2.6 Ethics Statement
The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to and the
appropriate ethical review committee approval has been received. Animals
were captured and biological samples were collected under licenses
numbers 27587-8 and 53798-7 from the Brazilian Federal Environmental
Protection Agency (ICMBIO), which regulates and protects wildlife in the
country. The study was approved by the Animal Care and Use Committee of
Tropical Medicine Institute - CEUA - 2013/182.
Results
In the Pantanal, forty-three armadillos of four species were captured
(P. maximus , E. sexcinctus , C. unicinctus andD. novemcinctus ), while, in the Cerrado, seven individuals of two
species were sampled (E. sexcinctus and D. novemcinctus ).
Individual age estimation, sex and average body mass for the species are
presented in Table 1. None of the individuals from the Pantanal showed
clinical signs of infection and were all considered to be in good
health. Ten road-killed armadillos of two species (E. sexcinctusand D. novemcinctus ) were recorded in the Cerrado and had
biological samples collected. However, due to the advanced level of
autolysis, only samples derived from seven of these were considered
suitable for molecular analysis: six E. sexcinctus and oneD. novemcinctus (Table 1). Blood samples were not obtained from
those animals, and serological and blood analysis were not performed
(T. gondii and Trypanosoma sp ).
Hemocultures performed for Trypanosoma sp . detection revealed
that two out of 43 armadillos (4.65%) from the Pantanal were positive.
These cultures were from two D. novemcinctus , one captured on
July 2013 (end of wet season), and the other captured on January 2014
(beginning of wet season). Both isolates were diagnosed asTrypanosoma cruzi and characterized as zymodeme 3 (Z3) in the
multiplex PCR. Further characterization using PCR-RFLP of H3/AluI
genotyped the isolates as belonging to DTU TcIII. The same isolates were
also submitted to characterization through the sequence analysis of the
PCR products obtained in the nested 18S PCR. One of them was confirmed
as Trypanosoma cruzi DTU TcIII/TcV (100% of coverage and
identity), because similarities in the sequences do not allow the
differentiation between these two T. cruzi DTUs using this
molecular target. The other isolate was characterized as T.
rangeli, subpopulation A (100% of coverage and 99.5% of identity),
demonstrating a probably mixed infection in this individual. Both
isolates are cryopreserved in COLTRYP and the sequenced deposited in the
GenBank, respectively Coltryp 566 (GenBank number MT253589 and Coltryp
542 (GenBank number MT253688).
Presence of anti-T. gondii antibodies were detected in 13
(30.2%) of the 43 individuals (Table 2), with presented titers ranging
from 1:25 to 1:100. Detection of T. gondii antibodies presented
no variation among species (p = 0.59), between sexes (p = 1) or age
classes (p = 1, CI = 0.06 – 5.02, odds ratio (OR) = 0.73). Even within
each species there were no differences between sexes (Pm – p =
1, Es – p = 1) or age class (Pm – p = 0.6, CI = 0.1 –
36.37, OR = 1.91; Es – p = 0.51, CI = 0 – 6.04, OR = 0).
All ear biopsy samples of the 43 individuals from the Pantanal and the
seven from Cerrado tested negative for both Leishmania sp. andM. leprae . In contrast, all lungs, spleen, and liver samples from
the Cerrado tested positive for P. brasiliensis (Table 2).
Discussion
Many wild mammal species can share armadillo burrows looking for food,
thermal refuges, to escape from predators or to use the sand in front of
burrows as a latrine or a rest place, as described for giant armadillos
by Desbiez and Kluyber (2013). It has been hypothesized that the use of
armadillos’ burrows by several vertebrate species and the constant mild
temperatures encountered inside these excavations, may favor survival
and proliferation of parasites and vectors (Desbiez and Kluyber, 2013).
Furthermore, armadillo burrows may act as shelters for living insects,
such as kissing bugs and the sandflies, which act as vectors of
protozoans such as T. cruzi and Leishmania sp.
respectively; besides some parasites that can resist long periods in the
environment, such as M. leprae, P. brasiliensis and T.
gondii oocysts.
Armadillos are considered important species for public health, given
that its meat is widely consumed in several regions, from the south of
the United States throughout South America. (Cardona-Castro et al.,
2009; Rodrigues et al., 2019). In Latin America, armadillos are
hunted for cultural practices such as medicinal use, manufacturing of
musical instruments but especially, for food (Capellão et al., 2008;
Rodrigues et al., 2019).
Although the hunting of wildlife is not allowed in Brazil (Environmental
Crime Law - 9.605/98), armadillos are still considered one of the
favorite bush meats and a main protein source, in rural areas (Deps et
al., 2008). Different leprosy studies in the United States and Brazil,
suggest that meat intake and direct contact with contaminated armadillos
may be considered an important source of infection (Kerr et al. 2015,
Truman et al., 2011). In general, studies highlight that contact with
contaminated environments and poor sanitary conditions are some of the
most important factors that favor zoonotic parasites transmission
(Sharma et al., 2015; Ker-Pontes et al., 2006).
