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Trypanosoma Found in Synanthropic Mammals from Urban Forests of Paraná,

Southern Brazil

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 VECTOR-BORNE AND ZOONOTIC DISEASES
Volume XX, Number XX, 2019
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2018.2433

Trypanosoma Found in Synanthropic Mammals

from Urban Forests of Paraná, Southern Brazil

Ricardo Nascimento Drozino,1 Flávio Haragushiku Otomura,2 Janaina Gazarini,3

Mônica Lúcia Gomes,1 and Max Jean de Ornelas Toledo1
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Abstract

Trypanosoma cruzi is a parasitic protozoan that infects a diversity of hosts constituting the cycle of enzootic
transmission in wild environments and causing disease in humans (Chagas disease) and domestic animals. Wild
mammals constitute natural reservoirs of this parasite, which is transmitted by hematophagous kissing bugs of
the family Reduviidae. T. cruzi is genetically subdivided into six discrete typing units (DTUs), T. cruzi (Tc)I to
TcVI. In Brazil, especially in the state of Paraná, TcI and TcII are widely distributed. However, TcII is less
frequently found in wild reservoirs and triatomine, and more frequently found in patients. The goal of this study
was to investigate the natural occurrence of T. cruzi in wild synanthropic mammals captured in urban forest
fragments of the Atlantic Forest of Paraná, southern Brazil. In this way, 12 opossums and 35 bats belonging to
five species were captured in urban forest parks of the city of Maringá, Paraná, an area considered endemic for
Chagas disease. PCR-kinetoplast DNA molecular diagnostic reveals Trypanosoma sp. infection in 12 (100%)
Didelphis albiventris and 10 (40%) Artibeus lituratus. In addition to demonstrating the presence of Trypano-
soma in the two groups of mammals studied, we obtained an isolate of the parasite genotyped as TcII by
amplification of the cytochrome oxidase II gene by PCR, followed by restriction fragment length polymorphism
with AluI, and confirmed by PCR of rDNA 24Sa. This is the first record of the encounter in wild mammals of
Trypanosoma DNA (in A. lituratus) and T. cruzi DTU TcII (in D. albiventris) in the state of Paraná.

Keywords: Trypanosoma cruzi II, Didelphis albiventris, Artibeus lituratus, natural infection, Paraná endemic area

Introduction Atlantic Forest biome, TcII is the most frequently isolated

(95%) DTU in human patients with chronic infection (Gas-

T rypanosoma cruzi (Kinetoplastida, Trypanosomati-

dae), the etiologic agent of Chagas disease or American
trypanosomiasis, is a parasitic protozoan capable of infecting
a diversity of hosts, constituting a wild enzootic transmission
cycle through vectors in humans and domestic animals. The
species is subdivided into discrete typing units (DTUs), from
T. cruzi (Tc)I to TcVI, as well as Tcbat (Marcili et al. 2009,
Zingales et al. 2009, 2012, Lima et al. 2015). Another re-
cently proposed classification system separates T. cruzi into
just three groups, from mtTcI to mtTcIII, based on mito-
chondrial genes (Barnabé et al. 2016). All DTUs are found in
a diverse array of mammalian hosts, representing seven dif-
ferent orders, and in triatomine insects (Reduviidae, Triato-
minae) throughout all states and biomes in Brazil ( Jansen
et al. 2015). However, in the state of Paraná, which is 97.8%
parim et al. 2018), as well as in the triatomine species Pan-
strongylus megistus and Triatoma sordida, but not in wild
mammals (Abolis et al. 2011). In this state, TcI has been
isolated, in these same species of triatomines, both in pure
and mixed infections with TcII, as well as the marsupial
Didelphis albiventris.
Mammals of the orders Chiroptera and Didelphimorphia
are well-known reservoirs of T. cruzi and other trypanoso-
matids. With ample population abundance, accentuated sy-
nanthropism, and high mobility, these animals play an
important role in the distribution of diverse species of path-
ogens (De Oliveira et al. 2010, Hamilton et al. 2012, Brook
and Dobson 2015, da Costa et al. 2015). These mammals’
species demonstrate the highly complex ecological interac-
tions found in the T. cruzi transmission cycle, being necessary

1
Department of Basic Health Sciences, Universidade Estadual de Maringá, Maringá, Brazil.
2
Biological Sciences Sector, Universidade Estadual do Norte do Paraná, Bandeirantes, Brazil.
3
College of Biological and Environmental Sciences, Universidade Federal da Grande Dourados, Dourados, Brazil.

