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Acute experimental Trypanosoma cruzi infection: Establishing a murine model that utilises non-invasive measurements of disease parameters


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Trypanosoma cruzi infection has a large public health impact in Latin American countries. Although the transmission 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 experimental model or descriptions of the clinical signs that occur during the course of acute murine infection are available. 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. cruzi. 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 physiological alterations. We hope that the results presented in this report provide parameters that complement the effective monitoring of trypanocidal treatment and other interventions used to treat experimental Chagas disease.
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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 moliveira@ioc.fiocru
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.
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
Figure legends.
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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was approximately 0 between 20-22 dpi. The non-infected group [N I
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Fig. 6: body temperatu re. From 1-5 days post-infection (dpi), Try-
panosoma cruzi-infected group [Inf (grey circles)] presented with hy-
perthermia. Between 21-26 dpi there was a sudden decrease in body
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... This analysis measured the following parameters at different times: (i) locomotor activity, i.e., covered distance (m) and average velocity (cm/s); and (ii) exploratory activity, the frequency of travel to the central region (number of events) every 5 min and the time spent in this region (seconds). The different groups were compared using the Student's t-test with the results considered statistically significant at p ≤ 0.05 [44]. The tests were performed twice for control groups (neither bled nor injected), AWB 20 μL and saline 20 μL analysis (n = 10 each group). ...
... Body weight variation and mortality rates in both healthy and T. cruzi-infected groups were checked individually weekly and daily, respectively. In the assays performed on healthy animals, at the each endpoint, the heart, spleen, liver and kidneys were collected and their respective weights measured [44]. In all assays, the different groups were compared using analysis of variance (ANOVA) and the results considered statistically significant when p ≤ 0.05. ...
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Background Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 μL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p = 0.02) in TNF-alpha levels than infected and untreated mice. Conclusions This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.
... Thus, we would have an association between acute kidney injury, histopathological analysis showed glomerular IgM deposits [29], changes in the cardiovascular and endothelial system, myocardial inflammatory response due to parasitic multiplication, evolution to cardiogenic shock and death of the animal [36]. Spironolactone suggests minimizing the effects of this possible parasite endo or exotoxin, decreased vasodilation, or loss of elasticity of the endothelium, balancing blood pressure in the vessels and avoiding cardiogenic shock [37,38]. Melnikov et al. [39] described that, during different phases, T. cruzi infection can be observed in lungs. ...
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Dr. Carlos Chagas, in 1909, published his notable discovery, a new pathology denominated Chagas disease. He was able to identify: etiologic agent, the protozoan Trypanosoma cruzi, its biological cycle and your pathogenesis: etiologic agent, the protozoan T. cruzi its biological cycle and your pathogenesis. However, to date, there is still no vaccine or effective treatment for the symptomatic chronic phase. In 2019, a new Severe Acute Respiratory Syndrome, promoted by a member of the Coronavirus family, emerged in Wuhan (China province), whose origin has not yet been totally elucidate. SARS-Cov-2 or COVID-19 is characterize by high transmissibility and high morbidity. Thus, in 2020 it became a global pandemic. Highlights into similarities between a neglected disease, which affects 40.000 new cases per year and intense research for a vaccine and treatment using experimental models and severe COVID-19 infection, with millions of victims, by evolution to cardiovascular disturbance, mainly through its target point to ACE2 enzyme. To compare acute T. cruzi experimental infection in mice, the cardiorenal axis involvement and suggest possible common points to research about serious course of the COVID- 19 infection and cardiovascular involvement
... endpoints that can predict death and can be used to avoid unnecessary suffering or distress in the experimental animals), which carries benefit for both researchers (e.g. they do not lose samples for histopathology, sera, etc) and animals (e.g. avoiding stressing death as result of sickness behavior and septic shock) [30]. ...
