Molecular identification of Trypanosoma cruzi in domestic animals in municipalities of the State of Rio Grande do Norte, Brazil

Abstract

Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.

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Parasitology Research
https://doi.org/10.1007/s00436-022-07719-w
RESEARCH
Molecular identification ofTrypanosoma cruzi indomestic animals
inmunicipalities oftheState ofRio Grande doNorte, Brazil
VicenteToscanodeAraújo‑Neto1· AndressaNoronhaBarbosa‑Silva1· NathanRaviMedeirosHonorato2·
LetíciaMikardyaLimaSales3· RenatadeCassiaPires1· CarlosRamondoNascimentoBrito4·
PauloMarcosdaMattaGuedes4· LúciaMariadaCunhaGalvão1,2· AntoniaClaudiaJácomedaCâmara1,4
Received: 3 August 2022 / Accepted: 5 November 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been
little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by
T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires),
22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state
of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the
rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in
phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60),
22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions
throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemio-
logical transmission scenario in an area where T. brasiliensis is the main vector.
Keywords Trypanosoma cruzi· Natural infection· Phylogenetic analysis· American trypanosomiasis
Introduction
Chagas disease (CD) or American trypanosomiasis, caused
by Trypanosoma cruzi Chagas, 1909, affects about 6 to 7
million people in the world and another 75 million lives in
areas at risk for transmission (WHO 2022). CD presents
high morbidity and mortality in endemic countries, with
approximately 12,000 deaths per year, and is considered
a public health problem in Latin America (PAHO 2022).
This continent presents a high density of insects of the Tri-
atominae subfamily (Hemiptera, Reduviidae), which are
Section Editor: Vyacheslav Yurchenko
* Antonia Claudia Jácome da Câmara
acjcamara@ufrnet.br
Vicente Toscano de Araújo-Neto
toscanorn1@gmail.com
Andressa Noronha Barbosa-Silva
noronha.andressa@gmail.com
Nathan Ravi Medeiros Honorato
nathanhonorato@hotmail.com
Letícia Mikardya Lima Sales
lmlsaless@gmail.com
Renata de Cassia Pires
renatacapires@gmail.com
Carlos Ramon do Nascimento Brito
crnbrito@yahoo.com.br
Paulo Marcos da Matta Guedes
guedespmm@gmail.com
Lúcia Maria da Cunha Galvão
luciabhster@gmail.com
1 Graduate Program inPharmaceutical Sciences, Federal
University ofRio Grande Do Norte, Natal59012-570, Brazil
2 Graduate Program inParasitology, Federal University
ofMinas Gerais, Belo Horizonte, BeloHorizonte31270-901,
Brazil
3 Undergraduate Course inPharmacy, Federal University
ofRio Grande Do Norte, Natal59012-570, Brazil
4 Graduate Program inParasite Biology, Federal University
ofRio Grande Do Norte, Natal59064-741, Brazil

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vectors of T. cruzi (Chagas 1909; Lent and Wygodzinsky
1979; Lima-Neiva etal. 2021).
Trypanosoma cruzi is a digenetic protozoan which
infects several animal orders, for example, Didelphi-
morphia, Carnivora, Rodentia, Chiroptera, and Primate,
including human beings, demonstrating the generalist and
successful character of this parasite (Jansen etal. 2018).
Transmission to vertebrate animals can happen in a classi-
cal way by contamination in the vector’s feces, or orally by
predation of infected reservoirs by susceptible ones. The
interaction dynamic between the host and parasite is com-
plex, and every animal has a unique role in the transmis-
sion cycle in different areas (Jansen and Roque 2010). The
infection prevalence of an animal, host capacity to infect
vectors, and the host–triatomine contact are factors which
determine the importance of a species as a pathogen res-
ervoir transmitted by a vector (Cohen and Gurtler 2001).