1
 2 DROZINO ET AL.
the conduction of studies that evaluate its dynamics in the
context of the One Health concept, triad that encompasses
humans, domestic animals, wildlife, and the changing eco-
systems in which they live (Thompson 2013, Pinazo and
Gascon 2015).
In this study, a preliminary investigation into the presence
of Trypanosoma in free-range wild mammals was accom-
plished in Atlantic Forest remnants situated in urban areas, an
environment not still explored in the state of Paraná.
Materials and Methods
Study area
Wild mammals were captured in two urban forests frag-
ments, in 2325¢39¢ of latitude south and 5155¢51† of lon-
gitude west, and in 2323¢27¢ of latitude south and 5156¢34†
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of longitude west. Both, remnants of the Atlantic Forest bi-
ome of the city of Maringá, located in the northeast of Paraná,
southern Brazil, and an area considered endemic for Chagas
disease (Fig. 1).
FIG. 1. Map highlighting the two capture points located in the urban area of the Municipality of Maringá, state of Paraná,
Southern Brazil.
Wild mammal capture
The campaigns were held once a month in the following
periods: from September 2014 to December 2015. Bats
were captured with mist nets, conditioned in cloth sacs, and
identified at the species level. Opossums of the species
D. albiventris were captured using Tomahawk Live Traps
baited with fruits and peanut butter. These methods were
approved by the Instituto Chico Mendes de Conservação da
Biodiversidade—Ministério do Meio Ambiente, Brazil (no.
42881) and by the Ethics Committee in Animal Experi-
mentation (CEUA) at Universidade Estadual de Maringá
(UEM) (no. 023/2014).
Blood collection and hemocultures
Opossums and bats were anesthetized with 4.0 mg/kg
chloridrate of xylazine and 20.0 mg/kg body weight chlori-
drate of ketamine for blood collection. Approximately
0.5 mL of whole blood obtained by cardiac puncture in the
bats and from the tail medium vein in the opossums was
 TRYPANOSOMA IN WILD MAMMALS OF URBAN FOREST 3

placed in Novy, McNeal, and Nicolle medium, and covered
with an overlay of liver infusion tryptose (LIT) medium
containing 10% fetal bovine serum. Another 0.2 mL was
stored in microtubes containing 400 lL of 0.2 M ethylene-
diaminetetraacetic acid (EDTA) and 6.0 M guanidine solu-
tion for the extraction and amplification of blood DNA
according to Miyamoto et al. (2006).
Molecular analysis for detection of Trypanosoma DNA
DNA was extracted by the phenol/chloroform method and
precipitated with addition of ethanol and sodium acetate (Ma-
cedo et al. 1992). PCR amplification was performed with oli-
gonucleotides number 121 (5¢-AAATAATGTACGGG(T/G)
GAGATGCATGA-3¢) and number 122 (5¢-GGTT CGATTG
GGGTTGGTGTAATATA-3¢) according to the protocol of
Gomes et al. (1998) modified by Miyamoto et al. (2006). These
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PCR of rDNA 24Sa
PCR amplification of rDNA 24Sa using D71 (5¢-AAGGT
GCGTCGACAGTGTGG-3¢) and D72 (5¢-TTTTCAGAATG
GCCGAACAGT-3¢) primers as previously described by
Souto et al. (1996). Amplified fragments were resolved on
6% polyacrylamide gel, stained with silver, and analyzed
according to Sá et al. (2016).
Results
Detection of the kDNA minicircle in Trypanosoma
PCR-kDNA revealed that all samples of captured
D. albiventris (n = 12, 100%) exhibited a fragment of
330 bp kDNA minicircle from Trypanosoma. Of the 35
examined bat species, PCR-kDNA also detected this
same fragment in 10/25 (40%) Artibeus lituratus caught.