Trypanosoma cruzi is a multihost wild parasite species
transmitted by contaminated feces of triatomines bugs and between
mammals through predation (Jansen et al., 2015). Curiously, it was in an
armadillo (D. novemcinctus ) that Carlos Chagas first observedT. cruzi infection in the wild (Chagas, 1912). After that, Yaeger
et al. (1988) and Forrester et al. (1992) described significant
prevalence of T. cruzi for D. novemcinctus in the United
States. In Brazil, this armadillo species is also considered a potential
reservoir of T. cruzi and several studies reported infected
individuals (Barret and Naiff, 1990; Herrera et al., 2004; Roque et al.,
2008). Few studies have been conducted focusing on T. cruzi in
other wild armadillo species, but there have been records of individuals
of E. sexcinctus, C. villosus, C. vellerosus, T. matacusand C. unicinctus exposed to T. cruzi (Yeo et al.,2005; Noireau et al., 2009; Telleria and Tibayrenc, 2010; Orozco
et al., 2013; Cardona-Castro et al., 2009). The high number of
studies focusing only on D. novemcinctus might be explained by
its wide geographic distribution and the absence of health surveillance
studies for other armadillo species.
According to Herrera et al. (2004), the burrowing habits of certain
species, such as armadillos, in combination with the vector’s ecology
(i.e. living inside burrows), may influence the transmission of T.
cruzi . In fact, underground burrows, as the ones used by armadillos,
are usually associated with triatomines from the Panstrongylusgenus (Jansen et al., 2017). Moreover, armadillo’s habits of bringing
grass and leaves to the inside of their burrows and to place plant
debris on the burrow’s entrance to cover it, (Loughry and McDonough,
2013), creates an ideal habitat for triatomines and; consequently, sets
place for an opportunistic behavior. This vector’s habitat use behavior
was also described by de Lima et al. (2015), who recorded the presence
of triatomine bugs in arboreal nests of Coatis (Nasua nasua ) in
the Pantanal wetlands of Brazil. Finally, D’Alessandro et al. (1984)
indicated the insectivorous diet of armadillos as a possible cause of
accidental ingestion of triatomines, and one of their main sources of
infection by T. cruzi.
After the first biochemical characterizations of T. cruziisolates, proposed by Miles et al. (1977), isolates derived from
armadillos were usually associated with zymodeme Z3, which lead Yeo et
al. (2005) to propose an association between armadillos and the T.
cruzi subpopulations currently recognized as DTUs TcII to TcVI
(Zingales et al., 2009). Nowadays, although the frequency of infections
by T. cruzi DTUs TcIII and TcIV (formerly described as Z3
zymodeme) are higher than other T. cruzi DTUs, armadillos have
also been found infected with DTUs TcI and TcII, demonstrating their
importance to maintain distinct transmission cycles in nature (Jansen et
al., 2017). It is likely that the scarcity of long-term studies, and
health monitoring programs of different armadillo species, might be
leading the field to an underestimation of the role of armadillos as
hosts for T. cruzi .
In the present study, four different species of armadillos were studied,
but T. cruzi could only be isolated from blood cultures of two of
them, D. novemcinctus and P. maximus . Both isolates were
characterized as T. cruzi TcIII based on PCR/RFLP (H3/AluI)
corroborating previous studies that demonstrate that this DTU is the
most common in armadillos (Barros et al., 2017; Jansen et al., 2018).
The characterization of one of these isolates was also confirmed by the
sequence analysis of the 18S rDNA PCR products. However, the other
isolate was characterized as T. rangeli, subpopulation A. Because
the Miniexon assay was positive only for T. cruzi Z3 (further
confirmed by the H3 PCR) and did not detect T. rangeli, although
it is able to Fernandes et al., 2001, two distinct results were
considered as an indicative of mixed infection, a trait probably more
common in nature than it is usually detected (Jansen et al., 2020).T. rangeli is also a multihost wild mammal parasite transmitted
by kissing bugs, but unlike T. cruzi, the transmission takes
place by the inoculative pathway and the only proven vectors are the
triatomine bugs of the genus Rhodnius . The subpopulation A is
commonly described in bats from distinct areas in South America, but has
also been described in primates and carnivores (Maia da Silva et al.,
2007; 2009; Dario et al., 2017). To our knowledge, this is the first
description of T. rangeli, subpopulation A, in armadillos.
This is the first report of armadillo exposure to T. gondii in
the Pantanal of Mato Grosso do Sul, and the first record of this
infection for the species C. unicinctus. Previous studies have
described the exposure to T. gondii for D. novemcinctus, E.
sexcinctus, and P. maximus (Sogorb et al., 1977; da Silva et
al., 2008a; da Silva et al., 2008b; Deem et al., 2009). In a study
performed by da Silva et al., (2008) and Vitaliano et al., (2014),T. gondii could be isolated from E. sexcinctus andD. novemcinctus. Consumption of carrion by omnivore armadillo
species, such as E. sexcinctus (Medri, 2008), can be an important
source of infection.
