Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the...

Experimental Parasitology 107 (2004) 78–88

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Trypanosoma cruzi in a caviomorph rodent: parasitologicaland 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, Brazilb 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, Venezuelad 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, specific 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 infiltrates independent of the inoculated T. cruzi isolate. T. cruzi of different

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, fluorescein isothiocyanate; IFA, immunofluorescence assay; HE, hematoxylin–eosin; IgG, immunoglobulin G; kDNA, kinetoplast de-

oxyribonucleic acid; LCSSP-PCR, low-stringency single specific primer PCR; LIT, liver infusion tryptose medium; NNN, Novy–Mc Neal–Nicole

medium; PCR, polymerase chain reaction; PI, post-infection; SSC, saline sodium citrate solution; TAE, trisma acetate buffer; Taq, thermostable

DNA polymerase

1. Introduction

American Trypanosomiasis (Chagas, 1909) is a au-

tochthonous and ancient protozoan infection of wildmammals in which the heteroxenic hemoflagellate Try-

panosoma cruzi (Kinetoplastida, Trypanosomatidae) is

transmitted by wild vectors (Hemiptera, Reduviidae,

* Corresponding author. Fax: +55-21-280-1589.

E-mail address: [email protected] (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, differing in genetic and biological attributesand different 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 subpop-

ulations defined by its isozymic patterns or zymodemes.

mail to: [email protected]

L. Herrera et al. / Experimental Parasitology 107 (2004) 78–88 79

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 cavi-

omorph rodents, was suggested by Briones et al. (1999)

and Buscaglia and Di Noia (2003). Indeed, caviomorph

rodents and primates have an ancient evolutive historyin South America, since they arrived at the Southern

continent, coming probably from Africa, in the Oligo-

cene (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 hu-man dwellings. The genus Trichomys has an ample dis-

tribution in savannahs (‘‘cerrado’’), white scrub

(‘‘caatinga’’), and marshland (‘‘pantanal’’) biomes, typ-

ical 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 diver-

gence of T. cruzi I and T. cruzi II and the importance ofcaviomorph 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 spec-

imens collected inside Serra da Capivara National Park

a biological reserve in semi-arid biome of ‘‘caatinga,’’ inPiau�ı 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 cavi-

omorph, a possible reservoir in endemic areas of Chagas

disease. In this study, the parasitological and histo-

pathological patterns of experimental infection of T.

apereoides with T. cruzi I and T. cruzi II genotypes werestudied. Our purpose was also to supply data that may

clarify the putative association of caviomorph rodents

with the T. cruzi II genotype.

Table 1

Trypanosoma cruzi in Trichomys apereoides: isolates from different origins a

Codea Host

MTRI/BR/1999/R4 Trichomys apereoides

MDID/BR/1999/M1 Didelphis albiventris

TBRA/BR/1999/JCA3 Triatoma brasiliensis

MLEO/BR/2000/M593 Leontopithecus rosalia

MHOM/BR/1957/Y Human

XXXX/BR/1971/F Unknown primary sourceb

aAnon. (1999).bDeane et al. (1984b).

2. Materials and methods

2.1. Parasites

Experimental infections were performed with four

recently obtained T. cruzi isolates, previously typified 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 dis-

tributed in six batches of six animals; (n ¼ 36). The

animals, born at animal facilities and gently supplied by

Dr. P. D’Andrea from the Tropical Medicine Depart-

ment, Oswaldo Cruz Institute, RJ, Brazil, were inocu-

lated subcutaneously with 200 metacyclics/g body

weight, for each isolate. A group of non-infected ani-mals was included as control (n ¼ 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 untilnegativation of parasitemia and the following parame-

ters were determined: pre-patent period, parasitemia (by

countings in a Neubauer hemocytometer) and pattern

and percentage of hematic cells (by differential countings

of 100 microscopic fields). 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).

nd molecular groups used in this study

Locality T. cruzi group

Piau�ı, Northeast Brazil II

Piau�ı, Northeast Brazil I

Piau�ı, Northeast Brazil II

Rio de Janeiro, Brazil II

Maintained in Brazil as reference isolate b II

Maintained in Brazil as reference isolateb I

80 L. Herrera et al. / Experimental Parasitology 107 (2004) 78–88

Hemocultures were performed in all animals at nec-ropsy, which was accomplished 5 months PI. Hemo-

cultures 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 sub-patent phase of infection (5 months PI) of all groups

were sacrificed by anesthetic overdose of ketamine

(Ketaset HCl 100mg/ml). Tissue samples were fixed 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 intes-tines, 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 pho-

tographed with a Nikon Microflex HPX-35 camera on

Agfa APX-100 films.