sequences amplify a specific fragment of *330 base pairs (bp)
of kinetoplast DNA (kDNA) from Trypanosoma sp. The am-
plification products were analyzed using 4.5% polyacrylamide
gel electrophoresis, stained with silver, and digitally recorded.
Isolation and genotyping of T. cruzi
Parasites from a unique positive hemoculture, with low
manipulation to avoid clonal selection, were amplified in LIT
medium until the exponential phase. This material was
centrifuged and washed with Krebs-Ringer-Tris/pH 7.2 so-
lution buffer three times. The obtained mass was then re-
suspended in 500 lL of lysis solution (0.5 M EDTA, 5 M
sodium chloride and 1% sodium dodecyl sulfate) supple-
mented with 10 mg/mL of proteinase K (Invitrogen) at 37C
overnight. DNA extraction was performed by the phe-
nol/chloroform method after Sá et al. (2016).
PCR/restriction fragment length polymorphism
cytochrome oxidase II
Differentiation of T. cruzi isolates (TcI to TcVI) was done
by amplification of the cytochrome oxidase II (COII) gene by
PCR, followed by restriction fragment length polymorphism
(RFLP) with AluI (de Freitas et al. 2006, Abolis et al. 2011).
The primers used were the Tcmit-10 (5¢-CCATATATTG
TTGCATTATT-3¢) and Tcmit-21 (5¢-TTGTAATAGGAGT
CATGTTT-3¢) primers with Taq DNA polymerase (Plati-
num, Invitrogen) and the restriction enzyme AluI (New
England BioLabs) in NEB 4 buffer. The PCR amplicons were
resolved on 6% polyacrylamide gel and products sizes ana-
lyzed as described by Sá et al. (2016).
In other species of captured bats, Sturnira lilium (n = 4),
Carollia perspicillata (n = 3), Pygoderma bilabiatum
(n = 2), and Platyrrhinus lineatus (n = 1), this fragment
was not detected (Table 1). This high rate of positivity in
PCR was not observed in hemocultures, and in only 1/12
(8.3%) opossums was positive in this technique, allowing
to obtain a T. cruzi isolate (Table 1).
Genotyping of DTU of T. cruzi isolate
The isolate was genotyped by analysis of mitochondrial
COII gene polymorphisms. The PCR/RFLP-COII showed
two bands, the first at *80 bp referring to the species T. cruzi
and the second at 250 bp, which consistently classifies the
isolate as TcII (Fig. 2A).
The result of PCR amplification of the rDNA 24Sa showed
a band of *125 bp compatible with the TcII and TcVI ge-
notypes. Although this marker is not able to distinguish these
two genotypes, it was used to confirm the DTU of the isolate
(Zingales et al. 2009, 2012). The profiles generated by the
genetic analyzes of COII and rDNA 24Sa together, allowed
to classify the T. cruzi isolate obtained as TcII (Fig. 2B).
Discussion
Studies of the dynamic population of T. cruzi in Paraná,
located in the Southern Region of Brazil, an endemic area for
Chagas disease (Gasparim et al. 2018), reveal TcII to be the
principal DTU isolated from chronic phase patients residents
in this state (Zalloum et al. 2005, Abolis et al. 2011). In con-
trast, a wild host of this DTU has yet to be registered in the
state. TcII was surprisingly found in this central urban forest
(Atlantic Forest biome) of the city of Maringá in the northwest

Table 1. Wild Mammals Captured and Examined for the Presence of Trypanosoma cruzi
(By Detection of Kinetoplast DNA PCR and Hemoculture) in Two Atlantic Forest Urban Parks
of Maringá City, Paraná, Southern Brazil
Species No. of specimens Prevalence (%) Hemoculture (%)
Didelphis albiventris 12 100.0 8.3
Artibeus lituratus 25 40.0 0.0
Sturnira lilium 4 0.0 0.0
Carollia perspicillata 3 0.0 0.0
Pygoderma bilabiatum 2 0.0 0.0
Platyrrhinus lineatus 1 0.0 0.0
Total 47 46.8 2.1
 4 DROZINO ET AL.
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FIG. 2. Amplification and restriction fragment profiles of PCR/RFLP-COII in silver-stained 6% polyacrylamide gel used
for genotyping Trypanosoma cruzi isolate (MDID/BR/2015/DALB01) obtained from Didelphis albiventris (A). Schematic
figure to discriminating and typing T. cruzi DTUs based in PCR RFLP-COII (de Sá et al. 2013) and rDNA 24Sa (Souto et al.
1996), and adapted to Sá et al. (2016) (B). *TcV and TcVI discriminating to second step (rDNA 24Sa); **does not amplify
T. rangeli DNA. COII, cytochrome oxidase II; DTUs, discrete typing units; RFLP, restriction fragment length polymor-
phism; Tc, Trypanosoma rangeli.

