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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(2): 211-216, March 2012
In Latin America, the transmission rates of the proto-
zoan parasite Trypanosoma cruzi, the causative agent of
Chagas disease, have steadily declined due to a series of
multinational initiatives aimed at both the interruption
of vector transmission (by Triatoma infestans) and the
practice of screening blood donors (Schofield et al. 2006,
Dias et al. 2008, Coura & Borges-Pereira 2010). The in-
cidence of Chagas disease has dropped from 700,000-
40,000 new cases per year and the annual number of
deaths has fallen from more than 45,000-12,500 (Mon-
cayo & Silveira 2009). However, the epidemiology of the
disease has become more complex due to the presence of
multiple vectors and reservoirs and the added effects of
geopolitical, economic and ecological upheaval (Coura
2006, Beltrão et al. 2009, Lescure et al. 2010).
The eminent Brazilian scientists Carlos Chagas and
Emanuel Dias have described the basic features of T.
cruzi infection in mice (Chagas 1909, Dias 1934). De-
pending on the mouse lineage and parasite st rain used
for the infection, there are subtle differences in the ki-
netics of parasitaemia, the intensity of the parasite load
and the mortality rates during the course of infection.
Until now, there has been no consensus model for the
various aspects of the disease due to the heterogeneity
of the results obtained from different studies (Araújo-
Jorge & De Castro 2000). This obstacle, along with
others, hampers a reproducible comparison of the ex-
perimental infection to the human disease as it occurs
in nature (Coura 2000).
Many aspects of T. cruzi infection have been studied
using mouse models, including the infectivity of vari-
ous tissues (Lenzi et al. 1996), the presence of cardiac
inf lammatory lesions (Marinho et al. 1999, Andrade et
al. 2006, Pavanelli et al. 2010), the disturbance of the
cardiac electrical conduction system (Eickhoff et al.
2010), acute kidney injury (AKI) (Oliveira et al. 2009b)
and the efficacy of drug treatment regimens (Soeiro et
al. 2009). However, there are significant differences be-
tween the natural disease and experimental models. The
disease models often hinge on the description of spe-
cific clinical symptoms observed during the course of
an experimental infection. Balb/c mice are susceptible
to T. cruzi infection (Araújo-Jorge & De Castro 2000)
and we have previously reported important alterations
in this mouse lineage during infection with the T. cruzi
Y strain based on electrocardiographic and arterial pres-
sure parameters (Oliveira et al. 2009a). In addition to the
non-invasive cardiac evaluation, we also observed AKI
in infected mice (before the parasitaemia peak and the
onset of inf lammatory myocardial damage), which was
independent of parasite load (Oliveira et al. 2009b).
In this context, the aim of this study was to use non-
invasive methods to monitor the clinical signs (disease
model) of Balb/c mice infected with T. cr uz i. Acutely
infected mice showed evident clinical changes begin-
ning in the third week of infection. The mice were
evaluated based on physical characteristics, spontane-
ous activity, explorator y behaviour and physiological
alterations. We hope that the data obtained in this study
will provide helpful parameters to monitor the efficacy
of trypanocidal chemotherapy and other treatments for
experimental Chagas disease.
Financial support: FAPERJ, CNPq, FIOCRUZ
+ Corresponding author: g firstname.lastname@example.org z.br
Received 3 June 2011
Accepted 11 January 2012
Acute experimental Trypanosoma cruzi infection:
establishing a murine model that utilises non-invasive
measurements of disease parameters
Diana Rodrigues da Silva, Solange Lisboa de Castro, Monique Castro da Silva Alves,
Wanderson da Silva Batista, Gabriel Melo de Oliveira/+
Laboratório de Biologia Celular, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
Trypanosoma cruzi infection has a large public health impact in Latin American countries. Although the trans-
mission rates via blood transfusions and insect vectors have declined sharply in the past 20 years due to policies of
the Southern Cone countries, a large number of people are still at risk for infection. Currently, no accepted experi-
mental model or descriptions of the clinical signs that occur during the course of acute murine infection are avail-
able. The aim of this work was to use non-invasive methods to evaluate the clinical signs of Balb/c mice infected with
the Y strain of T. c ru zi. The infected mice displayed evident clinical changes beginning in the third week of infection.