From an ecological point of view, CD is typical in rural
areas where the triatomine interacts with domestic and
sylvatic animals, transmitting the parasite (Oliveira-Lima
etal. 2000). This interaction is quite evident in peridomi-
cile environments, as they are important epidemiological
environments which act as a link between anthropized and
wild zones. Peridomicile transmission involves domestic
reservoirs and sylvatic triatomines attracted to the dwell-
ing by light searching for feeding sources, or well-adapted
vectors to human habitations which can establish their col-
onies in this environment (Dias etal. 2000). Synanthropic
vertebrates, like opossums, bats, and rodents also play an
important role in the transmission of T. cruzi to humans,
connecting both environments in a single epidemiological
cycle (Fernandes etal. 1989; Yefi-Quinteros etal. 2018;
Drozino etal. 2019).
Infestation and colonization of Triatoma brasiliensis
Neiva, 1911, the main vector in the state of Rio Grande do
Norte (RN), Northeast Region of Brazil, has been reported
in recent studies (Barbosa-Silva etal. 2019; Araújo-Neto
etal. 2019; Honorato etal. 2021; Lima-Neiva etal. 2021).
This species is frequently found in ecotopes like corrals,
brick/tile piles, and chicken coops (Barbosa-Silva etal.
2016; Lilioso etal. 2020), associated to domestic animals
such as sheep (Ovis aires), goats (Capra hircus), and
chickens (Gallus gallus), and synanthropic animals like
Galea spixii (Barbosa-Silva 2017; Bezerra etal. 2018;
Lima-Neiva etal. 2021). Moreover, high infection rates
in dogs (Canis familiaris) have also been demonstrated in
the same area (Araújo-Neto etal. 2019). However, the role
of domestic animals in establishing and maintaining the
transmission cycle of T. cruzi has not yet been completely
elucidated. In this context, we evaluated the natural infec-
tion occurrence of T. cruzi by molecular analysis in sheep,
goats, and horses in municipalities of RN State, Brazil.
Materials andmethods
Study area andpopulation
The state of RN is located in the Northeast Region of
Brazil and has an area of 52,809.6 km2, divided into 167
municipalities distributed in four mesoregions: West,
Central, Agreste, and East. A semi-arid, arid, or dry sub-
humid climate predominates in the state, ranging from
the Agreste mesoregion to the northern coast (IDEMA
2020). Livestock production mostly comprises small herds
of goats, sheep, and horses of resistant and well-adapted
breeds mainly used as food support (IBGE 2021).
The surveyed areas were selected according to the fol-
lowing criteria: (i) medium or high risk of vectoral trans-
mission of T. cruzi (Barbosa-Silva etal. 2019); (ii) infec-
tion occurrence in human, dogs, or sylvatic animals (Brito
etal. 2012; Martins etal. 2015; Araújo-Neto etal. 2019);
and (iii) occurrence of infected triatomines (Barbosa-Silva
etal. 2016, 2019; Vargas etal. 2018; Liliosoet al. 2020;
Honorato etal. 2021). This study was conducted in 17
rural communities in the following municipalities: João
Câmara in the Agreste mesoregion, Alexandria, Marcelino
Vieira, Upanema, Lajes, Caraúbas and Campo Grande in
the West mesoregion, and Afonso Bezerra in the Central
mesoregion (Table1).
Sampling andDNA extraction
Blood was initially collected from 60 sheep, 22 goats, and
14 horses and submitted to the microhematocrit method to
search for motile parasites (Woo 1970). A blood clot was
obtained from all samples and individually stored in abso-
lute ethanol until the moment of DNA extraction (Rodri-
gues etal. 2019). DNA extraction following the protocol
of Garcia etal (2018).
Nested PCR ofrRNA 18S SSU gene
The 18S SSUrRNA gene was detected as proposed by
Noyes etal. (1999) using two pairs of primers. In the first
step, the TRY927F (5′-GAA ACA AGA AAC ACG GGA
G-3′) and TRY927R (5′-CTA CTG GGC AGC TTGGA-3′)
primers were used, and then, the SSU561F (5′-TGG GAT
AAC AAA GGA GCA -3′) and SSU561R primers (5′-CTG
AGA CTG TAA CCT CAA AGC-3′) were used in the sec-
ond step (Smith etal. 2008). The amplicons between 600
and 1000bp were visualized by electrophoresis in a 6%
polyacrylamide gel stained with silver salts (Santos etal.
