Transactions of the Royal Society of Tropical Medicine and Hygiene (2005) 99, 379388

Trypanosoma cruzi infection in wild mammals of

the National Park Serra da Capivara and its
surroundings (Piau, Brazil), an area
endemic for Chagas disease
L. Herrera
a, b
, P.S. DAndrea
c
, S.C.C. Xavier
a
, R.H. Mangia
d
,
O. Fernandes
d
, A.M. Jansen
a,
a
Laboratory of Trypanosomatid Biology, Department of Protozoology, Oswaldo Cruz Institute, FIOCRUZ,
Av. Brasil 4365, Manguinhos, RJ, Brazil
b
Institute of Tropical Zoology, Science Faculty, Universidad Central de Venezuela 47058, Los
Chaguaramos 1041-A, Caracas, Venezuela
c
Laboratory of Biology and Control of Schistosomoses, Department of Tropical Medicine, Oswaldo Cruz
Institute, FIOCRUZ, Av. Brasil 4365, Manguinhos, RJ, Brazil
d
Department of Tropical Medicine, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Manguinhos, RJ,
Brazil
Received 14 April 2004; received in revised form 5 July 2004; accepted 7 July 2004
KEYWORDS
Chagas disease;
Trypanosoma cruzi;
Wild mammals;
Dogs;
Goats;
Brazil
Summary We studied the prevalence of Trypanosoma cruzi infection among eight
species of wild small mammals (n = 289) in an area where human cases of infec-
tion/disease have occurred. Dogs (n = 52) and goats (n = 56) were also surveyed. The
study was carried out inside a biological reserve, the National Park Serra da Capi-
vara and its surroundings in Piau State, Brazil. The marsupial Didelphis albiventris
and the caviomorph rodent Trichomys apereoides were found to be the most im-
portant reservoirs in the study area. Trichomys apereoides was the most abundant
species (80%) and D. albiventris the most frequently infected (61%). Both T. cruzi
I and T. cruzi II genotypes were isolated from these species. One specimen of Tr.
apereoides displayed a mixed T. cruzi I/zymodeme 3 infection. Serum prevalence
among dogs suggests that they may be involved in the maintenance of the parasite
in the peridomestic environment, in contrast to goats, which are not apparently of
any epidemiological importance. The distinct distribution and patterns of infection
observed in the study areas suggest that even in the same biome, epidemiological
* Corresponding author. Tel.: +55 21 25984324; fax: +55 21 25606572.
E-mail address: jansen@ioc.ocruz.br (A.M. Jansen).
0035-9203/$ see front matter 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.trstmh.2004.07.006
380 L. Herrera et al.
studies or determination of control measures must take into account ecological pe-
culiarities.
2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd.
All rights reserved.
1. Introduction
Trypanosoma cruzi (Kinetoplastida: Trypanosomati-
dae), the agent of Chagas disease, is a widely dis-
tributed trypanosomatid. It has been detected in
more than 100 mammalian species, belonging to
eight orders, which are spread through all phy-
togeographic regions of the Neotropics (Barretto,
1979).
Trypanosoma cruzi displays extreme genetic di-
versity. The geographic distribution of its genetic
variants and their association with particular hosts
remain unclear. The earliest studies, based on
the analysis of six isozymes, recognized three
main subpopulation assemblages or zymodemes:
zymodemes 1(Z1), 2(Z2) and 3(Z3) which, in Brazil,
were associated mainly with human infection (Z2)
and to the enzootic transmission cycle (Z1 and Z3).
Subpopulation Z3 is prevalent mainly in Amazonas
and is associated with acute or indeterminate hu-
man cases. Infrequent cases of human infection by
this zymodeme were also reported in Baha and Par a
states. In the wild, Z3 is associated with terrestrial
and burrowing animals, mainly armadillos (Miles,
1983).
Recent molecular studies (analysis of rRNA
genes, miniexon genes and microsatellites) recog-
nized two main subpopulations of T. cruzi: T. cruzi
I (TcI, corresponding to Z1) found in wild mammals,
particularly opossums of the genus Didelphis, and
T. cruzi II (TcII, corresponding to Z2), associated
with areas of Brazil endemic for Chagas disease
and recently linked to placental mammals (Devera
et al., 2003). It has been suggested that TcI origi-
nated from Didelphis marsupialis and TcII from pri-
mates and/or caviomorph rodents (Briones et al.,
1999; Miles, 1983). The phylogenetic relatedness of
Z3 subpopulation to TcI or TcII is still controversial
(Araujo et al., 2002).
Domestic and sylvatic transmission cycles of T.
cruzi may be isolated or connected (Miles et al.,
1977). Nevertheless, the so-called sylvatic trans-
mission cycle is far more complex than was formerly
understood, since distinct transmission cycles may
occur simultaneously in the same forest fragment
independently of the forest strata or behavior pat-
tern of the hosts (Fernandes et al., 1999; Jansen et
al., 2000; Lisboa et al., 2000). Brazil is considered
to be free from vectorial transmission of Chagas
disease. Nevertheless detailed studies of still un-
known aspects of the biology and ecology of T.
cruzi, including the variables that modulate the
transmission cycles among its reservoir hosts, are
of pivotal importance in a former endemic area, if
denitive control of the disease is proposed.
In the National Park Serra da Capivara (PARNA)
the signs of very ancient human colonization in
South America, attested by paintings on the walls
of caves and rock shelters (Guidon and Arnaud,
1991) suggest that an ancient interaction between
T. cruzi and primates, including humans, may have
occurred in this ecosystem.
This paper presents the results of a study on
the infection of wild small mammals and domestic
mammals by T. cruzi inside the PARNA and in two
neighboring districts.
2. Materials and methods
2.1. Study area
The region studied, located in the southwest of the
state of Piau, is one of the poorest and most un-
derdeveloped regions in Brazil. The climate is semi-
arid and the region is known as caatinga (white
scrub) due to the whitish color of the leaess vege-
tation during the dry season. The mammalian fauna
in the caatinga is actually a subset of the fauna of
Atlantic rainforest and Cerrado, a central region
of Brazil (FUNDMHAM, 1998). The wild small mam-
malian fauna consists mainly of marsupials and ro-
dents; they prefer the more humid microhabitats
among the rocks, but can also be found close to
human dwellings.
The PARNA is located between latitude 08

