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