of the state, in D. albiventris, a wild reservoir generally con-
sidered uncommon for harboring this DTU.
All previous attempts to associate T. cruzi populations with
specific mammalian host species have led to controversial
results ( Jansen et al. 2015). Even without an association
between DTUs and mammalian reservoirs, a majority of
isolates from D. albiventris are genotyped as TcI, an en-
counter with TcII being a rarity. When encountered, TcII is
generally associated with mixed infections of TcI, TcV, or
TcVI (Abolis et al. 2011, Jansen et al. 2015, 2018). However,
the ability of Didelphis spp. to rapidly control TcII infections
and reduce parasitemia to undetectable levels (Legey et al.
2003, Jansen et al. 2015) is a likely variable affecting isola-
tion efficiency of TcII from these animals.
 TRYPANOSOMA IN WILD MAMMALS OF URBAN FOREST
D. albiventris is characterized as a highly synanthropic
species, adaptable, opportunistic, and omnivorous, as well as
potential disseminators of disease and one of the main res-
ervoirs of T. cruzi and potentially other Trypanosoma spp.
(De Oliveira et al. 2010, Jansen et al. 2015, Dario et al. 2017),
which justifies our single encounter of TcII. Jansen et al.
(2015) reported the finding of TcII infecting wild animals in
all Brazilian biomes, including the Caatinga in the Northeast
Region of Brazil, where that species of marsupial has been
found harboring TcII, in both pure and mixed infections with
TcV and TcVI. However, its discovery was never recorded in
Paraná, a state that has 97.8% of its area inserted in the
Atlantic Forest biome (Muylaert et al. 2018). This is the first
registration of T. cruzi genotyped as TcII isolated from
D. albiventris in the state of Paraná.
The high positivity of PCR-kDNA for Trypanosoma spp.
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in D. albiventris and the nonencounter of triatomines in the
area of study could be related to the complexity of parasitic
cycles in the wild environment. Nouvellet et al. (2013) states
that the probability of transmission after contact with an in-
fected triatomine is 6.0 · 104, confirming that transmission is
still more difficult in free-range wild mammals were thick,
dense skin impedes contact with the metacyclic forms of the
parasite ( Jansen et al. 2015). The same holds true for infected
A. lituratus specimens, in addition to the large diversity of
Trypanosoma spp. with which they are subject to becoming
infected (Dario et al. 2017, Jansen et al. 2018, Lourenço et al.
2018).
Although no triatomine was found in the study area, the
TcII encounter with the high positivity of D. albiventris for
Trypanosoma sp. suggest an active wild cycle. Opossums and
bats are reservoirs of T. cruzi that are frequently found in the
peridomicile (Abolis et al. 2011, Jansen et al. 2015). There-
fore, surveillance work is required that encompasses synan-
thropic animals in urban and periurban forest areas to avoid
reintroduction or a spillover of T. cruzi to the domestic cycle
in controlled areas for Chagas disease (Pinazo and Gascon
2015, Gasparim et al. 2018). The encounter of TcII in
D. albiventris, the main isolate obtained in chronic patients in
Paraná, suggests that surveillance actions in these areas with
a focus on synanthropic wild animals should be considered.
PCR-kDNA analysis of T. cruzi is used in many experi-
mental studies, both for proving the infection and for moni-
toring the cure of treated animals due to its high sensitivity
and specificity to T. cruzi (Teston et al. 2013, Margioto et al.
2016, Zanusso Junior et al. 2018). This is why it has also been
used in the laboratory diagnosis of American trypanosomi-
asis in chronic human patients (Ramı́rez et al. 2015). Despite
the high sensitivity, its use is limited to the genus Trypano-
soma, when applied to the parasite’s detection in wild ani-
mals. This is because wild animals are exposed to a wide
range of Trypanosoma spp., some of which have not yet been
cataloged or even recently discovered (Lopes et al. 2018),
requiring the use of several molecular markers to identify
these species unequivocally. However, the use of more than
one molecular marker for blood samples obtained from wild
animals becomes infeasible, since the variety of Trypanoso-
ma spp. that can infect didelphids and chiroptera is immense
(Dos Santos et al. 2018, Jansen et al. 2018, Lourenço et al.
2018).
In bats, the presence of Trypanosoma infection detected
through PCR-kDNA accompanied by the difficulty in ob-