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Publication of accurate and detailed descriptions of methods in research articles involving animals is essential for health scientists to accurately interpret published data, evaluate results and replicate findings. Inadequate reporting of key aspects of experimental design may reduce the impact of studies and could act as a barrier to translation of research findings. Reporting of animal use must be as comprehensive as possible in order to take advantage of every study and every animal used. Animal models are essential to understanding and assessing new chemotherapy candidates for Chagas disease pathology, a widespread parasitic disease with few treatment options currently available. A systematic review was carried out to compare ARRIVE guidelines recommendations with information provided in publications of preclinical studies for new anti-Trypanosoma cruzi compounds. A total of 83 publications were reviewed. Before ARRIVE guidelines, 69% of publications failed to report any macroenvironment information, compared to 57% after ARRIVE publication. Similar proportions were observed when evaluating reporting of microenvironmental information (56% vs. 61%). Also, before ARRIVE guidelines publication, only 13% of papers described animal gender, only 18% specified microbiological status and 13% reported randomized treatment assignment, among other essential information missing or incomplete. Unfortunately, publication of ARRIVE guidelines did not seem to enhance reporting quality, compared to papers appeared before ARRIVE publication. Our results suggest that there is a strong need for the scientific community to improve animal use description, animal models employed, transparent reporting and experiment design to facilitate its transfer and application to the affected human population. Full compliance with ARRIVE guidelines, or similar animal research reporting guidelines, would be an excellent start in this direction.
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Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).
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Background Chagas disease (CD) is caused by Trypanosoma cruzi and affects 6–7 million people worldwide. Approximately 30% of chronic patients develop chronic chagasic cardiomyopathy (CCC) after decades. Benznidazole (BNZ), one of the first-line chemotherapy used for CD, induces toxicity and fails to halt the progression of CCC in chronic patients. The recombinant parasite-derived antigens, including Tc24, Tc24-C4, TSA-1, and TSA-1-C4 with Toll-like receptor 4 (TLR-4) agonist-adjuvants reduce cardiac parasite burdens, heart inflammation, and fibrosis, leading us to envision their use as immunotherapy together with BNZ. Given genetic immunization (DNA vaccines) encoding Tc24 and TSA-1 induce protective immunity in mice and dogs, we propose that immunization with the corresponding recombinant proteins offers an alternative and feasible strategy to develop these antigens as a bivalent human vaccine. We hypothesized that a low dose of BNZ in combination with a therapeutic vaccine (TSA-1-C4 and Tc24-C4 antigens formulated with a synthetic TLR-4 agonist-adjuvant, E6020-SE) given during early chronic infection, could prevent cardiac disease progression and provide antigen-specific T cell immunity. Methodology/ Principal findings We evaluated the therapeutic vaccine candidate plus BNZ (25 mg/kg/day/7 days) given on days 72 and 79 post-infection (p.i) (early chronic phase). Fibrosis, inflammation, and parasite burden were quantified in heart tissue at day 200 p.i. (late chronic phase). Further, spleen cells were collected to evaluate antigen-specific CD4 ⁺ and CD8 ⁺ T cell immune response, using flow cytometry. We found that vaccine-linked BNZ treated mice had lower cardiac fibrosis compared to the infected untreated control group. Moreover, cells from mice that received the immunotherapy had higher stimulation index of antigen-specific CD8 ⁺ Perforin ⁺ T cells as well as antigen-specific central memory T cells compared to the infected untreated control. Conclusions Our results suggest that the bivalent immunotherapy together with BNZ treatment given during early chronic infection protects BALB/c mice against cardiac fibrosis progression and activates a strong CD8 ⁺ T cell response by in vitro restimulation, evidencing the induction of a long-lasting T . cruzi -immunity.