The mice were evaluated based on physical characteristics, spontaneous activity, exploratory behaviour and physi-
ological alterations. We hope that the results presented in this report provide parameters that complement the effec-
tive monitoring of trypanocidal treatment and other interventions used to treat experimental Chagas disease.
Key words: Trypanosoma cruzi - clinical signs - non-invasive parameters - murine model
Clinical model of T. cruzi infection • Diana Rodrigues da Silva et al.
MATERIALS AND METHODS
Animals - Eight-week-old specific pathogen-free male
isogenic BALB/c mice were obtained from the Oswaldo
Cruz Foundation (Fiocruz) animal facility. The mice were
housed at the Department of Animal Experimentation,
Oswaldo Cruz Institute, Fiocruz, and maintained under
stable temperature conditions with 12 h light/dark cycles.
The mice were housed in the facility for at least one week
before T. cr uz i infection. All procedures were performed
in accordance with the guidelines established by the Fi-
ocruz Committee of Ethics for the Use of Animals (proto-
col 020/08). The number of animals used in each experi-
mental group is presented in the Figure legends.
Parasites and infection - The T. cruzi Y strain was
passaged in vivo in outbreed Swiss Webster mice and
trypomastigote forms were isolated from the blood-
stream as previously described (Araújo-Jorge 1989).
The parasites were resuspended in phosphate-buffered
saline (PBS) and counted using a haemocytometer. The
concentration of the inoculum was adjusted to 5 x 103
parasites/mL. The infection was performed with 200 µL
of this suspension (1 x 103 parasites) through intraperito-
neal injection (Inf group). The uninfected control mice
(NI) received only PBS.
Parasitaemia, weight loss and cumulative mortal-
ity - Parasitaemia was determined daily from six-15
days post-infection (dpi) using the Pizzi-Brener method
(Brener 1962). The body weight was evaluated weekly
from 0-30 dpi. The mortalities were noted daily and the
index of cumulative mortality was calculated at 30 dpi.
Physical characteristics and food consumption -
The animals were physically inspected daily during the
course of the infection. The following parameters were
evaluated: body posture, skin integrity (injury and/or
peeling), fur appearance (piloerection, dull fur and focal
or diffuse alopecia), infestation by ectoparasites and the
presence of clinical symptoms associated with second-
ary bacterial infections, such as dermatitis and conjunc-
tivitis. The changes were recorded daily with video and
photographs of each animal. The consumption of food
and water was measured daily (beginning 1 week prior
to infection) by calculating the differences between the
weight/volume offered to animals (250 g/250 mL) and
the amounts of food and water remaining in each cage
after 24 h. The individual consumption amounts were
estimated using the following formulae:
Constota l = weight/volume added - weight/volume after 24 h
Consind = Constot al/number of animals per cage
Ethogram - Using an adaptation of the previously
repor ted mouse phenotype characterisation method
(Keeney et al. 2006, Kalueff et al. 2007), a set of be-
havioural activities, including grooming, immobility
and rearing (vertical lifting), was registered. The etho-
grams were produced daily for each animal from 0-30
dpi. The number of behavioural activities (number of
events) in an open field test (interval of 5 min) and the
mean value for each experimental group (NI and Inf )
are expressed in the ethogram.
Motor and exploratory activity - To further charac-
terise the spontaneous activity of the mice, we used the
video-tracking tool Noldus EthoVision XT6 (Noldus In-
formation Technology, Leesburg, The Netherlands). The
arena was defined as 12 rectangles divided into lateral
and central areas. In the total arena, the rectangles were
calibrated to contain equal areas to ensure the consistency
of the parameters through which the Noldus EthoVision
XT6 apparatus detected transitional mouse movements.
This analysis was used to measure the following param-
eters: (i) locomotor activity, defined as covered distance
(cm) and average velocity (cm/s), and (ii) exploratory ac-
tivity, defined as the f requency of travel to the central re-
gion (number of events) per 5 min, which was measured
daily from 0-30 dpi. The video was recorded with a cam-
era placed 1.0 m away from the observation arena.
Body temperature - The body temperature of each
mouse was assessed daily throughout the course of the
infection (from 0-30 dpi). The temperatures were mea-
sured by physically restraining the mice and evaluating
the auricular temperature with a Braun ThermoScan
Digital Ear Thermometer (Braun Ind, São Paulo, BR).
