Experimental Parasitology 107 (2004) 7888

www.elsevier.com/locate/yexpr

Trypanosoma cruzi in a caviomorph rodent: parasitological

and pathological features of the experimental infection of Trichomys
apereoides (Rodentia, Echimyidae)
Leidi Herrera,a,b Samanta das Chagas Xavier,a Claudia Viegas,a Clara Martinez,c
Paulo Marcelo Cotias,d Hernan Carrasco,e Servio Urdaneta-Morales,b
and Ana Maria Jansena,*
a

Laboratory of Tripanosomatid Biology, Department of Protozoology, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Manguinhos, RJ, Brazil
b
Institute of Tropical Zoology, Science Faculty, Central University of Venezuela, 47058, Los Chaguaramos 1041-A, Caracas, Venezuela
c
Nutrition School, Medicine Faculty, Central University of Venezuela, Caracas, Venezuela
d
Evandro Chagas Research Institute, FIOCRUZ, RJ, Brazil
e
Tropical Medicine Institute, Medicine Faculty, Central University of Venezuela, Caracas, Venezuela
Received 1 December 2003; received in revised form 30 March 2004; accepted 21 April 2004
Available online

Abstract
To understand the interaction of Trypanosoma cruzi with caviomorph rodents, which supposedly have an ancient co-evolutionary
history with this parasite, experimental infection of laboratory reared Trichomys apereoides with several isolates of both genotypes
of the parasite was studied. Parasitemia, pattern of hematic cells, specic humoral immune response, histopathological features and
parasite clearance were appraised. T. apereoides maintained stable infections independent of the T. cruzi genotype as demonstrated
by positive PCR results in analyses of several tissues after a 5 months follow-up. The acute phase was characterized by abundant and
disseminated presence of amastigotes, vacuolization and/or myocytolysis. Lymphocytosis was a common feature. The chronic phase
was characterized mainly by lymphomacroeosinophilic inltrates independent of the inoculated T. cruzi isolate. T. cruzi of dierent
genotypes did not show any tissular preference in T. apereoides.
2004 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: Trypanosoma cruzi; Changes disease; Echimyidae; Trichomys apereoides; Histopathology; bp, base pairs;
DIG-labeled DNA, DNA probes with digoxygenin-labeled deoxynucleotides; DNA, deoxyribonucleic acid; Ethylene diamine tetra acetic acid;
FITC, uorescein isothiocyanate; IFA, immunouorescence assay; HE, hematoxylineosin; IgG, immunoglobulin G; kDNA, kinetoplast deoxyribonucleic acid; LCSSP-PCR, low-stringency single specic primer PCR; LIT, liver infusion tryptose medium; NNN, NovyMc NealNicole
medium; PCR, polymerase chain reaction; PI, post-infection; SSC, saline sodium citrate solution; TAE, trisma acetate buer; Taq, thermostable
DNA polymerase

1. Introduction
American Trypanosomiasis (Chagas, 1909) is a autochthonous and ancient protozoan infection of wild
mammals in which the heteroxenic hemoagellate Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) is
transmitted by wild vectors (Hemiptera, Reduviidae,

*
Corresponding author. Fax: +55-21-280-1589.
E-mail address: jansen@ioc.ocruz.br (A.M. Jansen).

0014-4894/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2004.04.008

Triatominae) in a primary sylvatic cycle (Pinto Dias,

2000).
Trypanosoma cruzi comprises complex multiclonal
populations, diering in genetic and biological attributes
and dierent epidemiological, pathological, and clinical
manifestations of the human disease. Two main T. cruzi
groups, T. cruzi I and T. cruzi II, were recognized in the
taxon, associated, in Brazil, with wild and domestic
transmission cycles, respectively. T. cruzi I and T. cruzi
II are described as correlated with phenotypic subpopulations dened by its isozymic patterns or zymodemes.