26

50

and 08

54

23

S and longitude 42

19

47

and
42

45

51

Win Piau; there has been no human inu-

ence, besides protection measures, in this area for
the last 2030 years. We studied three areas inside
the PARNA, Zabel e (Z), Sitio dos Oitenta (S80) and
Pedra Solta (PS) and two surrounding administra-
tive districts, Jo ao Costa (JC) and Coronel Jose Dias
(CJD). They are 40 km away from each other. Both
districts are subjected to much human activity and
the vegetation is highly degraded by deforestation,
burning and pressure from raising goats (Figure 1).
In JC, both the cardiac and digestive forms of the
Trypanosoma cruzi infection in wild mammals 381
Figure 1 Location of the National Park Serra da Capivara (PARNA) and its surroundings, Piau State, north-eastern
Brazil. ( ) PARNA, () Jo ao Costa, ( ) Coronel Jose Dias, ( ) S ao Jo ao de Piau, ( ) S ao Raimundo Nonato. PS, Pedra
Solta; Z, Zabele; S80, Sitio dos Oitenta.
human disease have been described, although no
new case has been reported in the last 10 years.
In CJD, in spite of 4% human serum prevalence, no
clinical case was observed (Borges-Pereira et al.,
2002; Ramos and Maul, 2001; Texeira, 1993).
2.2. Capture of wild small mammals
Mammals were collected with baited Tomahawk
and Sherman traps (Tomahawk Live Traps Co.,
Tomahawk, Winsconsin, USA). The traps (250) were
placed in linear transects, at 20-m intervals in rock
caves, near to water sources, natural pools and
canyons, on four nights, for each site. The total
capture effort was 5000 trap-nights. Animals were
collected on expeditions conducted in the dry sea-
sons between 1999 and 2001.
Since Trichomys apereoides was the most col-
lected species, a goodness-of-t test was applied in
order to evaluate its pattern of distribution in the
study areas. The variables used were the maximum
distance between the cumulative real distribution
of Tr. apereoides and the tted data for normal dis-
tribution in the program Statgraphics 2.0.
382 L. Herrera et al.
Blood for hemoculture and serum of wild mam-
mals was collected by venepuncture under anes-
thesia (50-mg/kg bodyweight of ketamine by intra-
muscular injection). After recovery from anesthe-
sia and in order to avoid repeated examinations of
the same specimen, the animals were individually
marked with ear tags before being released at the
capture sites. Depending on the volume of blood,
both hemoculture and serology were performed.
If insufcient material was collected, priority was
given to the hemoculture.
Two tubes containing NNN medium with a liver
infusion triptose (LIT) overlay were each inoculated
with 0.2 ml of blood from each animal. The tubes
were examined in the laboratory every other week
up to a maximumof ve months for the serologically
positive animals and two months for the indirect
immunouorescence assay (IFA)-negative animals.
Furthermore, a total of 108 domestic animals, 52
dogs and 56 goats, from JC and CJD respectively,
were examined with the informed consent of their
owners. Blood to test for anti-T. cruzi antibodies
by IFA was drawn by puncture of the cephalic vein
(dogs) or jugular vein (goats).
2.3. Culture of Trypanosoma cruzi isolates
Parasites from the positive hemocultures were am-
plied in LIT culture medium for a maximum of
ve passages. Subsequently the medium was cen-
trifuged (1400 g) and the cell pellet resuspended in
0.2 ml of TE buffer (10 mM Tris HCl, pH 8.0, 10 mM
EDTA) pH 8.0 for cryopreservation and molecular
characterization.
2.4. Serological tests
The IFA was performed according to Camargo
(1966). Briey, serial two-fold sera dilutions
(1:101:1280) were assayed against T. cruzi ax-
enic medium (LIT) epimastigote forms of strain
I00/BR/00F, TcI (Deane et al., 1984b).
Rodent sera, except that of Tr. apereoides,
were tested with a commercial uorescein isoth-
iocyanate labeled anti-rat IgG (FITC, Sigma

). Tri-
chomys apereoides and marsupial sera were tested
with specic intermediary antibody anti-opossum
and anti-Trichomys sera raised in rabbits; the reac-
tion was visualized using a commercial FITC labeled
anti-rabbit serum (Sigma

). The cut-off value for

the serological titers of rodents was 1:10, since
this was the lowest serum dilution of an animal
with positive hemoculture. For opossums, the cut-
off value was 1:40, as described elsewhere (Jansen
et al., 1985).
In addition to being assayed against T. cruzi,
and in order to evaluate possible cross-reactions,
dog sera were also assayed against Leishma-
nia infantum (syn. chagasi) promastigotes
(MHOM/BR/1996/RR050 strain); both reactions
were performed with a commercial FITC labeled
anti-dog IgG (Sigma

). Goat sera were tested

against T. cruzi and Phytomonas davidi (kindly
supplied by Dr Previato, Federal University of Rio
de Janeiro, Brazil). The reaction was visualized by
a commercial anti-goat FITC conjugate (Sigma