Statistical analysis - The Mann-Whitney non-para-
metric test was used to compare the two groups (SPSS
software, version 8.0) and p values are indicated in the
Our results demonstrated the reproducibility of the
infection course in Balb/c mice infected with the Y strain
of T. cruzi. The peak of parasitaemia was observed at
8 dpi, with approximately 4 x 106 parasites/mL (Fig. 1A).
A significant decrease in body weight was observed in
Inf mice compared to NI animals at 21 dpi (18.5 ± 0.5 g
vs. 23.0 ± 0.6 g), 28 dpi (17.7 ± 0.4 g vs. 23.6 ± 0.8 g) and
30 dpi (19.1 ± 0.6 g vs. 23.8 ± 0.7 g) (Fig. 1B). The cumu-
lative mortality of Inf mice was 80% at 30 dpi (Fig. 1C).
The analysis of the physical characteristics of each
mouse from 0-30 dpi is depicted in Fig. 2. The NI mice
(0 dpi) displayed lively and bright eyes, lined and brilliant
fur, good skin integrity and no signs of peeling or alopecia
(Fig. 2A). During the first two weeks of infection (13 dpi),
slight alterations in the fur were observed. These changes
included mild piloerection, especially in the dorsal region
(Fig. 2B), which increased during the second week. At 15
dpi, altered body posture was observed, which was char-
acterised by downward-tilted ears, matted fur and an in-
crease in piloerection (Fig. 2C). The alterations in posture
and fur texture became more pronounced between 19-25
dpi. At this stage, the mice displayed a hunched posture
(mainly in the thoracic region), closed eyes (conjunctivi-
tis was detected in some individuals), peeling skin and an
increase in piloerection (Fig. 2D). Animals that survived
this period showed clinical signs of remission at 30 dpi
and these signs were similar to those observed during the
second week of infection (Fig. 2E). At 21 dpi, the physical
characteristics of immobility, prostration and social isola-
tion became more evident (Fig. 2F).
We also measured the motor and exploratory activities
of the Inf mice (Fig. 3). The motor activity was measured
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(2), March 2012
as the distance covered by each animal in a delimited are-
na within 5 min (Fig. 3A). The exploratory activity was
measured based on the frequency with which each mouse
inhabited the central quadrant of the arena (Fig. 3B). Be-
fore the infection, both groups displayed similar levels of
motor activity (Inf animals moved 2,580 ± 390 cm and
NI mice moved 2,500 ± 280 cm) and exploratory activity
(Inf mice displayed 9.1 ± 1.8 events and NI mice displayed
9.0 ± 2.0 events). Interestingly, at 1 dpi, there was a sig-
nificant decrease in both motor (1,390 ± 320 cm vs. 2,250
± 369 cm) and exploratory activities (1.0 ± 0.4 events vs.
9.0 ± 1.9 events). The motor activity of Inf mice was de-
creased at 4 dpi (439 ± 59 cm), showed a partial recov-
ery at 8 dpi (1,890 ± 220 cm) and was decreased again at
10 dpi (1,310 ± 198 cm). The lowest motor activity value
was observed at 21 dpi (232 ± 23 cm). The exploratory
activit y decreased ste adi ly th rough ou t the c ou rse of in fec-
tion and at 21 dpi (0.5 ± 0.01 events) and 22 dpi (0.4 ± 0.01
events), the animals displayed practically no curiosity.
T. cruzi infection led to a decrease in the mobility
of the mice, as monitored with video footage (Fig. 4A).
There was a significant decrease in the number of rear-
ing events (16 ± 1.5 events), which reached minimal val-
ues at 21 dpi (1.0 ± 0.01 events) (Fig. 4B). For grooming
levels, there was a drop at 15 dpi (0.9 ± 0.01) and the low-
est levels were reached at 21 dpi (0.7 ± 0.01) (Fig. 4C).
With regard to food consumption (Fig. 5), the intake
levels were similar in the Inf and NI groups until 6 dpi (4.1
± 0.5 mg). After 6 dpi, there was a decrease in the food
consumption by Inf mice, which reached the lowest value
at 24 dpi (0.5 ± 0.01 mg). For water intake, there was a
gradual decrease in consumption by the infected animals
starting at 5 dpi (Inf 4.2 ± 0.8 mL vs. NI 6.1 ± 0.5 mL),
with a minimum measurement of 0.3 ± 0.01 mL at 22 dpi.