L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Zymodeme 1 and 2 are correlated with T. cruzi I and II,

respectively; zymodeme 3 is still a matter of debate
(Anon., 1999; Araujo et al., 2002; Fernandes et al., 1999;
Miles et al., 1977; Santos et al., 2002).
The origin of T. cruzi I and T. cruzi II, respectively, in
Didelphis marsupialis and in primates and/or caviomorph rodents, was suggested by Briones et al. (1999)
and Buscaglia and Di Noia (2003). Indeed, caviomorph
rodents and primates have an ancient evolutive history
in South America, since they arrived at the Southern
continent, coming probably from Africa, in the Oligocene (38 millions of years ago) (Flynn and Wyss, 1998;
Gaunt and Miles, 2000).
Trichomys apereoides (Rodentia, Echimyidae) is a
caviomorph rodent and therefore probably an ancient
host of T. cruzi. The taxon is associated to xeric and
rocky environments with frequent incursions into human dwellings. The genus Trichomys has an ample distribution in savannahs (cerrado), white scrub
(caatinga), and marshland (pantanal) biomes, typical of the Northeast and Central Brazil, occupying a
vast extension of the country (Bandouk and dos Reis,
1995; Neiva and Penna, 1916).
In spite of the recent hypothesis concerning divergence of T. cruzi I and T. cruzi II and the importance of
caviomorph rodents in the evolutionary history of T.
cruzi only scarce data on the interaction of this parasite
with these hosts are available. In addition, studies have
shown that T. apereoides may act as a good reservoir of
T. cruzi, as demonstrated (Jansen, A.M., non-published
data), by positive hemocultures of 50% (21/44) of specimens collected inside Serra da Capivara National Park
a biological reserve in semi-arid biome of caatinga, in
Piau
State, Brazil and an endemic area for Chagas
disease.
In this context, we considered it necessary to better
understand the association of T. cruzi with this caviomorph, a possible reservoir in endemic areas of Chagas
disease. In this study, the parasitological and histopathological patterns of experimental infection of T.
apereoides with T. cruzi I and T. cruzi II genotypes were
studied. Our purpose was also to supply data that may
clarify the putative association of caviomorph rodents
with the T. cruzi II genotype.

79

2. Materials and methods

2.1. Parasites
Experimental infections were performed with four
recently obtained T. cruzi isolates, previously typied by
analysis of the non-transcribed spacer of the mini-exon
gene, in agreement with Fernandes et al. (1999). Two
isolates considered as references for T. cruzi I (XXXX/
BR/1971/F) and T. cruzi II (MHOM/BR/1957/Y) were
also used (Deane et al., 1984b). Characteristics of the
inoculated isolates are listed in Table 1.
Inocula derived from spontaneous metacyclogenesis
in NNN medium with a LIT overlay were used
throughout.
2.2. Inoculation schedule
Trichomys apereoides (250 g mean weight) were distributed in six batches of six animals; (n 36). The
animals, born at animal facilities and gently supplied by
Dr. P. DAndrea from the Tropical Medicine Department, Oswaldo Cruz Institute, RJ, Brazil, were inoculated subcutaneously with 200 metacyclics/g body
weight, for each isolate. A group of non-infected animals was included as control (n 6).
2.3. Parasitological, hematological, and serological
follow-up
Fresh blood samples from the tail vein and Giemsa
stained thin smears of infected and control animals were
microscopically examined at 400
, thrice a week until
negativation of parasitemia and the following parameters were determined: pre-patent period, parasitemia (by
countings in a Neubauer hemocytometer) and pattern
and percentage of hematic cells (by dierential countings
of 100 microscopic elds). Mortality was recorded daily.
Humoral immune response was evaluated weekly by
IFA until 63 days PI and reevaluated at the end of the
follow-up, when necropsy was performed (5 months PI).
IFA was performed by an FITC anti-mouse IgG Sigma
conjugate and the test was performed as described by
Camargo (1966).