).
The cut-off value for the serological titers of goats
and dogs was 1:40. Two negative controls (sera
from laboratory-reared animals) from each tested
species were always included. Sera obtained from
experimentally infected animals from all tested
species were used as positive control in all assays.
Antigen was obtained from parasites harvested
from axenic culture in the exponential phase.
Parasites were centrifuged twice in buffer solution
and maintained in formolized phosphate buffer
solution (0.15 M-1%). The reaction was performed
on glass slides and antigen was diluted to yield
40 parasites/eld under 400 magnication.
Commercial FITC conjugates were always used as
recommended by the manufacturer (Sigma

).
2.5. Molecular characterization of
Trypanosoma cruzi isolates
The cryopreserved isolates were incubated with
0.5% sodium dodecyl sulfate and genomic DNA
was extracted from parasite lysates using phenol-
chloroform 1:1 and precipitated with sodium ac-
etate and ethanol.
A miniexon multiplex PCR assay was carried out
in order to type T. cruzi isolates as TcI, TcII, Z3 or
Trypanosoma rangeli. Four oligonucleotides were
used as forward primers: TcI 5

ACACTTTCTGT-
GGCGCTGATCG3

; TcII 5

TTGCTCGCACACTCGGC-
TGATCG3

; Z3 5

CCGCGWACAACCCCTMATAAAAATG3

and TR (T. rangeli) 5

CCTATTGTGATCCCCATC-
TTCG3

. A common reverse oligonucleotide (ME

5

TACCAATATAGTACAGAAACTG3

), corresponding
to sequences present in the most conserved re-
gion of the miniexon gene was used as a reverse
primer, according to Fernandes et al. (2001). The
amplied PCR products were analyzed in ethidium
bromide-stained agarose gels (3%) and visualized
under ultraviolet light.
2.6. Biological characterization of
Trypanosoma cruzi isolates
Five isolates of T. cruzi derived from Didelphis al-
biventris (MDID/BR/1999/M1, TcI; MDID/BR/2001/
Trypanosoma cruzi infection in wild mammals 383
2632, TcI; MDID/BR/1999/M2, TcII; MDID/BR/1999/
M3, TcII; MDID/BR/2001/1239, TcI) and one from Tr.
apereoides (MTRI/BR/1999/R4, TcII) were charac-
terized by the experimental infection pattern in
Swiss-Webster mice. For this purpose, 10
5
meta-
cyclic trypomastigote forms were obtained through
spontaneous metacyclogenesis in LIT medium of
each T. cruzi isolate and inoculated intraperi-
toneally in batches of six mice (n = 36; bodyweight
1820 g). The daily follow-up included fresh blood
preparations and Giemsa-stained smears, as well as
counting of parasites in a Neubauer hemocytome-
ter.
3. Results
3.1. Wild mammals
A total of 289 wild mammals, mainly marsupials
and rodents, representing eight species was sam-
pled and examined (Table 1). The relation between
the capture effort and number of collected ani-
mals, i.e. the capture success, was 5.7%. The num-
ber of species collected at the ve sites Z, S80,
PS, CJD and JC were 3, 5, 3, 7, and 5 respec-
tively. Trichomys apereoides was the most abun-
dant and most widely distributed species in the
areas with and without human inuence. A nor-
mal distribution of Tr. apereoides by the good-
ness of t KolmogorovSmirnov test (P 0.10,
plus = 0.428269; minus = 0.287387) was observed in
all study areas. The synanthropic species D. al-
biventris was more frequent in the areas where hu-
man activity was exerted (Table 1).
3.2. Trypanosoma cruzi infection
Data on faunal composition and T. cruzi infection
of small wild mammals in each locality are summa-
rized in Table 1.
3.2.1. Wild mammals
Trypanosoma cruzi infection was detected by
hemoculture and/or IFA in six mammalian species:
D. albiventris, Monodelphis domestica, Gracili-
nanus agilis, Galea spixii, Tr. apereoides and
Rhiphidomys macrurus. None of the mammals col-
lected displayed patent parasitemia as assayed by
fresh blood smear examinations. Positive hemocul-
tures were observed in Tr. apereoides, D. albiven-
tris, G. spixii and M. domestica. (Table 1).
The prevalence and infection pattern of small
wild mammals was distinct in the ve study areas.
Transmission of T. cruzi appears to be focal, with ar-
T
a
b
l
e
1
A
b
u
n
d
a
n
c
e
o
f
a
n
i
m
a
l
s
p
e
c
i
e
s
a
n
d
p
r
e
v
a
l
e
n
c
e
o
f
T
r
y
p
a
n
o
s
o
m
a
c
r
u
z
i
i
n
f
e
c
t
i
o
n
b
y
h
e
m
o
c
u
l
t
u
r
e
(
H
)
a
n
d
i
m
m
u
n
o

