Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae)
Trang 1Trypanosoma cruzi in a caviomorph rodent: parasitological
and pathological features of the experimental infection of Trichomys
apereoides (Rodentia, Echimyidae)
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, 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.
Ó2004 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 wild
mammals in which the heteroxenic hemoflagellate
Try-panosoma cruzi (Kinetoplastida, TryTry-panosomatidae) is
transmitted by wild vectors (Hemiptera, Reduviidae,
Triatominae) in a primary sylvatic cycle (Pinto Dias, 2000)
Trypanosoma cruzi comprises complex multiclonal
populations, differing in genetic and biological attributes and 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
*
Corresponding author Fax: +55-21-280-1589.
E-mail address:jansen@ioc.fiocruz.br (A.M Jansen).
0014-4894/$ - see front matter Ó 2004 Elsevier Inc All rights reserved.
doi:10.1016/j.exppara.2004.04.008
www.elsevier.com/locate/yexpr
Trang 2Zymodeme 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 history
in 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 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
spec-imens collected inside Serra da Capivara National Park
a biological reserve in semi-arid biome of ‘‘caatinga,’’ in
Piauı 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 were
studied Our purpose was also to supply data that may
clarify the putative association of caviomorph rodents
with the T cruzi II genotype.
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 until negativation 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)
Table 1
Trypanosoma cruzi in Trichomys apereoides: isolates from different origins and molecular groups used in this study
MTRI/BR/1999/R4 Trichomys apereoides Piau ı, Northeast Brazil II
MDID/BR/1999/M1 Didelphis albiventris Piau ı, Northeast Brazil I
TBRA/BR/1999/JCA3 Triatoma brasiliensis Piau ı, Northeast Brazil II
MLEO/BR/2000/M593 Leontopithecus rosalia Rio de Janeiro, Brazil II
MHOM/BR/1957/Y Human Maintained in Brazil as reference isolate b II
XXXX/BR/1971/F Unknown primary source b Maintained in Brazil as reference isolate b I
a
Anon (1999).
b Deane et al (1984b).
Trang 3Hemocultures 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 100 mg/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 of
acetone Tissue digestion was done with 100 ll of
di-gestion buffer (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 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-AAATAATGTACGG
Briefly, 5 ll of DNA template was added to 15 ll of PCR
mixture to give a final 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
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 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
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
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 (0.5 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 fixed 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 ran-dom 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 anti-digoxygenin phosphatase alkaline conjugated Fab fragments and visualized with a chemiluminescent sub-strate and recorded on X-ray Films (10 min 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 to
efficiently 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
Trang 4with 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
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
105parasites/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 cells
displayed 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 huhu-moral
im-mune response varied according to the inoculated
isolate: a precocious humoral immune response
(de-tectable from the fifth 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 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 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 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)
Table 2
Trypanosoma cruzi in Trichomys apereoides: parasitological follow-up of laboratory reared animals inoculated with isolates with different origin and
genotypes
Isolates code a Pre-patent period (days) Patent period (days) Peak of parasitemia
(tripomastigotes/ml blood)/day
Mortality (%)
MTRI/BR/1999/R4 (T cruzi II) 18 1.6 10 7.4 2.9 10 6 /27 50
MDID/BR/1999/M1(T cruzi I) 22 1.83 14 3.2 1.8 10 5 /22 0
MHOM/BR/1957/Y(T cruzi II) 29 0 b
— 0.2 10 5 /32 0
TBRA/BR/1999/JCA3 (T cruzi II) 45 2.5 b — 0.3 10 5 /46 0
MLEO/BR/2000/M593 (T cruzi II) 38 12.2 b — 0.6 10 5 /44 0
XXXX/BR/1971/F (T cruzi I) 22 1.83 17 10.6 2.3 10 5 /26 0
a
Anon (1999).
b
Intermittent patent parasitemia expressed by recurring parasitemia, Fig 1.
Trang 5The 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 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 log2of 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.
Trang 64 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 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 ge-notypes, T apereoides displayed stable infections with
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).
Trang 7hardly any mortality and a significant humoral immune
response 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 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 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 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 (Monteon 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 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 (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
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
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
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.
Trang 8Fig 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 sarcoplasm of fibers (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 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, with myocytolysis 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 bar ¼ 15 lm).
Trang 9Mortality 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 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 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
consid-ering that infection by T cruzi II has already been described 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 origin and 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 is
probably 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
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.
Trang 10The authors are thankful to Marlene Rodriguez,
Estefanıa Flores, Carlos Arde, 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,
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
Fundacß~ao Oswaldo Cruz—FIOCRUZ, RJ, Brazil
Registration No P0007
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