Table 1
Trypanosoma cruzi in Trichomys apereoides: isolates from dierent origins and molecular groups used in this study
Codea

Host

Locality

T. cruzi group

MTRI/BR/1999/R4
MDID/BR/1999/M1
TBRA/BR/1999/JCA3
MLEO/BR/2000/M593
MHOM/BR/1957/Y
XXXX/BR/1971/F

Trichomys apereoides
Didelphis albiventris
Triatoma brasiliensis
Leontopithecus rosalia
Human
Unknown primary sourceb

Piau
, Northeast Brazil

Piau
, Northeast Brazil
Piau
, Northeast Brazil
Rio de Janeiro, Brazil
Maintained in Brazil as reference isolate b
Maintained in Brazil as reference isolateb

II
I
II
II
II
I

a
b

Anon. (1999).
Deane et al. (1984b).

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L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Hemocultures were performed in all animals at necropsy, which was accomplished 5 months PI. Hemocultures were maintained at 28 C and examined
fortnightly during 5 months.
2.4. Necropsy
Two animals in the patent phase infected with MTRI/
BR/1999/R4 isolate and two animals/batch in the subpatent phase of infection (5 months PI) of all groups
were sacriced by anesthetic overdose of ketamine
(Ketaset HCl 100 mg/ml). Tissue samples were xed in
Formalin-Millonig (Carson et al., 1973) and routinely
processed for paraplast embedding and hematoxylin
eosin staining.
At least two 5 lm thick sections, at intervals of 60 lm,
from heart, skeletal muscle, skin, small and large intestines, liver, spleen, lungs, kidneys, pancreas, urinary
bladder, and brain were microscopically examined
(1000
) in a double blind test. The tissular parasitism
and histopathologic features were determined and photographed with a Nikon Microex HPX-35 camera on
Agfa APX-100 lms.
2.5. Extraction of DNA from T. apereoides tissue sections
Sections (510 lm thick) from blocks of xed embedded heart, smooth muscle from urinary bladder,
skeletal muscle and pancreas of subpatent animals (5
months PI) were dispensed with sterile toothpicks in
Eppendorf tubes. Each section was treated twice with
octane or mixed xylene to remove the paraplast, washed
twice with 100% ethanol, and rinsed with 23 drops of
acetone. Tissue digestion was done with 100 ll of digestion buer (50 mM Tris, pH 8.5, 1 mM EDTA, and
0.5% Tween 20) and 2 ll of Proteinase K (200 lg/ml)
incubating at 37 C (Wright and Manos, 1990).
DNA of the digested tissue was extracted using the
Wizard Genomic DNA Purication System (Promega,
Maddison, WI).

An initial denaturizing step at 94 C, for 4 min, was

followed by 35 cycles of 94, 55, and 72 C, for 1 min
each, and an extension at 72 C for 10 min in a Programmable Thermal Controller (Lane et al., 1997).
To ensure that the product of DNA tissue isolation
was amenable to DNA amplication, the b-actin protein
for mice was simultaneously amplied with the primers
50 -GCTGTGCTATGTTGCCCTAGAATTCGAGC-30
and 50 -CGTACTCCTGCTTGCTGATCCACATGTG
C-30 (Herwaldt et al., 2000), determining the integrity
of the constitutive DNA. A positive control of 5 lg of
T. cruzi DNA; DNA of non-infected animals and negative control in absence of DNA template were included
with every PCR run.
The PCR products were analyzed by electrophoresis
on a 2.5% ethidium bromide stained agarose gel and
visualized under ultraviolet light.
2.7. Southern blot, labeling, and hybridization of PCR
products
The Southern blot was carried according to Holtke
(1995). In short: the PCR products of tissular kDNA
amplication, electrophorized in 2.5% agarose gel, were
submitted to alkali denaturizing step (0.5 N NaOH;
1.5 M NaCl) and, subsequently, transferred to nylon
membranes (capillary transfer). The membrane was
neutralized with 1.5 M NaCl and 0.5 M NaOH, washed
with SSC 10
, and the transferred products were xed
with 120,000 mJ of ultraviolet light, using an ultraviolet
cross-linking apparatus. DIG-labeled DNA probes were
generated with DIG-High Prime, according to the random primed labeling technique (DIG High Prime: DNA
labeling and detection Starter Kit II, Roche) and used
for hybridization with membrane blotted nucleic acids,
according to standard methods, with high stringency.
The hybridized probes were immunodetected with antidigoxygenin phosphatase alkaline conjugated Fab
fragments and visualized with a chemiluminescent substrate and recorded on X-ray Films (10 min exposure
time).