1993). In addition, RN28 (Câmara etal. 2013) and 3188

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(Martins etal. 2015) strains of T. cruzi were used as posi-
tive controls. The samples had their final DNA concentra-
tion adjusted from 5 to 20ng/μL.
Purification, sequencing, andphylogenetic analysis
Purification was performed using the Exo SAP-IT™ PCR
Product Clean up (Thermo Fisher Scientific, Waltham, USA)
following the manufacturer’s protocol. Sequencing reactions
were carried out using the BigDye® Terminator v 3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, USA).
The sequencing reactions were performed in an ABI 3500
DNA Analyzer (Thermo Fisher Scientific, USA).
Electropherograms were analyzed, and consensus
sequences were generated using the Mega X software pro-
gram (Kumar etal. 2018). The consensus sequences for
phylogenetic analysis were compared to samples depos-
ited in the NCBI/GenBank database (National Center for
Table 1 Distribution of goats,
sheep, and equine infected by
Trypanosoma cruzi identified
with kDNA and 18S SSU
in rural communities of the
mesoregions and municipalities
in the State of Rio Grande do
Norte
Mesoregions/Municipality Hosts species
Capra hircus Ovis aries Equus caballus
NkDNA 18S NkDNA 18S NkDNA 18S
Agreste
João Câmara 3 3 3 15 11 11 1 1 1
Central
Afonso Bezerra 2 2 2 - - - - - -
West
Marcelino Vieira 6 0 2 15 3 5 - - -
Alexandria 3 0 0 17 2 9 12 1 0
Campo Grande 7 0 6 2 0 2 - - -
Caraúbas - - - 6 0 5 - - -
Lajes 1 0 1 4 1 1 1 0 0
Upanema - - - 1 0 1 - - -
Total (%) 22 5 (22.7) 14 (63.6) 60 17 (28.3) 34 (56.6) 14 2 (35.4) 1 (7.7)
Table 2 Sample, molecular identification, discrete typing unit (DTU), hosts species, GenBank code, and reference of samples used in genetic
sequencing analysis
DTU discrete typing unit by molecular identification of gene 18S SSU; *ingroup; #outgroup
Sample ID Molecular identification DTU Host species GenBank code Reference
bCth 743*T. cruzi TcI Cerdocyon thous MH411618 Rodrigues etal. 2019
bCth 744*T. cruzi TcI Cerdocyon thous MH411619 Rodrigues etal. 2019
CPAPGM 855*T. cruzi TcI Cerdocyon thous MH411626 Rodrigues etal. 2019
APAGM 850*T. cruzi TcI Cerdocyon thous MH411625 Rodrigues etal. 2019
CPAGM 796*T. cruzi TcI Cerdocyon thous MH411623 Rodrigues etal. 2019
CPAGM 848*T. cruzi TcI Cerdocyon thous MH411624 Rodrigues etal. 2019
Colombiana*T. cruzi TcI Homo sapiens AF239980 Kawashita etal., 2001
LBT 6867*T. cruzi TcII Artibeus lituratus MH411627 Rodrigues etal. 2019
YL 881*T. cruzi TcII Marmosopsincanus MH558662 Rodrigues etal. 2019
Y*T. cruzi TcII Homo sapiens AF301912 Kawashita etal., 2001
LBCE 19,688*T. cruzi TcII Didelphis marsupialis MH411637 Rodrigues etal. 2019
LBCE 19,675*T. cruzi TcII Didelphis marsupialis MH411635 Rodrigues etal. 2019
PCE 383*T. cruzi TcII Marmosa sp. MH411630 Rodrigues etal. 2019
EM 897*T. cruzi TcII Artibeus fimbriatus MH411629 Rodrigues etal. 2019
EM 712*T. cruzi TcIII Artibeus lituratus MH558661 Rodrigues etal. 2019
EM 693*T. cruzi TcIII Carollia perspicillata MH411642 Rodrigues etal. 2019
RN02*T. cruzi TcIII Triatoma brasiliensis OP132415 Câmara etal. 2010
SH022 T. cruzi TcII Ovis aries OP132414 Araujo-Neto etal. in
press
PCE 51# T. cascavelli Didelphis albiventris MH411650 Rodrigues etal. 2019

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Biotecnology Information https:// www. ncbi. nlm. nih. gov/
BLAST/) using the BLAST tool, program selection optimize
for highly similar sequences (megablast). Table2 shows the
sequences of ingroups and outgroups. These sequences
were submitted to multiple alignments with the MAFFT v.