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BACKGROUND Chagas disease, which is caused by the protozoan Trypanosoma cruzi, is endemic to Latin America and mainly affects low-income populations. Chemotherapy is based on two nitrocompounds, but their reduced efficacy encourages the continuous search for alternative drugs. Our group has characterised the trypanocidal effect of naphthoquinones and their derivatives, with naphthoimidazoles derived from β-lapachone (N1, N2 and N3) being the most active in vitro. OBJECTIVES In the present work, the effects of N1, N2 and N3 on acutely infected mice were investigated. METHODS in vivo activity of the compounds was assessed by parasitological, biochemical, histopathological, immunophenotypical, electrocardiographic (ECG) and behavioral analyses. FINDINGS Naphthoimidazoles led to a decrease in parasitaemia (8 dpi) by reducing the number of bloodstream trypomastigotes by 25-50% but not by reducing mortality. N1 protected mice from heart injury (15 dpi) by decreasing inflammation. Bradycardia was also partially reversed after treatment with N1 and N2. Furthermore, the three compounds did not reverse hepatic and renal lesions or promote the improvement of other evaluated parameters. MAIN CONCLUSION N1 showed moderate trypanocidal and promising immunomodulatory activities, and its use in combination with benznidazole and/or anti-arrhythmic drugs as well as the efficacy of its alternative formulations must be investigated in the near future.
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Experimental animals have contributed to basic and translational research on Chagas disease. Although many species and models have been used depending on the main experimental objective, there is a lack of uniformity and harmonization in preclinical in vivo studies. This chapter focuses on the description of relevant T. cruzi-infected animal models for drug discovery, and results are discussed in a translational research perspective, while some key concepts are presented in order to choose and establish a suitable animal model of T. cruzi infection to assess new chemotherapies’ efficacy. Also, some strategies related to the 3Rs principles (replacement, reduction, and refinement) are proposed to apply on Chagas disease research to achieve scientific aims while ensuring animal well-being.
Aim: To evaluate the effects of dynamized ethyl alcohol (Ethylicum)6cH and 30cH in mice infected with T. cruzi. Methods: In a blind, randomized and controlled assay, 63 eight-week-old, Swiss, male mice, infected with IP (1400 trypomastigotes, T. cruzi-Y-strain), were allocated into groups: CNI-non-infected (n=12), CI-infected and non-treated (n=17), Et6cH-infected, treated with Ethylicum 6cH (dilution 1:10(12)) (n=17), Et30cH-infected, treated with Ethylicum 30cH (dilution 1:10(60)) (n=17). Treatment was administered 48h before and after infection, followed by 56h/56h periods, until the 9th day after infection (a.i), for 16 h. Survival and mortality were assessed until the 82nd day after infection (a.i.). TNF-α, IL-6, IL-10, IL-5 and IL-17A cytokines were assessed in serum (3-4 animals/group), at time T0 (before infection), T8 and T12 (8th and 12th a.i), using the Mouse Cytokine 20-Plex Panel Magnetic Kit (Invitrogen, USA). Inflammation was determined in heart sections (eosin-hematoxylin staining) and behavior was analyzed with ANY-maze® software. The study was approved by the Animal Ethics Committee/UEM. Statistica 8.0 and R 3.0.2 software were used for statistical analyses. Results: The greater survival observed in the Et6cH group was related to decreased inflammation in heart tissue and increased IL-5 at T0 (p<0.05) and IL-10 at T8 (p<0.05), characterizing the Th2 response. It was also related to shorter periods of immobility, observed on day 12 a.i. The higher mortality in the Et30cH group was related to increased inflammation in the heart and a higher concentration of IL-6 and TNF-α cytokines, characterizing the Th1 response. Conclusion: The results demonstrate the beneficial effect of Ethylicum 6cH in acute murine infection by T. cruzi.
