plating.
mice at day 13, dissection of the same animals showed a clear
at SERIALS CONTROL Lane Medical Library on September 19, 2007
The nondestructive and noninvasive nature of biolumines-
signal in the brain (Fig. 4C). Moreover, once the brain was
cent imaging overcomes many of these problems. For example,
dissected, the signal could be seen to be coming from speci?c
the procedure can be performed repeatedly, which allows each
parts of the brain. This degree of resolution could be critical
animal to be used as its own control. Thus, averaging effects
when the neurobiology of Toxoplasma infection is studied, as it
are eliminated at the same time that the overall number of
allows correlations to be made between particular symptoms
animals required is greatly reduced. The noninvasive approach
and the focus of the infection in the brain (e.g., in the well-
described here is signi?cantly more rapid than conventional
described phenomenon of inhibition of neophobia in rats by
techniques and should not be subject to variations in recovery
Toxoplasma [3, 25, 26]). These results also show the danger of
or to sampling of an organ that is not the actual focus of
using one side of the brain for plaque assays and the other side
infection in an animal. In the present study we observed re-
for initiating an oral infection and assuming that the same
markable differences in dissemination between two T. gondii
numbers of parasites are present.
strains (S22-luc2 and S23-luc7) that was apparent by day 8 p.i.
The bioluminescence detected in mice infected with S22-
Given the large differences in the virulence of these two sibling
luc2 was contained in the peritoneal area, and the signal dis-
strains, this dissemination pattern could serve as an early in-
appeared abruptly and completely between day 8 and day 10,
dicator of the eventual outcome of the infection. Of course,
presumably due to an effective immune response. However,
there are limitations to this approach. First, while T. gondii is
because the ?re?y luciferase gene was cloned behind the Tox-
highly amenable to genetic manipulation, it is not trivial to
oplasma DHFR promoter, the loss of signal could also be
engineer strains that stably express GFP and luciferase, and
explained by a loss of promoter activity for this gene (e.g.,
there is no way to be absolutely sure that the resulting strains
because the parasites had switched from the tachyzoite stage to
have no phenotypic differences from the wild-type strains. In
the bradyzoite stage). Consistent with this, all ESTs for DHFR
these experiments, we found an extremely small difference in
are from tachyzoite libraries, suggesting that the DHFR pro-
growth in vitro, and both the bioluminescent strains had LD s
moter is speci?c for this parasite stage. If this is true, the
50
in the previously published range, suggesting that the lucif-
luciferase expression might be turned off upon stage conver-
erase and GFP had little if any effect in vivo. The genomic
sion. This limitation could be overcome by using a constitutive
insertion site could also affect the phenotype independent of
promoter, although metabolic differences between the two
the luciferase gene, but again we saw no evidence of this in
forms might still give different sensitivities of detection. On the
these experiments. The potential problem of a phenotypic ef-
other hand, this phenomenon could be exploited to distinguish
fect of the insertion site could be solved either by using mul-
between the two asexual forms of the parasite by, for example,
tiple strains with insertions in different loci or by targeting the
engineering parasites to express luciferase driven by a brady-
insertion to the same, neutral locus in all strains. Second, for
zoite-speci?c promoter. Ultimately, it may prove to be possible
detection of parasites in a tissue of interest, our assay is much
to engineer strains in which luciferases which use different
less sensitive than plaque assays or PCR, both of which have a
substrates (e.g., luciferases from ?re?y and renilla) are under
potentially lower limit of detection (a single organism). This
control of tachyzoite- or bradyzoite-speci?c promoters in order
limitation can be partially overcome by sacri?cing the animal
to simultaneously distinguish the two forms.
and exposing the organs, which results in a substantial increase
Bioluminescent imaging of Toxoplasma infection was prob-
in sensitivity but obviously ends the time course with that
ably most valuable in the studies reported here when reactiva-
animal unless the study is done with nonlethal, surgical proce-
tion was investigated. After the start of treatment, it is dif?cult

702
SAEIJ ET AL.
INFECT. IMMUN.
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1976.
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treatment regimens. More animals need to be studied, but it is
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ACKNOWLEDGMENTS
Molecular tools for genetic dissection of the protozoan parasite Toxoplasma
gondii
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We thank Chris Contag, Timothy Doyle, and Jonathan Hardy for
20. Sibley, L. D., and J. C. Boothroyd. 1992. Virulent strains of Toxoplasma
many helpful discussions.,
gondii comprise a single clonal lineage. Nature 359:82–85.
21. Sibley, L. D., A. J. LeBlanc, E. R. Pfefferkorn, and J. C. Boothroyd. 1992.
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Editor: J. F. Urban, Jr.