u
o
r
e
s
e
n
c
e
a
s
s
a
y
(
I
F
A
)
i
n
t
h
e
N
a
t
i
o
n
a
l
P
a
r
k

S
e
r
r
a
d
a
C
a
p
i
v
a
r
a

(
P
A
R
N
A
)
a
n
d
i
t
s
s
u
r
r
o
u
n
d
i
n
g
s
,
P
i
a
u

S
t
a
t
e
,
B
r
a
z
i
l
S
p
e
c
i
e
s
A
b
u
n
d
a
n
c
e
I
n
s
i
d
e
P
A
R
N
A
S
u
r
r
o
u
n
d
i
n
g
P
A
R
N
A
T
o
t
a
l
Z
a
b
e
l
e
S
i
t
i
o
d
o
s
O
i
t
e
n
t
a
P
e
d
r
a
S
o
l
t
a
C
o
r
o
n
e
l
J
o
s
e
D
i
a
s
J
o
a
o
C
o
s
t
a
H
I
F
A
H
I
F
A
H
I
F
A
H
I
F
A
H
I
F
A
H
I
F
A
T
r
i
c
h
o
m
y
s
a
p
e
r
e
o
i
d
e
s
2
3
1
(
8
0
%
)
1
/
3
9
1
/
2
9
0
/
3
9
1
6
/
4
2
1
9
/
4
2
3
4
/
4
2
3
/
6
9
1
7
/
3
7
1
/
3
9
6
/
2
9
2
4
/
2
2
8
(
1
1
%
)
7
4
/
1
7
9
(
4
1
%
)
R
h
i
p
h
i
d
o
m
y
s
m
a
c
r
u
r
u
s
1
5
(
5
%
)
0
/
0
0
/
0
0
/
1
0
6
/
1
0
0
/
1
0
/
1
0
/
4
0
/
2
0
/
0
0
/
0
0
/
1
5
(
0
%
)
6
/
1
3
(
4
6
%
)
D
i
d
e
l
p
h
i
s
a
l
b
i
v
e
n
t
r
i
s
1
7
(
6
%
)
0
/
0
0
/
0
2
/
3
1
/
3
0
/
0
0
/
0
3
/
4
3
/
4
2
/
1
0
4
/
6
7
/
1
7
(
4
1
%
)
8
/
1
3
(
6
2
%
)
M
o
n
o
d
e
l
p
h
i
s
d
o
m
e
s
t
i
c
a
9
(
3
%
)
0
/
0
0
/
0
1
/
2
0
/
2
0
/
0
0
/
0
0
/
4
0
/
4
0
/
3
0
/
3
1
/
9
(
1
1
%
)
0
/
9
(
0
%
)
G
r
a
c
i
l
i
n
a
n
u
s
a
g
i
l
i
s
6
(
2
%
)
0
/
0
0
/
0
0
/
3
0
/
2
0
/
0
0
/
0
0
/
1
1
/
1
0
/
2
0
/
0
0
/
6
(
0
%
)
1
/
3
(
3
3
%
)
G
a
l
e
a
s
p
i
x
i
i
7
(
2
%
)
0
/
4
0
/
4
0
/
0
0
/
0
1
/
1
0
/
1
0
/
2
1
/
2
0
/
0
0
/
0
1
/
7
(
1
4
%
)
1
/
7
(
1
4
%
)
C
o
n
e
p
a
t
u
s
s
e
m
i
s
t
r
i
a
t
u
s
2
(
1
%
)
0
/
1
0
/
1
0
/
0
0
/
0
0
/
0
0
/
0
0
/
1
0
/
0
0
/
0
0
/
0
0
/
2
(
0
%
)
0
/
1
(
0
%
)
C
a
l
l
o
m
y
s
c
a
l
l
o
s
u
s
2
(
1
%
)
0
/
0
0
/
0
0
/
0
0
/
0
0
/
0
0
/
0
0
/
0
0
/
0
0
/
1
0
/
2
0
/
2
(
0
%
)
0
/
2
(
0
%
)
T
o
t
a
l
2
8
9
1
/
4
4
(
2
%
)
1
/
3
4
(
3
%
)
3
/
5
7
(
5
%
)
2
3
/
5
9
(
3
9
%
)
2
0
/
4
4
(
4
5
%
)
3
4
/
4
4
(
7
7
%
)
6
/
8
5
(
7
%
)
2
2
/
5
0
(
4
4
%
)
3
/
5
5
(
5
%
)
1
0
/
4
0
(
2
5
%
)
3
3
/
2
8
5
(
1
2
%
)
9
0
/
2
2
7
(
4
0
%
)
384 L. Herrera et al.
eas Z and PS representing the opposite ends of the
spectrum. Animals collected in Z displayed negligi-
ble infection rates in contrast to the animals col-
lected in PS, which displayed signicant high serum
prevalence and positive hemocultures, indicating
higher parasitemia and presumably higher infectiv-
ity for the vectors (Table 1).
Didelphis albiventris was the most commonly in-
fected species as assessed by IFA (62%); parasitemia
was detected by hemoculture in 41% of the speci-
mens examined.
Trichomys apereoides, the most abundant and
widely distributed species, displayed two distinct
patterns of T. cruzi infection: in PS, 45% of the
trapped animals displayed positive hemocultures
in contrast to the 4% (mean values) with positive
hemocultures observed in the other areas; sero-
logical titers with positive hemocultures ranged
from 1:10 to 1:160. No direct correlation could be
established between serological titer and positive
hemocultures; T. cruzi was isolated from infected
Tr. apereoides with serological titers of 1:10
Figure 2 Agarose gel electrophoresis of the amplied PCR products corresponding to the hypervariable region of the
non-transcribed spacer of mini-exon gene of Trypanosoma cruzi isolates from trapped wild mammals of the National
Park Serra da Capivara (PARNA) and its surroundings, Piau State, north-eastern Brazil. (A) Isolates fromPedra Solta vil-
lage (inside PARNA). Lane 1: negative control; 2: IOO/BR/OO/F.T. cruzi I pattern; 3: Y strain MDM/BR/OO/Dm28-T. cruzi
II pattern; 4: MHOM/BR/OO/JJ Z3 pattern; 5: T. rangeli out group; 619: T. cruzi isolates from Trichomys apereoides;
20: T. cruzi isolate from Galea spixii; 21: molecular weight marker corresponding to X DNA digested with Hae III.
(B) Isolates from Jo ao Costa and Coronel Jose Dias administrative districts (surrounding the PARNA). Lane 1: negative
control; 2: IOO/BR/OO/F.T. cruzi I pattern; 3: Y strain MDM/BR/OO/Dm28-T. cruzi II pattern; 4: MHOM/BR/OO/JJ Z3
pattern; 5: T. rangeli out group; 6: MDID/BR/1999/M1 isolate from Didelphis albiventris from Coronel Jos e Dias; 7:
MDID/BR/1999/M2 isolate from Didelphis albiventris from Coronel Jos e Dias; 8: MDID/BR/1999/M3 isolate from Didel-
phis albiventris trapped in Coronel Jos e Dias; 9: MDID/BR/2001/1239 isolate from Didelphis albiventris from Jo ao
Costa; 10: MDID/BR/2001/2632 isolate from Didelphis albiventris from Jo ao Costa; 11: MTRI/BR/1999/R4 isolate from
Trichomys apereoides from Coronel Jos e Dias; 12: molecular weight marker corresponding to X DNA digested with
HaeIII.
Trypanosoma cruzi infection in wild mammals 385
T
a
b
l
e
2
B
i
o
l
o
g
i
c
a
l
c
h
a
r
a
c
t
e
r
i
z
a
t
i
o
n
(
i
n
f
e
c
t
i
o
n
p
a
t
t
e
r
n
i
n
S
w
i
s
s
W
e
b
s
t
e
r
m
i
c
e
)
o
f