Finally, the body temperature measurements were
consistent with the profiles of toxaemia and circulatory
shock models (Fig. 6). The body temperature of NI mice
was 36.5 ± 0.3ºC, whereas fever was detected in Inf mice
beginning at 1 dpi (37.5 ± 0.2ºC). A temperature peak oc-
curred at 3 dpi (38.5 ± 0.3ºC) and there was a severe tem-
perature decrease (33.0 ± 0.4ºC) at 21 dpi. The minimum
body temperature of 30.5 ± 0.3ºC was reached at 23 dpi.
The surviving animals recovered a normal body tempera-
ture at approximately 27 dpi.
There are several experimental models of T. cruzi
infection; however, there is no single murine model that
accurately recapitulates the parasitological parameters
Fig. 2: physical characteristics of the animals. A: the healthy mice
showed characterist ics such as normal posture, lined and brilliant fur,
bright eyes and erect ears; B: at 8 days post-infection (dpi), only subtle
alterat ions in the fur were observed; C: at 15 dpi, f ur alterations in-
creased, including piloerection and alterations in the ea r position; D:
at 21 dpi, the animals showed prostrat ion, piloerect ion and cachexia;
E: at 30 dpi, the surviving animals exhibited a physical appearance
similar to that obser ved at 7 dpi; F: with regard to social behaviou r, at
21 dpi there was social isolation a nd decreased mobility.
Fig. 1: parasitological parameters a nd body weig ht. Balb/c mice were
inoculated by intraperitoneal injection of 1 x 103 parasites of the Tr y -
panosoma cruzi Y strain in 20 0 µL. A: parasitaemia curve for the
infected animals with a peak of 400 x 104 parasites/m L at 8 days post-
infection (dpi); B: k inetic study of body weight, showing a statisti-
cally significant decrease for the infected group after 14 dpi; C: curve
of the cumulative mortality which reached 80% at 30 dpi. Values cor-
respond to means ± sta ndard deviation of three independent experi-
ments performed wit h 10 mice each; NI (white squa res): non-infected
group; Inf (grey circles): T. cruzi-infected group.
Clinical model of T. cruzi infection • Diana Rodrigues da Silva et al.
and the pathological and clinical aspects observed in
humans (Araújo-Jorge & De Castro 2000). The devel-
opment of a murine disease model using non-invasive
parameters enables the translation of pathophysiologic
alterations induced by the parasite into an experimental
model that can be used for research.
The characteristics of the model presented in this
report (Balb/c mice infected with the Y strain of T. cru-
zi) reproduced the observations of parasitaemia, body
weight and cumulative mortality in our previous publi-
cations (Oliveira et al. 2007, 2009a, b).
Medical (or veterinary) semiology defines a clinical
sign as any objective disturbance that can be perceived by
the examiner for the purpose of establishing a diagnosis
(Porto 2010). Several murine models of human diseases,
including cancer (Kalamarides et al. 2010), asthma (Bates
et al. 2009) and diabetes (Babad et al. 2010), can be found
in the literature. However, studies evaluating the clinical
signs of infectious diseases are mostly associated with the
routine sanitary control of animal colonies (Lapchik et
al. 2009). Research focusing on human behavioural dis-
orders, such as depression and anxiety, employs murine
models and non-invasive techniques, including the mea-
surement of motor and exploratory activities, to evaluate
the effectiveness of experimental therapies (Cryan & Hol-
mes 2005, Matsumoto et al. 2005, Taylor et al. 2010). We
believe that these parameters are also useful for the clini-
cal evaluation of our experimental model.
The results presented here demonstrate that the physi-
cal appearance and activity of mice are markedly altered
during the course of acute T. cr uz i infection. The altered
characteristics included body posture, piloerection, fa-
tigue, weakness, weight loss, reduction in food consump-
tion and, most importantly, hypothermia. Each of these
signs is characteristic of progressive cachexia (Cerami et
al. 1985). Starting at one day after infection, a reduction
in spontaneous physical activity and a loss of exploratory
interest were observed in the mice. These data were con-
firmed through an alteration of normal behaviours, such
as vertical lifting (rearing) and grooming, which began
during the first week of infection. Moreover, these re-
sults correlated with alterations in body temperature. In
the first week of infection, an increase in body tempera-
ture was observed (fever), which could be the cause of the
decrease in spontaneous activity (Melo et al. 2010).