2.5. Extraction of DNA from T. apereoides tissue sections

Sections (5–10 lm thick) from blocks of fixed em-

bedded 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 2–3 drops ofacetone. Tissue digestion was done with 100 ll of di-

gestion buffer (50mM Tris, pH 8.5, 1mM 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 Purification System (Promega,

Maddison, WI).

2.6. Detection of T. cruzi DNA by polymerase chain

reaction amplification

Specific polymerase chain reaction (PCR) amplifica-

tion of a nucleotide sequence of the 330 bp corre-

sponding to the four variable regions of T. cruzi kDNA

minicircle in the digested tissue of T. apereoides was

performed with primers 121—50-AAATAATGTACGGGKGAGATGCATGA-30 and 122—50-GGTTCGATT

GGGTTGGTGTAATATA-30 (Britto et al., 1995).

Briefly, 5 ll of DNA template was added to 15 ll of PCRmixture to give a final concentration of 20mM Tris–

HCl, pH 8.8; 50mM KCl; 1.5mM MgCl2; 10 lMdNTPs, 10 pM of each primer, and 1U of Taq DNA

Polymerase.

An initial denaturizing step at 94 �C, for 4min, wasfollowed by 35 cycles of 94, 55, and 72 �C, for 1min

each, and an extension at 72 �C for 10min in a Pro-

grammable Thermal Controller (Lane et al., 1997).

To ensure that the product of DNA tissue isolation

was amenable to DNA amplification, the b-actin protein

for mice was simultaneously amplified with the primers

50-GCTGTGCTATGTTGCCCTAGAATTCGAGC-30

and 50-CGTACTCCTGCTTGCTGATCCACATGTGC-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 neg-

ative 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

amplification, electrophorized in 2.5% agarose gel, were

submitted to alkali denaturizing step (0.5N NaOH;1.5M NaCl) and, subsequently, transferred to nylon

membranes (capillary transfer). The membrane was

neutralized with 1.5M NaCl and 0.5M NaOH, washed

with SSC 10�, and the transferred products were fixed

with 120,000mJ of ultraviolet light, using an ultraviolet

cross-linking apparatus. DIG-labeled DNA probes were

generated with DIG-High Prime, according to the ran-

dom primed labeling technique (DIG High Prime: DNAlabeling 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 anti-

digoxygenin phosphatase alkaline conjugated Fab

fragments and visualized with a chemiluminescent sub-

strate and recorded on X-ray Films (10min exposure

time).

3. Results

3.1. Parasitological follow-up

Data concerning parasitological follow-up are sum-

marized in Table 2. In short, T. apereoides was able toefficiently 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

Table 2

Trypanosoma cruzi in Trichomys apereoides: parasitological follow-up of laboratory reared animals inoculated with isolates with different origin and

genotypes

Isolates codea Pre-patent period (days) Patent period (days) Peak of parasitemia

(tripomastigotes/ml

blood)/day

Mortality (%)

X SD X SD

MTRI/BR/1999/R4 (T. cruzi II) 18 1.6 10 7.4 2.9� 106/27 50

MDID/BR/1999/M1(T. cruzi I) 22 1.83 14 3.2 1.8� 105/22 0

MHOM/BR/1957/Y(T. cruzi II) 29 0 b — 0.2� 105/32 0

TBRA/BR/1999/JCA3 (T. cruzi II) 45 2.5 b — 0.3� 105/46 0

MLEO/BR/2000/M593 (T. cruzi II) 38 12.2 b — 0.6� 105/44 0

XXXX/BR/1971/F (T. cruzi I) 22 1.83 17 10.6 2.3� 105/26 0

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


with MTRI/BR/1999/R4, XXXX/BR/1971/F, andMDID/BR/1999/M1 isolates and those that survived

MTRI/BR/1999/R4 inoculation displayed higher para-

sitemias and longer patent periods. When inoculated

with MHOM/BR/1957/Y, TBRA/BR/1999/JCA3, and

MLEO/BR/2000/M593 isolates, respectively, T. apereo-

ides displayed intermittent patent parasitemias ex-

pressed by recurring parasitemia waves that reached

105 parasites/ml (Table 2; Figs. 1A–C).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. 1A–C,respectively.