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Until now, there has been neither an agreed-upon experimental model nor descriptors of the clinical symptoms that occur over the course of acute murine infection. The aim of this work is to use non-invasive methods to evaluate clinical signs in Swiss Webster mice that were experimentally infected with the Y strain of Trypanosoma cruzi during acute phase (Inf group). Infected mice showed evident clinical changes beginning in the second week of infection (wpi): (i) animals in hunched postures, closed eyes, lowered ears, peeling skin, increased piloerection, prostration and social isolation; (ii) significant decrease in the body weight (Inf: 26.2±2.6 g vs. non-infected group (NI) 34.2±2.5 g), chow (1.5±0.3 vs. 6.3±0.5 mg) and water (2.4±0.5 vs. 5.8±0.7 ml) intake; (iii) significant decrease of the spontaneous activity as locomotor parameters: distance (0.64±0.06 vs. 1.8±0.13 m), the velocity (1.9±0.3 vs. 6.7±1.5 cm/s) and exploratory behaviour by the frequency (1.0±0.5 vs.5.7±1.0 events) and the duration (1.4±0.3 vs. 5.1±0.5 s in central arena region); (iv) significant increase in the PR (41.7±8.7 vs. 27.6±1.9 ms) and QT intervals (39.7±2.0 vs. 27.5±4.0 ms), and a decreased cardiac frequency (505±52.8 vs. 774±17.8 ms), showing a marked sinus bradycardia and an AV block. At 3 and 4 wpi, the surviving animals showed a tendency of recovery in body weight, food intake, locomotor activity and exploratory interest. Through the use of non-invasive parameters, we were able to monitor the severity of the infection in individuals prior to death. Our perspective is the application of non-invasive methods to describe clinical signs over the course of acute infection complementing the preclinical evaluation of new agents, alone or in combination with benznidazole.
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The existence of the nervous form of Chagas disease is a matter of discussion since Carlos Chagas described neurological disorders, learning and behavioural alterations in Trypanosoma cruzi-infected individuals. In most patients, the clinical manifestations of the acute phase, including neurological abnormalities, resolve spontaneously without apparent consequence in the chronic phase of infection. However, chronic Chagas disease patients have behavioural changes such as psychomotor alterations, attention and memory deficits, and depression. In the present study, we tested whether or not behavioural alterations are reproducible in experimental models. We show that C57BL/6 mice chronically infected with the Colombian strain of T. cruzi (150 days post-infection) exhibit behavioural changes as (i) depression in the tail suspension and forced swim tests, (ii) anxiety analysed by elevated plus maze and open field test sand and (iii) motor coordination in the rotarod test. These alterations are neither associated with neuromuscular disorders assessed by the grip strength test nor with sickness behaviour analysed by temperature variation sand weight loss. Therefore, chronically T. cruzi-infected mice replicate behavioural alterations (depression and anxiety) detected in Chagas disease patients opening an opportunity to study the interconnection and the physiopathology of these two biological processes in an infectious scenario.
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Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.
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Experimental acute infection with Trypanosoma cruzi in mice promotes an intense myocarditis and other systemic changes. However, the network of pathophysiological disorders and renal injury caused by the infection has not been elucidated. Our previous results with a murine model observed a discrete acute myocarditis and high mortality with significant inflammatory kidney injury with T. cruzi infection. The aim of this study was to investigate the mechanisms of kidney injury caused by the parasite in mice during the experimental acute phase. Results employing BALB/c mice infected with T. cruzi of Y strain showed renal injury on the 6th day postinfection (dpi) caused by a transitory decrease of renal blood flow. Acute kidney injury (AKI) was also observed similar to the model of ischemia/reperfusion lesion in these infected mice. The injury was not related to the presence (or multiplication) of parasites. Only rare nests were microscopically detected, and the presence of scattered parasites in renal parenchyma was seen on the 15th dpi. Thus, it was observed that during the acute phase of the disease, AKI in infected mice is linked to early cardiovascular effects, including heart failure, caused by striking inflammatory lesions in the myocardium, which lead to the high mortality rate of animals.
Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing.