7 online program (Katoh and Standley 2013). The genome
alignments were inspected and corrected on Mega X; then,
the most suitable evolutionary model was tested using the
Model Finder plugin on the PhyloSuite v. 1.2.2 (Zhang etal.
2020). Next, phylogenetic analysis was performed using two
maximum likelihood models: (I) MrBayes v. 3.2.6 (Ronquist
etal. 2012) was used for phylogenetic analysis by Bayesian
inference with GTR + F + I model, two parallel runs, and
10,000 bootstrap replicates. About 25% of the initial data
sampled were discarded as burn-in; (II) IQ-TREE (Nguyen
etal. 2015) using the TN + I model for 10,000 standard
bootstraps (Minh etal. 2013), as well as the Shimodaira-
Hasegawa approximate likelihood-ratio test (Guindon etal.
2010). A phylogenetic tree was generated, evaluated, and
interpreted using Figtree v. 1.4.4 and edited in Inkscape.
T. cruzi kDNA detection
PCR for detecting T. cruzi kDNA was performed as pre-
viously described (Gomes etal. 1998), targeting constant
regions of minicircles using primers described by Vallejo
etal (1999). Amplicons of 330bp were visualized in poly-
acrylamide gels as previously described. The SM76 strain
(Martins etal. 2015) of T. cruzi was used as positive control
and non-infected human blood as negative control.
Results
Trypanosoma spp. infection
Motile parasites were not detected in the samples by the
microhematocrit technique. Table1 shows a wide distri-
bution of 18S-positive samples throughout RN State. All
animal species in the municipality of João Câmara were
Fig. 1 a Representative polyacrylamide gel electrophoresis show-
ing the amplification of rRNA 18S SSU gene of Trypanosoma spp
stained by silver from blood clot samples from goats (GOA), sheep
(SH), and controls. Fragments between 600 and 1000bp were con-
sidered positive. M: Molecular size marked. Lines 2–7: Trypanosoma
spp. samples SH056, SH057, SH058, GOA012, GOA013, GOA014.
Line 8, 10 and 11: T. cruzi strain-positive controls RN2, 3188. Line 9,
SH004-negative sample, NC. Line 12, NC: negative control. b Rep-
resentative polyacrylamide gel electrophoresis showing the ampli-
fication of kDNA of T. cruzi from blood clot samples from goats
(GOA) and sheep (SH). Fragment of 330bp was considered positive.
M Molecular size marked. Line 2: T. cruzi strain-positive controls:
SM76. Lines 3–10: T. cruzi-positive samples: GOA003. GOA004,
SH008, SH009, SH010, SH011, SH012, SH013. NC: negative control

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infected, while only sheep and goats were found positive in
the municipalities of Marcelino Vieira, Campo Grande, and
Lajes. On the other hand, only goats were infected in Afonso
Bezerra, while only sheep were infected in Alexandria,
Caraúbas, and Upanema. In total, Trypanosoma spp. DNA
was detected in more than 50% of the sheep clot samples.
rRNA 18S SSU gene amplified fragments between 600
and 1000bp in 48.9% (48/96) of samples collected in all
investigated areas. A representative polyacrylamide gel of
electrophoresis to show the amplicon pattern produced in
this reaction can be seen in Fig.1a.
Trypanosoma cruzi identification
From 48 positive samples for 18S amplification, only
8.3% (4) were submitted to DNA sequencing procedures,
and only one (ovine sample SH022) presented an electro-
pherogram with good quality. This sample was collected
in João Câmara, and its consensus sequence was submit-
ted to BLASTn tool, which matched 99.8% identity with
DTU II Y strain (AF301912) of T. cruzi. The Bayesian
phylogenetic tree grouped SH022 in DTU II clade was also
associated with Y and other strains of this genotype with a
bootstrap value higher than 0.98 (Fig.2). The RN02 sam-
ple, genotyped by Câmara etal. (2010) as TcIII from a T.
brasiliensis specimen captured in RN State, was grouped
with the sequences from GenBank forming a DTU III clade.