3.2. Hematological findings

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 cellsdisplayed increased nuclei with an amoeboid appear-

ance.

Number of lymphocytes in infected animals increased

significantly in relation to controls (95% of confidence

by Student’s 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. 2A–C.

3.3. Humoral immune response

Infected T. apereoides responded with a strong hu-

moral immune response. The onset of the humoral im-

mune response varied according to the inoculated

isolate: a precocious humoral immune response (de-

tectable from the fifth day PI onwards) was observed inthe 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. 1A–C).

High serological titers (1:80–1:320) were observed in

all animals at necropsy, performed 5 months PI.

3.4. Histopathological findings

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 pan-

creas acini, adipocytes, and macrophages of the con-

nective tissue adjacent to skeletal muscle was found.Liver and spleen were also parasitized. Parasites sur-

rounded by diffuse myocarditis and abundant inflam-

matory lymphoeosinophylic infiltrates with extensive

damage in cardiac fibers and different grades of vacu-

olization and/or myocytolysis in muscular tissue were

observed.

No correspondence was observed between the profile

of parasitemia and parasite load in the subpatent phaseas 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 findings are

summarized in Table 3 and Fig. 3.

3.5. Stability of the infection

The stability of infections of T. apereoides inoculated

with the T. cruzi isolates was evidenced by amplified T.

cruzi kDNA (330 bp band) in at least one of the fol-

lowing tissues: heart, skeletal muscle, and pancreas

(Fig. 4).

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

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). SD

(standard deviation); and , IgG titer, 5 months PI.


The Southern blot analyses of amplified PCR prod-

ucts confirmed that specific T. cruzi kDNA sequences

were present in all tissue samples studied. Consistently,

the amplified 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 follow-

up 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 hemocul-

ture performed at necropsy.

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 difficulties in breeding wild

mammals in captivity, they are rarely used as model

hosts for T. cruzi infection studies. Nevertheless, pecu-

liarities of a given host–parasite interaction may be

better clarified through studying the original host.

Herein we are evaluating the interaction of T. cruzi withT. 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 ge-

notypes, T. apereoides displayed stable infections with

Table 3

Trypanosoma cruzi in Trichomys apereoides: tissular parasitism and histopathology in microscopy of H/E viscera sections (5lm thick, at intervals of

60 lm, 1000�)

Isolates/organ MTRI/BR/

1999/R4

MTRI/BR/

1999/R4

TBRA/BR/

1999/JCA3

MDID/BR/

1999/M 1

MLEO/BR/2000/

M593

MHOM/BR/

1950/Y

XXXX/BR/

1971/F

(patent

phase)

(sub-patent

phase)

(chronic phase) (chronic phase) (chronic phase) (chronic

phase)

(chronic

phase)

Heart a, e, f, g, i d, m, n d, l d, m, n d, m, n c, l, n d, m, n

Skeletal muscle c, h, i d, m d d d, m, n d d

Urinary bladder b, g, j, m d, m, n d d d, l d d

Pancreas c, l d d d d d d

Duodenum c, g, h c, l d d d d d

Colon c, g, h d d d d d d, m

Liver c d d d d d d

Spleen c, k, l d d d d d d

a–c, Abundant, moderate or scarce pseudocysts with amastigotes and/or trypomastigotes.

d, No pseudocysts observed.

e, Diffuse myocarditis; extensive degeneration of myocardial fibers.

f, Lymphomacroeosinobasophilic inflammatory infiltration. Sarcoplasmic substitution.

g, Vacuolization and/or focal or diffuse 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 inflammatory infiltrates.

m, Abundant focal or disseminated infiltrates in myocytic cells or in interstitia.

n, Focal or diffuse myocytolysis and or vacuolization.