v
e
i
s
o
l
a
t
e
s
d
e
r
i
v
e
d
f
r
o
m
w
i
l
d
m
a
m
m
a
l
s
i
n
t
h
e
N
a
t
i
o
n
a
l
P
a
r
k

S
e
r
r
a
d
a
C
a
p
i
v
a
r
a

(
P
A
R
N
A
)
,
P
i
a
u

S
t
a
t
e
,
B
r
a
z
i
l
I
s
o
l
a
t
e
a
O
r
i
g
i
n
G
e
n
o
t
y
p
e
P
r
e
p
a
t
e
n
t
-
p
e
r
i
o
d
(
d
)
T
i
m
e
o
f
p
a
t
e
n
c
y
(
d
)
P
a
r
a
s
i
t
a
e
m
i
c
p
e
a
k
(
t
r
i
p
o
m
a
s
t
i
g
o
t
e
s
/
m
l
o
f
b
l
o
o
d

1
0
5

S
E
M
)
D
a
y
o
f
p
a
r
a
s
i
t
a
e
m
i
c
p
e
a
k
P
e
r
c
e
n
t
a
g
e
o
f
m
o
r
t
a
l
i
t
y
M
D
I
D
/
B
R
/
1
9
9
9
/
M
1
D
.
a
l
b
i
v
e
n
t
r
i
s
T
.
c
r
u
z
i
I
0
0
0
0
0
M
D
I
D
/
B
R
/
1
9
9
9
/
M
2
D
.
a
l
b
i
v
e
n
t
r
i
s
T
.
c
r
u
z
i
I
I
0
0
0
0
0
M
D
I
D
/
B
R
/
1
9
9
9
/
M
3
D
.
a
l
b
i
v
e
n
t
r
i
s
T
.
c
r
u
z
i
I
I
0
0
0
0
0
M
D
I
D
/
B
R
/
2
0
0
1
/
1
2
3
9
D
.
a
l
b
i
v
e
n
t
r
i
s
T
.
c
r
u
z
i
I
1
0
5
0
0
.
6
3

0
.
2
5
2
5
0
M
T
R
I
/
B
R
/
1
9
9
9
/
R
4
T
.
a
p
e
r
e
o
i
d
e
s
T
.
c
r
u
z
i
I
I
1
0
2
5
8
.
4
5