2.6. Detection of T. cruzi DNA by polymerase chain

reaction amplication
3. Results
Specic polymerase chain reaction (PCR) amplication of a nucleotide sequence of the 330 bp corresponding to the four variable regions of T. cruzi kDNA
minicircle in the digested tissue of T. apereoides was
performed with primers 12150 -AAATAATGTACGG
GKGAGATGCATGA-30 and 12250 -GGTTCGATT
GGGTTGGTGTAATATA-30 (Britto et al., 1995).
Briey, 5 ll of DNA template was added to 15 ll of PCR
mixture to give a nal concentration of 20 mM Tris
HCl, pH 8.8; 50 mM KCl; 1.5 mM MgCl2 ; 10 lM
dNTPs, 10 pM of each primer, and 1 U of Taq DNA
Polymerase.

3.1. Parasitological follow-up

Data concerning parasitological follow-up are summarized in Table 2. In short, T. apereoides was able to
eciently control the number of circulating parasites
and maintain the infection with both T. cruzi I and T.
cruzi II subpopulations. Mortality (50%) was observed
only in the rodents inoculated with MTRI/BR/1999/R4
isolate. Death of the animals infected with MTRI/BR/
1999/R4 isolate occurred, respectively, after 22, 25, and
27 days (mean value 25 days). The animals inoculated

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81

Table 2
Trypanosoma cruzi in Trichomys apereoides: parasitological follow-up of laboratory reared animals inoculated with isolates with dierent origin and
genotypes
Isolates codea

MTRI/BR/1999/R4 (T. cruzi II)

MDID/BR/1999/M1(T. cruzi I)
MHOM/BR/1957/Y(T. cruzi II)
TBRA/BR/1999/JCA3 (T. cruzi II)
MLEO/BR/2000/M593 (T. cruzi II)
XXXX/BR/1971/F (T. cruzi I)
a
b

Pre-patent period (days)

Patent period (days)

SD

18
22
29
45
38
22

1.6
1.83
0
2.5
12.2
1.83

10
14

SD
7.4
3.2

17

10.6

Peak of parasitemia
(tripomastigotes/ml
blood)/day

Mortality (%)

2.9
106 /27

1.8
105 /22
0.2
105 /32
0.3
105 /46
0.6
105 /44
2.3
105 /26

50
0
0
0
0
0

Anon. (1999).
Intermittent patent parasitemia expressed by recurring parasitemia, Fig. 1.

with MTRI/BR/1999/R4, XXXX/BR/1971/F, and

MDID/BR/1999/M1 isolates and those that survived
MTRI/BR/1999/R4 inoculation displayed higher parasitemias and longer patent periods. When inoculated
with MHOM/BR/1957/Y, TBRA/BR/1999/JCA3, and
MLEO/BR/2000/M593 isolates, respectively, T. apereoides displayed intermittent patent parasitemias expressed by recurring parasitemia waves that reached
105 parasites/ml (Table 2; Figs. 1AC).
Positive hemocultures (performed at necropsy) were
observed only in animals infected with MTRI/BR/1999/
R4 and MHOM/BR/1957/Y isolates. Positive PCR of
viscera was observed in all infected animals (5 months
PI).
The kinetics of parasitemia in the animals infected
with MTRI/BR/1999/R4, MDID/BR/1999/M1, and
MHOM/BR/1957/Y isolates are shown in Figs. 1AC,
respectively.
3.2. Hematological ndings
Leukocytosis with high numbers of lymphocytes and
neutrophils was the main feature of all infected animals.
An increase of 4% in the size of myeloid cells with
presence of basophile granules was observed. Band cells
displayed increased nuclei with an amoeboid appearance.
Number of lymphocytes in infected animals increased
signicantly in relation to controls (95% of condence
by Students t test, p < 0:05), regardless of parasitemia
levels. The kinetics of the lymphocyte populations in
relation to parasitemia in the animals infected with
MTRI/BR/1999/R4, MDID/BR/1999/M1, and MHOM/
BR/1957/Y isolates are shown in Figs. 2AC.