hardly any mortality and a significant humoral immuneresponse. A trend for an inverse association between the

rise of humoral immune response and the fall of para-

sitemia could be observed. This was an expected feature

since correspondence between humoral immune re-

sponse 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 anti-mouse (Muridae) conjugate. Higher serological titers

would be probably observed if a specific Trichomys

(Echymidae) conjugate had been used. The majority of

the rodents was able to efficiently control the parasite

population as confirmed by the scarce parasite burden

observed in HE stained tissues after necropsy. However,

infection was not eliminated, as demonstrated by the

persistence of specific antibodies as well as positive PCRtests 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 confirmed par-asite 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 confirms 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 otherexperimentally 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 inflammatory infil-

trates of the colonized tissues were also present in the

chronic phase, when the number of amastigote nests was

scarce or absent, suggesting that inflammation acts as a

control mechanism of tissular parasitism also in thisrodent 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 (Schofield,

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 ob-

served 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.

Fig. 3. Parasitological and histopathological features from Trichomys apereoides experimentally infected with different isolates and strains of Trypan-

osoma cruzi. (1) Degeneration of myocardial fibers showing inflammatory infiltrate, vacuolization, and myocytolysis; pseudocysts with amastigotes (a)

and trypomastigotes (t), in the sarcoplasmof fibers (MTRI/BR/1999/R4,T. cruzi II isolate, acute phase); (2) nestwith amastigotes in smoothmuscle (sm)

of urinary bladder near to the lumen (lu). Myocytolysis and lymphocytic diffuse infiltrates are observed (MTRI/BR/1999/R4 T. cruzi II isolate; acute

phase); (3) nest with amastigotes, lymphocytic infiltrate, and cellular lysis in pancreas (MTRI/BR/1999/R4, T. cruzi II isolate; acute phase); (4) urinary

bladder: intense lymphocytic infiltrate and lysis in myocytic cells and interstitium, without parasites (MTRI/BR/1999/R4 T. cruzi II isolate; chronic

phase); (5) amastigotes’ nest in heart,withmyocytolysis and infiltrate (MHOM/BR/1950/Y,T. cruzi II reference strain; chronic phase); and (6) interstitial

inflammatory infiltrate in skeletal muscle; parasites absents (MLEO/BR/2000/M593 T. cruzi II isolate; chronic phase) (HE; scale bar¼ 15lm).


Fig. 4. PCR amplification 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). Mo-

lecular size from the bottom up: 506, 1118–1115, and 1600–1300 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 amplification.


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, isprobably the consequence of the complexity of T. cruzi

transmission cycles in nature, where the parasite ran-domly infects several mammalian species through dif-

ferent 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 methodol-

ogies (LCSSP-PCR, RAPD, mini-exon polymorphism)have emphasized the importance of the genomic vari-

ation of T. cruzi in definition 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

filters of T. cruzi subpopulations, with a consequentparasite 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 consid-

ering that infection by T. cruzi II has already beendescribed in Procyon lotor (Procyoniidae—Carnivora),

T. apereoides (Echimyidae—Rodentia) (data not

shown), Philander frenata, and Didelphis albiventris

(Didelphidae—Marsupialia) (Pietrzak and Pung, 1998;

Pinho et al., 2000). One should rather take into ac-

count that each animal species may exert distinct and

particular selective pressures on the several T. cruzi

subpopulations according to factors such as originand size of inocula, health status of the host animal,

co-infection with other parasites, and even other sub-

populations of T. cruzi. Here, laboratory reared ani-

mals submitted to one single inoculum did not display

T. cruzi tissue dependent tropism, since no striking

differences in the pathological picture in the chronic

phase could be associated to parasite genotype or

isolate. Consequently, histotropism in T. cruzi isprobably a non-fixed peculiarity influenced by several

macro- and microenvironmental parameters in addi-

tion 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 efficient natural reservoir, since it main-

tains long-lasting infections by different T. cruzi sub-

populations 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.


Acknowledgments

The authors are thankful to Marlene Rodriguez,

Estefan�ıa Flores, Carlos Ard�e, and Alcidineia Ivo for

the technical support, to Dr. Paulo Sergio D’Andrea for

the supply and management of the experimental cavi-

omorph rodents, and to Dr. Vera Bongertz for many

helpful comments on the English version of the manu-

script. 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. 0331-

4729-2000, 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 Cruz—FIOCRUZ, RJ, Brazil.

Registration No. P0007.

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