Food and water intake declined gradually during the
first week of infection, which could be associated with the
decrease in rearing (Siegfried et al. 2003); this character-
istic behaviour of caged mice allows for feeding (among
other activities) (Johnstone & Higuchi 2001). The decrease
in food and water consumption could also be due to the in-
creased body temperature and cachexia (Emery 1999).
One study evaluating the involvement of molecules
from Gram-negative bacteria in the promotion of sep-
sis showed that mice injected with outer membrane
vesicles from Escherichia coli presented with clinically
relevant symptoms, such as piloerection, eye exudates,
hypothermia, tachypnea, leukopenia, hypotension and
the systemic induction of tumour necrosis factor-alpha
(TNF-α) and interleukin-6 (Park et al. 2010).
Fig. 3: physical activity. Spontaneous activity of the animals was
assessed in an arena divided into central and lateral quadrants. A:
motor act ivity was measured as the total dist ance covered (cm); B:
explorator y interest was measured by the number of visits (events) in
the central quadrant. Values correspond to means ± stand ard devia-
tion of three independent experiments performed with 10 mice each.
Asterisk mea ns st atistically signif icant differences between infected
and control mice (p < 0.05). NI (white squares): non-infected g roup;
Inf (grey circles): Trypanosoma cruzi-infected group.
Fig. 4: behaviour and animal mobility. A: animal mobility was de-
tected in a black arena recorded by the EthoVision XT6 system. The
white spots represent the animal’s position and the lines represent
their displacement; B: rear ing; C: grooming. NI [non-infected group
(white squares)] showed no significant alterations during the 30 days
of observation. Values correspond to means ± standard deviation of
three independent experiments performed with 10 mice each. Asterisk
means statist ically significant difference between infected and control
mice (p < 0.05). Inf (grey circles): Trypanosoma cruzi-infected group.
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(2), March 2012
Pro-inflammatory cytokines, especially TNF-α, are
associated with the development of cachexia (Silva et al.
2001, Porto 2010). Cachexia is characterised by the physi-
cal weakening of patients to a state of immobility stem-
ming from appetite loss, asthenia and anaemia (Bossola et
al. 2007, Lainscak et al. 2007). Tarleton (1988) suggested
that T. c ru zi -infected mice were primed for the production
of TNF-α and referred to this cytokine as cachectin. Ad-
ditionally, interferon-γ-activated macrophages produced
TNF-α following T. cruzi infection, which suggests that
TNF-α plays a role in both the amplification of nitric ox-
ide production and parasite killing (Silva et al. 1995). In
an experimental murine model of infection with T. cr uzi,
cachexia was reported to be caused by Chagas toxin (a
molecule with similar characteristics to lipopolysaccha-
ride). Mice given lethal doses of Chagas toxin became
sluggish, stopped eating and drinking, dragged their hind
legs, gradually developed both paralysis and breathing
difficulties and died five-seven days after infection (Sen-
eca 1969, Seneca & Peer 1966).
In conclusion, the application of non-invasive methods
is an effective way to evaluate and describe the clinical
signs of mice during the course of an acute experimental
infection with T. cr uz i and these methods will be evaluated
using other mouse lineages and parasite strains. Based on
the parameters of physical activity (motor and exploratory
activities), behaviour (rearing and grooming), food con-
sumption and body temperature, it is possible to describe
a murine model for Chagas disease with our results. The
comparison of our results with those in the literature sug-
gests that mice with acute T. cr uz i infection have similar
clinical signs to mice with toxaemia (sepsis).
To Dr Maria de Nazaré Corrêa Soeiro, head of the Labora-
tório de Biologia Celular (IOC/Fiocruz), for logistical support.
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Fig. 6: body temperatu re. From 1-5 days post-infection (dpi), Try-
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Clinical model of T. cruzi infection • Diana Rodrigues da Silva et al.
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