tectable from the fth day PI onwards) was observed in

the rodents infected with MDID/BR/1999/M1 isolate. In
contrast, animals infected with the MTRI/BR/1999/R4
strain displayed positive IFA tests, only from the 22th
day of infection onwards (Figs. 1AC).
High serological titers (1:801:320) were observed in
all animals at necropsy, performed 5 months PI.
3.4. Histopathological ndings
Histological analyses of in extremis killed animals,
during the acute infection phase with MTRI/BR/1999/
R4 isolate showed parasites in viscera, muscles, and
glands (8/12 organs examined) with pseudocysts in
heart, skeletal, and smooth muscle. Invasion of pancreas acini, adipocytes, and macrophages of the connective tissue adjacent to skeletal muscle was found.
Liver and spleen were also parasitized. Parasites surrounded by diuse myocarditis and abundant inammatory lymphoeosinophylic inltrates with extensive
damage in cardiac bers and dierent grades of vacuolization and/or myocytolysis in muscular tissue were
observed.
No correspondence was observed between the prole
of parasitemia and parasite load in the subpatent phase
as accessed by the examination of HE stained tissues.
Indeed, the necropsied animals displayed only low
numbers of amastigote nests found only in duodenum
and heart of rodents inoculated with the MTRI/BR/
1999/R4 isolate that resulted in high parasitema and the
MHOM/BR/1957/Y isolate that induced intermittent
parasitemia. Additional histopathological ndings are
summarized in Table 3 and Fig. 3.
3.5. Stability of the infection

3.3. Humoral immune response

Infected T. apereoides responded with a strong humoral immune response. The onset of the humoral immune response varied according to the inoculated
isolate: a precocious humoral immune response (de-

The stability of infections of T. apereoides inoculated

with the T. cruzi isolates was evidenced by amplied T.
cruzi kDNA (330 bp band) in at least one of the following tissues: heart, skeletal muscle, and pancreas
(Fig. 4).

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L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Fig. 1. Trypanosoma cruzi in Trichomys apereoides. Kinetics of parasitemia () and humoral immune response (IFA)
. Each point represents
the mean values of parasite numbers/ml peripheral blood or IgG values plotted as log2 of the dilution titers of six animals infected with isolates
SD
MTRI/BR/1999/R4, T. cruzi II genotype (A); MDID/BR/1999/M1, T. cruzi I genotype (B); MHOM/BR/1957/Y, T. cruzi II genotype (C).
(standard deviation); and
, IgG titer, 5 months PI.

The Southern blot analyses of amplied PCR products conrmed that specic T. cruzi kDNA sequences
were present in all tissue samples studied. Consistently,
the amplied kDNA also hybridized with the probe. No
hybridization was observed in DNA from non-infected
animals and negative controls from the PCR (data not

shown). That T. apereoides may display stable infections

by T. cruzi was also demonstrated by a two-year followup of one exemplar experimentally infected with MDID/
BR/1999/M1 (T. cruzi I) isolate that remained infected
during two years PI, as observed by positive hemoculture performed at necropsy.

L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

83

Fig. 2. Trypanosoma cruzi in Trichomys apereoides. Kinetics of parasitemia () and mean values of peripheral blood lymphocytes of infected (gray
columns) and control animals (intermittent line). Infection with isolates: MTRI/BR/1999/R4, T. cruzi II genotype (A); MDID/BR/1999/M1, T. cruzi I
genotype (B); and MHOM/BR/1957/Y, T. cruzi II genotype (C). Each point represents the mean value of parasite numbers/ml peripheral blood on
peripheral blood lymphocytes of six animals infected with isolates
SD (standard error).