In the past 15 years, there have been major advances in the control of Chagas disease in most of the countries endemic for this infection. Attention now turns to the future continuity of surveillance and control interventions - especially in regions where control has been so successful that the epidemiological significance of Chagas disease is in steep decline. The effort and expenditure of the recent past cannot continue indefinitely, but a degree of surveillance and selective intervention will be required because of the risk of new infestations and infections resulting from adventitious silvatic vectors accidentally entering houses. In this review, we summarize the progress of multinational control initiatives against Chagas disease. In addition, we suggest that the most sustainable approach to future surveillance involves both the primary healthcare system and university-based teams, with progressively greater attention given to case detection and treatment. Such an idea is not new, but we believe that it merits extensive discussion because of the different ways that research and health interventions are financed and because of the need to establish clearer reporting links between the research communities and the national health authorities.
LENZI, H. L., OLIVEIRA, D. N., LIMA, M. T., and GATTASS, C. R. 1996.Trypanosoma cruzi:Paninfectivity of CL strain during murine acute infection.Experimental Parasitology84,16–27. A systematic study of the distribution of intracellular parasites in the organs and tissues of mice acutely infected (15 days) with the CL strain ofTrypanosoma cruziwas performed. Almost all tissues and organs were parasitized with different intensities, including several epithelial cell types. In addition to striated, cardiac, and smooth muscles a very high parasitism of fat cells, pancreas, and genital adnexa was observed. A smaller number of parasites was found in all other structures studied except in highly vascularized structures such as in the penile corpora cavernosa, pulmonary and renal parenchyma, islets of Langerhans, hepatic sinusoids, and in atrial endothelium. This paper also shows, for the first time in the literature, the parasitism of milky spots, cornea epithelium, cornea stroma, retroorbital fibroblasts, seminal vesicles, and coagulative, Cowper's, urethral, preputial, sebaceous anal, and clitoris glands. The results indicated that CL strain is highly invasive, being able to infect cells derived from the three embryonic layers (ectoderm, mesoderm, and endoderm), suggesting that the paninfectivity may influence the outcome of immunological and pathological events.
Two strains of mice genetically selected for extreme phenotypes of immunological tolerance to ovalbumin, susceptible (TS) and resistant (TR), were experimentally infected with Sporothrix schenckii. The objective was to observe whether the genetic modifications produced by the selection might be associated with interstrain differences in adaptive immune and innate responses to infection. Therefore, we evaluated the LD50, CFU, phagocytic index, fungicidal activity, pro-inflammatory cytokines, specific antibody titres, and the delayed-type hypersensitivity reactivity. TR mice were tenfold more susceptible to infection than TS mice, as shown by LD50 (5 × 106 conidia i.v.). In TS mice, the resistance was a consequence of the tissue fungal load reduction, consistent specific T-cell-mediated immunity, and tumour necrosis factor (TNF)-α activity at onset of infection. In TR mice, these responses were not precociously detected. Therefore, the absence of CD4+ T-cell response in the first week of infection might explain the non-clearance of pathogen in TR mice. However, TR mice did show an increase in TNF level and delayed-type hypersensitivity response after the first week post-infection; there was also expansion and increase in granulomatous foci and CFU in the spleen. The expansion of granulomatous foci and the increase in TNF-α and tissue fungal load to damaging levels induced severe tissue destruction, general failure of the organs, cachexy and death in TR mice. The results show that genetic selection for extreme phenotypes of immunological tolerance also modified the responses to S. schenckii infection.
The non-obese diabetic (NOD) mouse model of autoimmune (type 1) diabetes has contributed greatly to our understanding of disease pathogenesis and has facilitated the development and testing of therapeutic strategies to combat the disease. Although the model is a valuable immunological tool in its own right, it reaches its fullest potential in areas where its findings translate to the human disease. Perhaps the foremost example of this is the field of T-cell antigen discovery, from which diverse benefits can be derived, including the development of antigen-specific disease interventions. The majority of NOD T-cell antigens are also targets of T-cell autoimmunity in patients with type 1 diabetes, and several of these are currently being evaluated in clinical trials. Here we review the journeys of these antigens from bench to bedside. We also discuss several recently identified NOD T-cell autoantigens whose translational potential warrants further investigation.