Sequences from DTU I were grouped all together in a father
branch. The outgroup, represented by Trypanosoma cas-
cavelli Pessoa and De Biasi, 1972 (MH411650), formed an
external clade (Fig.2).
T. cruzi kDNA detection
PCR detected T. cruzi kDNA in 28.3% (17/60), 22.7%
(5/22), and 15.4% (2/14) of samples of sheep, goats, and
horses, respectively, demonstrating a high infection rate. A
representative polyacrylamide gel of electrophoresis to show
the amplicon pattern of 330bp produced in this reaction
can be seen in Fig.1b. All samples of horses and goats and
73.3% (11/15) of sheep were infected in João Câmara. All
municipalities presented at least one positive animal species,
except for Campo Grande (Table1, Fig.3).
Discussion
The occurrence of natural infection and phylogenetic analy-
sis of T. cruzi was reported in sheep, goats, and horses in
municipalities of medium and high risk of vectorial trans-
mission of this parasite in the state of RN, Brazil. A broad
distribution of infected animals was observed, as confirmed
by T. cruzi kDNA identification and detection of DTU II by
sequencing and phylogeny of rRNA 18S SSU gene.
Fig. 2 Bayesian phylogenetic tree based on gene rRNA 18SSSU
using the sequences of SH022, RN02 isolated from T. brasiliensis
(CAMARA etal., 2010) and the sequence obtained from GenBank.
The numbers in branches correspond to support values, with 1.0
being the highest similarity value. The numbers in nodes are the sup-
port values ordered as Bayesian inference/IQ-TREE. T. cascavelli was
used as the outgroup

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Infection of farm animals by T. cruzi is poorly reported
using molecular or serological techniques. Infection in
sheep was described in Argentina by amplification of sat-
ellite DNA (Sat-DNA) by real-time PCR (Wehrendt etal.
2019). A high percentage of infected goats was detected in
locations with hot and dry climates in the Northeast Region
of Brazil, constituting areas with similar conditions of the
present work (Muñoz-San Martín etal. 2020). In addition,
only horses in Venezuela have been found positive by kDNA
and Sat-DNA (Herrera etal. 2022).
Studies regarding blood surveys have shown high infec-
tion rates in goats and sheep by the indirect hemagglutina-
tion test in Chile (Schenone etal. 1991). Goats in Northeast
Brazil were highly infected in Paraíba State (Fuentes-Cas-
tillo etal. 1988), while sheep and goats in Ceará State dem-
onstrated low reactivity percentages by indirect immunofluo-
rescence (Bezerra etal. 2014).
The role of these animals in spreading or maintaining
T. cruzi seems to be variable in different transmission
cycles. These animals are mostly part of a semi-intensive
livestock production system in the Northeast Region (i.e.,
they are free in the pasture during the day and collected
and put into corrals at night), increasing their chance of
exposure to the vectors. These ecotopes were already
reported sheltering triatomines in the same area sur-
veyed in the present study (Barbosa-Silva etal. 2016)
and in other areas of South America (Cecere etal. 1997;
Chartier and Crocco 2007; Gürtler etal. 2014; Carbajal-
de-la-Fuente etal. 2017). The DNA of these animals was
detected in vectors found in several ecotopes of peridom-
icile areas in Ceará (Valença-Barbosa etal. 2015) and in
Caraúbas (Barbosa-Silva, 2017) and Caicó (Lima-Neiva
etal. 2021), respectively located in the West and Cen-
tral mesoregions of RN State. Moreover, these animals
have also been found as a food source of triatomines
in sylvatic areas of RN (Barbosa-Silva, 2017). From an
anthropocentric point of view, these data suggest that
goats and sheep can act as a link between sylvatic and
peridomiciliar cycles (Ashford 1997), not only in RN
State but also in other endemic areas.