4. Discussion
Probably due to the diculties in breeding wild
mammals in captivity, they are rarely used as model
hosts for T. cruzi infection studies. Nevertheless, peculiarities of a given hostparasite interaction may be

better claried through studying the original host.

Herein we are evaluating the interaction of T. cruzi with
T. apereoides, a caviomorph rodent, a group claimed to
be ancient hosts of this parasite (Briones et al., 1999).
When inoculated with either of the two T. cruzi genotypes, T. apereoides displayed stable infections with

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L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Table 3
Trypanosoma cruzi in Trichomys apereoides: tissular parasitism and histopathology in microscopy of H/E viscera sections (5 lm thick, at intervals of
60 lm, 1000
)
Isolates/organ

MTRI/BR/
1999/R4
(patent
phase)

MTRI/BR/
1999/R4
(sub-patent
phase)

TBRA/BR/
1999/JCA3
(chronic phase)

MDID/BR/
1999/M 1
(chronic phase)

MLEO/BR/2000/
M593
(chronic phase)

MHOM/BR/
1950/Y
(chronic
phase)

XXXX/BR/
1971/F
(chronic
phase)

Heart
Skeletal muscle
Urinary bladder
Pancreas
Duodenum
Colon
Liver
Spleen

a, e, f, g, i
c, h, i
b, g, j, m
c, l
c, g, h
c, g, h
c
c, k, l

d, m, n
d, m
d, m, n
d
c, l
d
d
d

d, l
d
d
d
d
d
d
d

d, m, n
d
d
d
d
d
d
d

d, m, n
d, m, n
d, l
d
d
d
d
d

c, l, n
d
d
d
d
d
d
d

d, m, n
d
d
d
d
d, m
d
d

ac, Abundant, moderate or scarce pseudocysts with amastigotes and/or trypomastigotes.

d, No pseudocysts observed.
e, Diuse myocarditis; extensive degeneration of myocardial bers.
f, Lymphomacroeosinobasophilic inammatory inltration. Sarcoplasmic substitution.
g, Vacuolization and/or focal or diuse myocytolysis, adjacent to parasite nests.
h, Focal or interstitial lymphocytic myocytis.
i, Scarce smooth connective tissue.
j, Extensive peri-vascular smooth connective tissue.
k, Focal vacuolization in red pulp; apparent normal strome.
l, Scarce focal inammatory inltrates.
m, Abundant focal or disseminated inltrates in myocytic cells or in interstitia.
n, Focal or diuse myocytolysis and or vacuolization.

hardly any mortality and a signicant humoral immune

response. A trend for an inverse association between the
rise of humoral immune response and the fall of parasitemia could be observed. This was an expected feature
since correspondence between humoral immune response and decrease of parasitemia has already been
described in other placental and marsupial mammals
(dos Reis and Lopes, 2000; Jansen et al., 1991). It is
worth mentioning that IFA was performed with an antimouse (Muridae) conjugate. Higher serological titers
would be probably observed if a specic Trichomys
(Echymidae) conjugate had been used. The majority of
the rodents was able to eciently control the parasite
population as conrmed by the scarce parasite burden
observed in HE stained tissues after necropsy. However,
infection was not eliminated, as demonstrated by the
persistence of specic antibodies as well as positive PCR
tests in all inoculated rodents. It is worth mentioning
that when infected with the MHOM/BR/1957/Y isolate,
recognized as extremely virulent and pathogenic to Mus
musculus, T. apereoides displayed only intermittent
parasitemia and no mortality.
The T. apereoides muscle microhabitat was the most
appropriate niche for all T. cruzi isolates, as shown by
the frequency of colonization and PCR conrmed parasite persistence. T. cruzi was found colonizing almost
every tissue of this rodent including pancreatic cells and
adipocytes. This pan-infectivity of the parasite in T.
apereoides conrms the parasite eclecticism, regarding
its wide range of mammalian reservoirs and parasitized
tissues (Hoare, 1972; Lenzi et al., 1996).