3
.
7
0
2
0
5
0
%
M
D
I
D
/
B
R
/
2
0
0
1
/
2
6
3
2
D
.
a
l
b
i
v
e
n
t
r
i
s
T
.
c
r
u
z
i
I
0
0
0
0
0
a
F
o
r
d
e
t
a
i
l
s
o
f
c
o
d
e
,
s
e
e
A
n
o
n
y
m
o
u
s
(
1
9
9
9
)
.
and negative hemocultures in animals with high
serological titers could also be observed.
3.2.2. Domestic mammals
Antibodies to T. cruzi only were observed in 11%
(6/52) of dogs; 10% (5/52) of dogs displayed only
anti-L. infantum-positive IFA titers. Two dogs dis-
played both L. infantum and T. cruzi positive IFA
titers. The serological survey of goats revealed that
2% (1/56) of the tested animals presented only anti-
T. cruzi antibodies, 32% (18/56) displayed only anti-
P. davidi antibodies. Forty-three per cent (24/56) of
the specimens reacted against both T. cruzi and P.
davidi. Of these goats, 58% (14/24) had higher titers
for P. davidi.
3.3. Molecular and biological
characterization of Trypanosoma cruzi
samples
Electrophoretic analysis of the products of the am-
plied miniexon gene showed that both genotypes
TcI and TcII were circulating in the study areas.
In PS, all infected animals except one were in-
fected only by genotype TcI. One specimen of Tr.
apereoides displayed a mixed infection (TcI and
Z3), evidenced by the presence of two amplied
products corresponding to 200 bp (TcI) and 150 bp
(Z3) (Figure 2A, lane 13). The T. cruzi isolate de-
rived from D. albiventris collected in JC had the
TcI genotype (200 bp band). Both genotypes, TcI and
TcII, were found infecting D. albiventris collected
in CJD. The only Tr. apereoides isolate derived from
CJD also had the TcII genotype (Figure 2B, lane 11).
Swiss mice were able to control the infection by
both TcI and TcII isolates efciently. Mortality was
observed only in mice inoculated with the isolate
MTRI/BR/1999/R4 TcII (Table 2).
4. Discussion
An animal species is dened as a reservoir when it is
essential for the long-term maintenance of a given
parasite in a specic ecosystem (Ashford, 1996).
Our results indicate that within the temporal and
spatial framework of this study, the caviomorph ro-
dent Tr. apereoides and the opossum D. albiven-
tris, very ancient hosts of T. cruzi, are acting
as important reservoirs although presenting dis-
tinct features that must be considered. Trichomys
apereoides and D. albiventris were eclectic reser-
voirs since they were found harboring both the ma-
jor phylogenetic lineages of T. cruzi including Z3.
The other species that were found to be infected,
386 L. Herrera et al.
R. macrurus, G. agilis, M. domestica and G. spixii,
may be considered as incidental hosts.
Didelphis albiventris was the host that displayed
the highest serum prevalence of infection (62%),
but was not very abundant (6%). Didelphis spp.
are nomadic mammals that easily adapt to human
dwellings and, in the present case, they harbored
both T. cruzi genotypes, TcI and TcII. Parasitemia in
D. albiventris was noteworthy given that the par-
asite was recovered by hemoculture from 41% of
the animals. These data show that in spite of its
low abundance, D. albiventris may be considered
as an important reservoir for T. cruzi in this study
area.
Only 11% of specimens of Tr. apereoides, the pre-
vailing species (80% of all trapped wild mammals),
tested by hemoculture gave positive results, com-
pared with 41% testing positive by serology. Tak-
ing into account the abundance of this mammal
species, Tr. apereoides may also be considered an
important reservoir species in the area.
In PS, a moist forest enclave in the semiarid
caatinga, the transmission cycle of T. cruzi dis-
played a distinct pattern, as 45% of the animals
examined displayed positive hemocultures, a much
higher value than found in the other study areas.
Trichomys apereoides and G. spixii were probably
involved in the same transmission cycle of the par-
asite since isolates from both species presented the
same genotype.
It was rather surprising to nd that 40% of iso-
lates derived from D. albiventris were character-
ized as TcII. Our previous observations and data
of other authors (Deane et al., 1984a; Jansen et
al., 1991, 1997) indicated that the closely related
species D. marsupialis was a reservoir for TcI and
tended to control and even eliminate TcII subpop-
ulations. The present ndings may be explained by
the peculiarities of the interaction of T. cruzi with
D. marsupialis and D. albiventris. However, the set
of reservoir species and distribution of TcII in the
wild environment are still poorly understood. TcII
infections of wild mammals were described in the
Atlantic Coastal Rain Forest, Brazil (Lisboa et al.,
2000; Pinho et al., 2000).
Both T. cruzi genotypes (TcI and TcII) were
found infecting Tr. apereoides, and in PS, all Tr.
apereoides were infected with TcI, except one
specimen with mixed infection (TcI/Z3). Z3 is de-
scribed as being mainly associated with infection
of humans and animals in the Amazon Basin and
northern region of Brazil (Miles et al., 1977; Santos
et al., 2002). The presence of Z3 in the caatinga
may be explained by the remnant enclaves of moist
forest relics that resisted the climatic changes in
the region (Vanzolini, 2002). Indeed, desertica-
tion process in the region started 12 000 years ago
(Guidon and Arnaud, 1991).
Goats do not seem to be important in the main-
tenance of T. cruzi in the study area. Positive sero-
logical titers were mainly specic for P. davidi,
reecting their exposure to other kinetoplastids
and consequently cross-reaction with T. cruzi. Eu-
phorbiacea, common plants on the rocks where
the goats graze and remain during the day, are
rather frequently infected by Phytomonas. The in-
gestion of these plants by the goats is the probable
cause of the non-specic antibodies. Cross-reaction
with Phytomonas due to ingestion of Phytomonas-
infected vegetables has already been suggested
(Bregano et al., 2003).
Trypanosoma cruzi serum prevalence in dogs was
low (11%). Nevertheless, it suggests that these an-
imals are somehow involved in the maintenance of