Fig. 3 Distribution of animal samples positive for T. cruzi infection
by kDNA detection in Rio Grande do Norte State. Municipalities sur-
veyed are highlighted in grey and represented by the following num-
bers: 1: João Câmara; 2: Lajes; 3: Afonso Bezerra; 4: Upanema; 5:
Campo Grande; 6: Caraúbas; 7: Alexandria; and 8: Marcelino Vieira

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Herein, we have reported the natural infection of T. cruzi
in equines in Brazil for the first time. Ikenga and Richer-
son (1984) identified positive horses by serology in Texas,
USA. Molecular markers posteriorly identified infection in a
10-year-old quarter horse with neurological disturbances in
the same state, which also presented amastigote-like forms
in its spinal cord (Bryan etal. 2016). Taken together, our
findings and the literature data still cannot provide enough
information to suggest that horses have a role in the cycle of
T. cruzi. Thus, studies about parasite load and susceptibility
to blood meal of triatomines need to be performed.
Literature data in the last decade have also demonstrated
the diversity of T. cruzi populations which circulates through
anthropic and sylvatic cycles in RN State. Until this moment,
DTUs I, II, and III were found in humans and also in vectors
such as T. brasiliensis, Triatoma pseudomaculata Corrêa and
Espínola, 1964, and Panstrongylus lutzi Neiva and Pinto,
1923(Câmara etal. 2010; Martins etal. 2015; Honorato etal.
2021). Identification of DTU II in sheep adds a new variable
to understanding the interaction of animals and vectors in
the peridomicile areas in the surveyed region.
Amplification of 18S to identify Trypanosoma spp.
revealed some positive samples with T. cruzi. However, oth-
ers which were not identified can suggest the occurrence of
other trypanosomes like Trypanosoma vivax Ziemann, 1905,
and Trypanosoma evansi Evans, 1880. Both species have
been reported in South America (Guerra etal. 2008; Colpo
etal. 2005). The former is a protozoan with great economic
and veterinary importance which can cause abortion and
was identified in bovines in RN State (Batista etal. 2018).
The latter has been described in the Central-West Region of
Brazil (Costa etal. 2019), which is endemic for this infec-
tion, and in the states of Rio Grande do Sul (Rodrigues etal.
2005), Amazonas (Filgueiras etal. 2019), and Bahia (Costa
etal. 2019), but with no reports in RN State.
Conclusion
Identifying the high distribution of natural infection by T.
cruzi in farm animals contributes to amplify the epidemio-
logical transmission scenario in the areas where we sug-
gest that sheep and goats can be a link between sylvatic and
anthropic cycles. It is necessary to perform studies on horses
to deeper investigate the aspects about infection and its rel-
evance as a reservoir.
Acknowledgements The authors are grateful to the Secretariat of State
for Public Health, represented by the health authorities and health
agents of the Municipal Health Secretariats of Alexandria, Caraúbas,
Campo Grande, Marcelino Vieira, Upanema, Lajes, and João Câmara,
for their indispensable support in field activities and for the provision
of data during the development of this study.
Author contribution Vicente Toscano de Araújo-Neto, Andressa
Noronha Barbosa-Silva, Nathan Ravi de Medeiros Honorato, Antonia
Claudia Jácome da Câmara, and Renata de Cassia Pires designed the
experiments and wrote the original draft. Carlos Ramon do Nascimento
Brito and Letícia Micardya Lima Sales contributed to the study design.
Antonia Claudia Jácome da Câmara, Paulo Marcos da Matta Guedes,
and Lúcia Maria da Cunha Galvão conceptualized and reviewed the
manuscript. All authors have read the paper and approved the final
version.
Funding This study was supported by the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) (MCTI/CNPq/
Edital Universal 2016 grant no. 423966/2016–2). LMCG is a CNPq
fellow, and NRMH, ANBS, RCP, and LMLS thank the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for grant-
ing scholarships.
Data availability The sequences were submitted to GenBank under the
following access numbers: OP132414–SH022 and OP132415–RN02.
The isolate RN02 in all blood samples are in the LABIOPAR collection
at Department of Clinical and Toxicological Analyses/UFRN.
Declarations
Ethical approval This study was approved by Ethics Committee on the
Use of Animals of Federal University of Rio Grande do Norte (CEUA-
UFRN), protocol no. 134.062/2018, Natal, RN.
Consent for publication All authors gave their consent for publication.
Conflict of interest The authors declare there are no conflicts of inter-
est.
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