Leukocytosis has already been described in other

experimentally infected mammal species. Furthermore,
the high number of polymorphonuclear cells is probably
a consequence of the rupture of parasitized cells and the
subsequent liberation of chemoattractants as similarly
proposed for other mammals (Monte

on et al., 1996).
The high number of neutrophils recorded in the initial
phase of T. apereoides infection is most likely explained
by intense parasite phagocytosis.
The histopathological framework observed in T.
apereoides was comparable to other experimental animal
models, in which myocytosis prevailed and myocytolysis
was observed. The characteristic inammatory inltrates of the colonized tissues were also present in the
chronic phase, when the number of amastigote nests was
scarce or absent, suggesting that inammation acts as a
control mechanism of tissular parasitism also in this
rodent species, as well as in other hosts (Molina and
Kierszenbaum, 1988; Soares et al., 2001; Teixeira et al.,
2002). Given that the described pathological features
observed in T. apereoides were also noticed in other
mammalian hosts including opossums, considered to be
the most ancient T. cruzi reservoir host (Schoeld,
2000), these characteristics are, probably, ancient traits
of the T. cruzi survival strategy.
Higher parasitemia could not be associated to the
parasite genotype since positive hemocultures were observed only in the animals infected with HOM/BR/1957/
Y and MTRI/BR/1999/R4 isolates, both characterized
as T. cruzi II and not with MLEO/BR2000/M593 and
TBRA/BR/1999/JCA3 isolates, also of this genotype.

L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

85

Fig. 3. Parasitological and histopathological features from Trichomys apereoides experimentally infected with dierent isolates and strains of Trypanosoma cruzi. (1) Degeneration of myocardial bers showing inammatory inltrate, vacuolization, and myocytolysis; pseudocysts with amastigotes (a)
and trypomastigotes (t), in the sarcoplasm of bers (MTRI/BR/1999/R4, T. cruzi II isolate, acute phase); (2) nest with amastigotes in smooth muscle (sm)
of urinary bladder near to the lumen (lu). Myocytolysis and lymphocytic diuse inltrates are observed (MTRI/BR/1999/R4 T. cruzi II isolate; acute
phase); (3) nest with amastigotes, lymphocytic inltrate, and cellular lysis in pancreas (MTRI/BR/1999/R4, T. cruzi II isolate; acute phase); (4) urinary
bladder: intense lymphocytic inltrate and lysis in myocytic cells and interstitium, without parasites (MTRI/BR/1999/R4 T. cruzi II isolate; chronic
phase); (5) amastigotes nest in heart, with myocytolysis and inltrate (MHOM/BR/1950/Y, T. cruzi II reference strain; chronic phase); and (6) interstitial
inammatory inltrate in skeletal muscle; parasites absents (MLEO/BR/2000/M593 T. cruzi II isolate; chronic phase) (HE; scale bar 15 lm).

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L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Fig. 4. PCR amplication of the 330 bp fragment (black long arrow)

from the conserved regions of kDNA extracted from tissues of
Trichomys apereoides experimentally infected (chronic phase, 5 months
PI) with Trypanosoma cruzi isolates and reference strains, in agarose
gel 2.5% electrophoresis (ethidium bromide stain): (A) heart; (B)
skeletal muscle; and (C) pancreas. Lane 1, migration of the markers of
1 kb ladder (Gibco-BRL Life Technologies, Gaithersburg, MD). Molecular size from the bottom up: 506, 11181115, and 16001300 bp
(black short arrow). Lane 2, negative animal control; lane 3, nude T.
cruzi DNA; lane 4, MTRI/BR/1999/R4, T. cruzi II isolate; lane 5,
MHOM/BR/1950/Y, T. cruzi II reference strain; lane 6, TBRA/BR/
1999/JCA3, T. cruzi II isolate; lane 7, MDID/BR/1999/M1, T. cruzi I
isolate; lane 8, XXXX/BR/1971/F, T. cruzi I reference strain; lane 9,
MLEO/BR/2000/M593, T. cruzi II isolate; and lane 10, no DNA in the
reaction mixture for PCR amplication.