T. cruzi in the area close to peoples homes. The
importance of dogs in the transmission cycle of T.
cruzi has already been suggested by other authors
(Cohen and G urtler, 2001). Cross-reaction and high
serological titers to L. infantum were predictable,
since visceral leishmaniasis is endemic in Piau.
Our data show that natural infection of small
mammals in the study area was much lower than
that observed in several Atlantic Coastal rainfor-
est fragments in the state of Rio de Janeiro (south-
east Brazil), where autochthonous Chagas disease
has never been recorded (Lisboa et al., 2000; Pinho
et al., 2000). The current infection pattern found
in the study area may have changed compared with
the time when active transmission of T. cruzi to hu-
mans was still observed. Marked climatic seasonal-
ity, with severe drought periods, in the caatinga has
probably affected the density and survival of mam-
malian populations and, consequently, the trans-
mission cycle of the parasite, as suggested for other
vector-borne diseases (Franke et al., 2002; Walsh
et al., 1993). This feature would explain the low
prevalence of T. cruzi infection in the mammals of
all localities except PS (a humid enclave) and S80
both localized inside PARNA.
Alternatively, the present enzootic condition in
the wild may reect the past conditions, when ac-
tive transmission to humans still occurred and the
human cases originated as a consequence of human
invasion into an enzootic focus of high transmission.
This may explain the local epidemiology of Chagas
disease as already hypothesized by Ramos and Maul
(2001). They indicated that one of the most
important activities of the inhabitants of the re-
gion was the collection of rubber fromthe manic oba
tree (Manihot glaziovii). During the period of rub-
ber collection (almost eight months of the dry sea-
son) whole families lived in rock caves with an
Trypanosoma cruzi infection in wild mammals 387
environment similar to PS. In some of these shel-
ters, an active transmission cycle was established.
Returning to their houses at the end of the dry sea-
son, infected persons and their domestic animals
might have introduced a transmission cycle to their
villages. This activity ceased in recent decades and
as a consequence, transmission of the disease di-
minished signicantly; today there is only a resid-
ual cycle among dogs, rodents and opossums near
to human dwellings.
A transmission focus of TcII is very probable in
the area since both genotypes TcI and TcII were
found infecting Tr. apereoides and D. albiventris.
In experimental conditions, Tr. apereoides is able
to maintain stable infections of both genotypes
(Herrera et al., 2004).
The detection of TcII in marsupials and TcI in ro-
dents and the absence of correlation between these
genotypes and virulence for Swiss mice shows that,
at least in the present case, there was no strict as-
sociation of a given T. cruzi genotype with a given
group of mammals or virulence pattern as proposed
by Devera et al. (2003).
Different infection patterns of the same host
species in different localities of the same biome,
as was the case for Tr. apereoides in the present
study, indicates that this species displays distinct
infectivity for the vector according to the mi-
croenvironment colonized, and that this will in-
uence the dispersion of the parasite. This sug-
gests that the transmission cycle of T. cruzi in the
wild is more complex than has been assumed and
that demonstration of a given mammal species as
a reservoir is only possible when representative
microenvironments of the biome under study are
considered.
The increasingly overlapping distribution of
wildlife, livestock, domestic animals and humans
observed in recent decades is a feature that must
be considered in public health studies as well as
in conservation programs. An understanding of the
ecological role of hostparasite relationships and
their impact on the host population dynamics has
to be reached in order to prevent the risk of a dis-
ease spreading or re-emerging.
Ethical clearance
The present work has the endorsement of the
Ethical Commission for Experimentation with Ani-
mal Models (CEUA) from Fundac ao Oswaldo Cruz
FIOCRUZ, RJ, Brazil, registration number: P0007.
Conicts of interest statement
The authors have no conicts of interest concerning
the work reported in this paper.
Acknowledgements
The authors thank Carlos Ard e and Alcidineia
Ivo for technical support, and Dr Pavel Borodin
for English revision. This study was supported
by: IRD/CNPq No. 910157-00-6, PAPES No.
01250250108, CAPES, FUNDMHAM, FIOCRUZ-Brazil,
CONICIT No. S198000388, FONACIT S1-98000388,
C.D.C.H.-U.C.V. No. 0331-4729-2000 and No.
0934-4097-2001.
References
Anonymous, 1999. Recommendations from a Satellite Meeting.
International Symposium to commemorate the 90th anniver-
sary of the discovery of Chagas disease, April 1116 1999,
Rio de Janeiro, Brazil. Mem. Inst. Oswaldo Cruz 94 (Suppl.
1), 429432.
Araujo, C.A.C., Mello, C.B., Jansen, A.M., 2002. Trypanosoma
cruzi I and Trypanosoma cruzi II: recognition of sugar struc-
tures by Arachis hypogaea (peanut agglutinin) lectin. J. Par-
asitol. 88, 582586.
Ashford, R.W., 1996. Leishmaniasis reservoirs and their signi-
cance in control. Clin. Derm. 14, 523532.
Barretto, M.P., 1979. Epidemiologia. In: Brener, Z., Andrade, Z.
(Eds.), Trypanosoma cruzi e Doenc a de Chagas, Guanabara.
Koogan, Brasil, pp. 90151.
Borges-Pereira, J., Fonseca de Castro, J., Furtado Campos, J.,
de Souza Nogueira, J., Lago Souza, P., Marques, P., Cardoso,
M., Britto, C., Gonc alves de Ara ujo, A., 2002. Estudo da
infecc ao e morbidade da doenc a de Chagas no municpio Jo ao
Costa-Parque Nacional Serra da Capivara, Piau, Brasil. Rev.
Soc. Bras. Med. Trop. 35, 315322.
Bregano, J., Picao, R., Graca, V., Menolli, R., Itow Jankevicius,
S., Filho, P., Jankevicius, J.V., 2003. Phytomonas serpens,
a tomato parasite, shares antigens with Trypanosoma cruzi
that are recognized by human sera and induce protective