5
taining parasite isolates could be directly related to these
mammals’ capacity to incubate pathogens (Brook and
Dobson 2015), or still to the presence of Trypanosoma spp.
infection not grown in axenic medium (Dario et al. 2017,
Jansen et al. 2018). In the same way, D. albiventris has
already been found to be opportunistically predating on
A. lituratus (Gazarini et al. 2008), which may be the tip of the
iceberg for a semiopen cycle or spillover between niches in
urban and periurban areas. Whereas bats weakened by con-
comitant infections, together with poor dietary supply, may
be the source of infection for this didelphid. The encounter of
A. lituratus infected by Trypanosoma sp., a species that
presents high population densities and high synanthropism,
reinforces the importance of studies that focus on synan-
thropic species in the wild cycle of T. cruzi. This register
contrasts with the finding of a migratory bat (Tadarida bra-
siliensis) found infected with TcV in the southern United
States (Nichols et al. 2019). The authors report that even
with a low prevalence, T. brasiliensis may contribute to fu-
ture enzootic expansion of T. cruzi, playing a unique role in
the epidemiology of T. cruzi through its annual migrations
(Nichols et al. 2019).
PCR-RFLP that targets the COII gene restricted by the
enzyme AluI is able to accurately separate the T. cruzi ge-
notypes into five groups: TcI, TcII, TcIII, TcIV, and
TcV/TcVI (de Sá et al. 2013, Sá et al. 2016). In its turn, the
TcV/TcVI pattern can be differentiated using a second ge-
netic marker based on the 24Sa gene sequence of Trypano-
soma ribosomal DNA (de Sá et al. 2013). These markers
together still allow identification of T. rangeli (a very com-
mon species and frequently isolated from the wild environ-
ment), and the separation of its major genotypes: KP1+ and
KP1- (Sá et al. 2016). This pair of techniques, therefore,
allows to classify stocks of T. cruzi within the six genetic
lineages previously proposed with a high resolution, hence its
use being of great value in population studies with obtaining
isolates. This pair of techniques used allowed the isolate
obtained from D. albiventris to be genotyped as DTU TcII
(Fig. 2).
In previous studies, T. cruzi isolates obtained from humans
living in Paraná, all in the chronic phase of infection, were
genotyped as TcI, TcII, and TcIII, with TcII being the most
prevalent DTU (Abolis et al. 2011). In contrast, TcII had
previously been found in triatomines in genetically pure or
mixed infections with TcI, whereas the isolates obtained from
marsupial D. albiventris presented only TcI profiles (Zalloum
et al. 2005, Abolis et al. 2011). These data allow the first
record of TcII isolate from a wild D. albiventris from an
urban forest fragment of the state of Paraná.
Conclusion
PCR-kDNA, despite detecting the presence of kDNA re-
stricted to the Trypanosoma genus, is shown as an alternative
for the evaluation of groups of animals that act as reser-
voirs in areas with potential of transmission. Because it is a
low-cost technique and requires a small amount of blood,
PCR-kDNA has proved to be of great value in the monitoring
of infection, allowing more efficient isolation strategies to be
used in places where active cycles of Trypanosoma spp. are
detected. This strategy may potentiate the number of isolates
obtained in forest areas yet to be explored as to the genetic
 6 DROZINO ET AL.
diversity of the parasites. The results show a relation of the
synanthropic mammals sampled with persistent infections by
one T. cruzi DTU (TcII), or even by Trypanosoma spp.,
which may be circulating in the wild environment of the
region, but are more difficult to detect or isolate by more
conventional techniques, such as hemoculture. Infection by
Trypanosoma spp. was recorded in A. lituratus that inhab-
its and forages urban forest areas and their surrounds in the
city of Maringá. The TcII genotype was also observed in
D. albiventris, an unusual finding that had not yet been re-
corded in a wild mammal in the state of Paraná.
Acknowledgments
The authors thank Prefeitura Municipal de Maringá and
Secretaria de Meio Ambiente e Bem Estar Animal (SEMA)
for the support.
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Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Max Jean de Ornelas Toledo
Department of Basic Health Sciences
Universidade Estadual de Maringá
Avenida Colombo 5790, Bloco I-90, Sala 11
Maringá, Paraná CEP 87020-900
Brazil
E-mail: mjotoledo@uem.br