The habit of armadillos to dig burrows, live in organic and inorganic
matter and forage for food on the ground, in conjunction with the fact
that T. gondii oocysts can resist long periods in the environment
(Dubey, 2010), might favor the contact of armadillos with soil
contaminated by T. gondii sporulated oocysts, eliminated by
felids that share the same environment. Wild felids are the only
definitive hosts of T. gondii in the wild (Wang, 2002). The
ocelot (Leopardus pardalis ) is one of the main species of feline
that frequently uses giant armadillo burrows to rest or as a thermal
refuge. Due to its size, Puma concolor is not able to explore
inside burrows, but, like L. pardalis , have been using the sand
in front of giant armadillo burrows, to rest or as a latrine
(Desbiez and Kluyber, 2013).
Furthermore, the infectiousness of T. gondii from P.
maximus has been demonstrated through the isolation of T. gondiiin a mouse that had been inoculated with lungs and brain fragments from
an infected P. maximus (Sogorb et al., 1977). Hence, armadillos
may act as intermediate hosts and should be considered relevant species
for the maintenance of T . gondii cycle, which is directly
connected to their role in ecological food-chains (Kin et al.,
2014).
At least seven Leishmania species have been described infecting
xenarthras, especially sloths and tamanduas (Roque and Jansen, 2014).
However, few descriptions are available concerning Leishmaniainfection in armadillos and, infections had been detected only inD. novemcinctus (reviewed by Roque and Jansen, 2014). Two
distinct species of Leishmania , L. naiffi and L.
guyanensis were described in D. novemcinctus. Importantly,D. novemcinctus is the only non-human host in which L.
naiffi has been isolated (Naiff et al., 1991, Roque and Jansen, 2014).
Serology can indicate the exposure of a given mammal host toLeishmania infection, but the lack of available commercial
reagents for armadillo impairs this kind of investigation (Jansen et
al., 2015). In this sense, cultures or molecular diagnosis on punctures
or fragments of hematopoietic tissues are the frequently chosen methods
to identify Leishmania infections in armadillos (Roque and
Jansen, 2014).
Molecular tests like polymerase chain reaction (PCR) targeting the kDNA
of these parasites may provide sensitivity and specificity of almost
100%. Richini-Pereira et al. (2014) detected Leishmania sp. in
two armadillo’s species studied (1 D. novemcinctus and 1 E.
sexcinctus ) in Brazil. However, in the present study, all the samples
analyzed using PCR analysis were negative for Leishmania sp. As
described by Roque and Jansen, (2014), few Leishmania studies,
and lack of species- specific standard tests, are still a challenge for
wild mammal diagnosis.
In 1971, armadillos were experimentally infected with M. lepraefor the first time by Kirchheimer et al. (1971). Later, Walsh et al.
(1975) reported that wild armadillos could be naturally infected,
demonstrating that M. leprae infection was not limited to man as
previously thought. In the USA, Truman et al. (2011) described the
potential role of armadillos in the zoonotic transmission of M .leprae to humans. Mycobacterium leprae infects two kinds
of human body cells: skin macrophages and Schwann cells (Kaplan, 1986).
The ideal body temperature for M. leprae is about 30°C and that
is the reason the bacteria and its clinical signs are encountered in
colder parts of the body such as the extremities. This characteristic
explains, in part, the infection in armadillos, which present average
body temperature between 30 and 35°C (Shepard, 1965). Kerr et al.
(2015), proposed that ecological characteristics of armadillos, such as
its burrowing habits, and M. leprae ’s viability in the soil and
water, could result in a possible infection route (inhalation) among
armadillos. Transmission might occur through aerosol, direct contact,
antagonist encounters or while they are copulating (Truman et al.,
2011). Mohanty et al. (2016) also described that some species build
their burrows close to rivers or in humid soil, possibility creating a
new route of infection through the contact or exposure to different
parasites, including M. leprae .
Studies performed by Truman et al. (2011) and Loughry et al. (2009) in
the USA recorded wild D. novemcinctus infected with M.
leprae , mainly in Texas and Louisiana. Moreover, according to Sharma et
al. (2015) when M. leprae was investigated in 645 armadillos from
8 locations in the southern USA, 106 (16.4%) individuals were positive
using serologic (anti-PGL-I antibody) and/or PCR (RLEP and 18 kDa)
techniques. This indicates a continued spread of M. lepraeinfection in these animals as their range has expanded East and North
throughout the USA.
In Brazil, recent studies have shown D. novemcinctus and E.
sexcinctus individuals naturally infected by M. leprae in an
hyperendemic area in the Ceará state, northeastern Brazil (Frota et al.,
2012; Kerr et al., 2015). In the north, studies performed in Pará state
by da Silva et al. (2018) found 62% (10/16) of D. novemcinctuspositive for M. leprae. In the southeastern Brazil, Deps et al.
(2002), studied 14 D. novemcinctus in a rural area of Espírito
Santo state and none of the animals examined presented macroscopic
lesions suggesting infection, but five of them were PCR positive for