immunity in mice. FEMS Immunol. Med. Microbiol. 39, 257
264.
Briones, M.R.S., Souto, R.P., Stolf, B., Zingales, B., 1999. The
evolution of two Trypansoma cruzi subgroups inferred from
rRNA genes can be correlated with the interchange of Amer-
ican mammalian faunas in the Cenozoic and has implications
to pathogenicity and host specicity. Mol. Biochem. Parasitol.
104, 219232.
Camargo, M.E., 1966. Fluorescent antibody test for the sero-
diagnosis of American trypanosomiasis. Technical modica-
tion employing preserved culture forms of Trypanosoma cruzi
in a slide test. Rev. Inst. Med. Trop. S ao Paulo 8, 227
234.
Cohen, J., G urtler, R., 2001. Modeling household transmission of
American trypanosomiasis. Science 299, 694698.
Deane, M.P., Jansen, A.M., Lenzi, H.L., 1984a. Trypanosoma
cruzi: vertebrate and invertebrate cycles in the same mam-
mal host the opossum Didelphis marsupialis. Mem. Inst Os-
waldo Cruz. 79, 513515.
Deane, M.P., Sousa, M.A., Pereira Gonc alves, A., Momem, H.,
Morel, C., 1984b. Trypanosoma cruzi: inoculation schedules
and re-isolation methods demonstrated by schizodeme and
zymodeme analyses. J. Protozool. 31, 276280.
Devera, R., Fernandes, O., Coura, J.R., 2003. Should Try-
panosoma cruzi be called cruzi complex? A review of the
388 L. Herrera et al.
parasite diversity and the potential of selecting population
after in vitro culturing and mice infection. Mem. Inst. Os-
waldo Cruz. 98, 112.
Fernandes, O., Mangia, R.H., Lisboa, C.V., Pinho, A.P., Morel,
C.M., Zingales, B., Campbell, D.A., Jansen, A.M., 1999. The
complexity of the sylvatic cycle of Trypanosoma cruzi in Rio
de Janeiro State revealed by non-transcribed spacer of the
mini exon gene. Parasitology 118, 161166.
Fernandes, O., Santos, S.S., Cupolillo, E., Mendonc a, B., Derr e,
R., Junqueira, A.C.V., Santos, L.C., Sturm, N.R., Naiff, R.D.,
Barrett, T.V., Campbell, D.A., Coura, J.R., 2001. A mini-exon
multiplex polymerase chain reaction to distinguish the major
groups of Trypanosoma cruzi and T. rangeli in the Brazilian
Amazon. Trans. R. Soc. Trop. Med. Hyg. 95, 9799.
Franke, C.R., Ziller, M., Staubach, C., Latif, M., 2002. Impact
of the El Ni no/Southern oscillation on visceral leishmaniasis,
Brazil. Emerg. Infect. Dis. 8, 914917.
FUNDMHAM (Fundac ao Museu do Homem Americano), 1998. Par-
que Nacional Serra da Capivara. Piau-Brasil. Typelaser De-
senvolvimento Edit., S ao Paulo.
Guidon, N., Arnaud, B., 1991. The chronology of the New World:
two faces of one reality. World Arch. 23, 524529.
Herrera, L., Xavier, S.C.C., Viegas, C., Martinez, C., Cotas, P.M.,
Carrasco, H., Urdaneta-Morales, S., Jansen, A.M., 2004. Try-
panosoma cruzi in a caviomorph rodent: parasitological and
pathological features of the experimental infection of Tri-
chomys apereoides (Rodentia, Echimyidae). Exp. Parasitol.
107, 7888.
Jansen, A.M., Moriearty, P.L., Castro, B.G., Deane, M.P., 1985.
Trypanosoma cruzi in the opossum Didelphis marsupialis:
an indirect uorescent antibody test for the diagnosis and
follow-up of natural and experimental infections. Trans. R.
Soc. Trop. Med. Hyg. 79, 474477.
Jansen, A.M., Le on, L., Machado, G.M., da Silva, M.H., Souza-
Le ao, S.M., Deane, M.P., 1991. Trypanosoma cruzi in the
opossum Didelphis marsupialis: parasitological and serolog-
ical follow-up of the acute infection. Exp. Parasitol. 73,
249259.
Jansen, A.M., Madeira, M.F., Carreira, J.C.A., Deane, M.P., 1997.
Trypanosoma cruzi in the opossum Didelphis marsupialis: a
study of the correlations and kinetics of the systemic and
scents gland infection in naturally and experimentally in-
fected animals. Exp. Parasitol. 86, 3744.
Jansen, A.M., Lisboa, C.V., Pinho, A.P., Marchewsky, R.S., Man-
gia, R.H., Cupolillo, E., Fernandes, O., 2000. A ecologia e
a complexidade dos ciclos de transmiss ao do Trypanosoma
cruzi na natureza, In: Araujo-Jorge, T., Lisboa de Castro, S.
(Orgs.), Doenc a de Chagas. Manual para experimentac ao an-
imal. Editora Fiocruz, Rio de Janeiro, pp. 3339.
Lisboa, C.V., Dietz, J., Baker, A.J., Russel, N.N., Jansen, A.M.,
2000. Trypanosoma cruzi infection in Leonthopithecus rosalia
at the reserva biol ogica de Poc o das Antas, Rio de Janeiro,
Brazil. Mem. Inst. Oswaldo Cruz. 95, 445452.
Miles, M., 1983. The epidemiology of South American trypanoso-
miasis, biochemical and immunological approaches and their
relevance to control. Trans. R. Soc. Trop. Med. Hyg. 77, 523.
Miles, M.A., Toy e, P.J., Oswald, S.C., Godfrey, D.G., 1977. The
identication by isoenzyme patterns of two distinct strains
groups of Trypanosoma cruzi circulating independently in
a rural area of Brazil. Trans. R. Soc. Trop. Med. Hyg. 71,
217225.
Pinho, A.P., Cupolillo, E., Mangia, R.H., Fernandes, O., Jansen,
A.M., 2000. Trypanosoma cruzi in the sylvatic environment:
distinct transmission cycles involving two sympatric marsu-
pials. Trans. R. Soc. Trop. Med. Hyg. 94, 16.
Ramos, N.A., Maul, D.C., 2001. Os diferentes signicados da
certicac ao conferida ao Brasil como estando livre da Doenc a
de Chagas. Cad. Sa ude P ublica. 1, 14031412.
Santos, S.S., Cupolillo, E., Junqueira, A., Coura, J.R., Jansen,
A., Sturm, D.A., Campbell, D.A., Fernandes, O., 2002. The
genetic diversity of Brazilian Trypanosoma cruzi isolates and
the phylogenetic positioning of zymodeme 3, based on the
internal transcribed spacer of the ribosomal gene. Ann. Trop.
Med. Parasitol. 96, 755764.
Texeira, M.A.G., 1993. Potencial de transmiss ao da tripanos-
somase americana nas localidades do sitio de Moc o e Borda,
municpio S ao Raimundo Nonato. Sudeste do Piau. MS thesis.
Fundac ao Oswaldo Cruz, Escola Nacional de Sa ude P ublica,
Rio de Janeiro, Brazil, 170 pp.
Vanzolini, P.E., 2002. A second note on the geographical dif-
ferentiation of Amphisbaena fuliginosa L., 1758 (Squamata,
Amphisbaenidae), with a consideration of the forest refuge
model of speciation. An. Acad. Bras. Cienc. 74, 609648.
Walsh, J.F., Molyneux, D.H., Birley, M.H., 1993. Deforestation:
effects on vector-borne disease. Parasitology 106 (Suppl.),
S55S75.