Mortality and an overall severe scenario observed in T.

apereoides infected with MTRI/BR/1999/R4, an isolate
derived from a naturally infected T. apereoides, is
probably the consequence of the complexity of T. cruzi

transmission cycles in nature, where the parasite randomly infects several mammalian species through different routes and distinct inocula sizes. Indeed, as
observed in experimentally infected opossums, the oral
route resulted always in milder infections in comparison
to subcutaneously infected opossum (Jansen et al.,
1991).
Recent studies in light of more sensitive methodologies (LCSSP-PCR, RAPD, mini-exon polymorphism)
have emphasized the importance of the genomic variation of T. cruzi in denition of human disease. Also,
a putative association of T. cruzi strains with their
hosts, including humans, has been claimed (Andrade
et al., 2002; Vago et al., 1996, 2000; Zingales et al., 1999).
Indeed, it has been proven that living systems, i.e.,
animals and culture media, frequently act as biological
lters of T. cruzi subpopulations, with a consequent
parasite subpopulation selection (Deane et al., 1984a;
Jansen et al., 1991; Zingales et al., 1999). Moreover, at
least in Brazil, T. cruzi II is described as associated to
primates and to the domestic transmission cycle, while
T. cruzi I is associated to the sylvatic transmission
cycle (Fernandes et al., 1998, 1999). Nevertheless, this
does not seem to be a very strict association considering that infection by T. cruzi II has already been
described in Procyon lotor (ProcyoniidaeCarnivora),
T. apereoides (EchimyidaeRodentia) (data not
shown), Philander frenata, and Didelphis albiventris
(DidelphidaeMarsupialia) (Pietrzak and Pung, 1998;
Pinho et al., 2000). One should rather take into account that each animal species may exert distinct and
particular selective pressures on the several T. cruzi
subpopulations according to factors such as origin
and size of inocula, health status of the host animal,
co-infection with other parasites, and even other subpopulations of T. cruzi. Here, laboratory reared animals submitted to one single inoculum did not display
T. cruzi tissue dependent tropism, since no striking
dierences in the pathological picture in the chronic
phase could be associated to parasite genotype or
isolate. Consequently, histotropism in T. cruzi is
probably a non-xed peculiarity inuenced by several
macro- and microenvironmental parameters in addition to parasite and host genetic background. The
observed pathological picture of T. cruzi infection in
T. apereoides indicates that extreme caution is necessary
when forecasting the outcome of infection or attributing
virulence, morbidity and mortality to a given geno- or
phenotype of T. cruzi.
The presented results demonstrate that T. apereoides
may act as an ecient natural reservoir, since it maintains long-lasting infections by dierent T. cruzi subpopulations of both genotypes T. cruzi I and T. cruzi II.
In addition, this host, gradually becoming synanthropic,
may represent an important parasite source for human
infection.

L. Herrera et al. / Experimental Parasitology 107 (2004) 7888

Acknowledgments
The authors are thankful to Marlene Rodriguez,
Estefan
a Flores, Carlos Ard
e, and Alcidineia Ivo for
the technical support, to Dr. Paulo Sergio DAndrea for
the supply and management of the experimental caviomorph rodents, and to Dr. Vera Bongertz for many
helpful comments on the English version of the manuscript. Supported by: IRD/CNPq No. 910157-00-6,
PAPES No. 01250250108, CAPES, FUNDMHAM,
FIOCRUZ-Brazil,
CONICIT
No.
S198000388,
FONACYT S1-98000388, C.D.C.H.-U.C.V. No. 03314729-2000, and No. 0934-4097-2001. The present work
has the endorsement of the Ethical Commission for
Experimentation with Animal Models (CEUA) from
Fundac~
ao Oswaldo CruzFIOCRUZ, RJ, Brazil.
Registration No. P0007.

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