Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil.

Xenarthra mammals can be found from southern North America to southern South America, including all Brazilian biomes. Although it has been shown that Xenarthra mammals can play a role as reservoirs for several zoonotic agents, few studies investigate the diversity of piroplasmids (Apicomplexa: Piroplasmida) in this group of mammals. Taking into account that piroplasmids can cause disease in animals and humans, understanding the prevalence and diversity of piroplasmids in Xenarthra mammals would contribute to conservation efforts for this group of animals as well as to infer risk areas for transmission of emergent zoonosis. The present study aimed to investigate the occurrence and molecular identity of piroplasmids in free-living mammals of the Superorder Xenarthra from four Brazilian states (Mato Grosso do Sul, São Paulo, Rondônia, and Pará). For this, DNA was extracted from blood or spleen samples from 455 animals. A nested PCR based on the 18S rRNA gene was used as screening for piroplasmids. Of the 455 samples analyzed, 25 (5.5%) were positive. Additionally, PCR assays based on 18S rRNA near-complete, cox-1, cox-3, hsp70, cytB, ß-tubulin genes and the ITS-1 intergenic region were performed. Five out of 25 positive samples also tested positive for ITS-1-based PCR. The phylogenetic analysis positioned three 18S rRNA sequences detected in Priodontes maximus into the same clade of Babesia sp. detected in marsupials (Didelphis albiventris, Didelphis marsupialis, and Monodelphis domestica) and Amblyomma dubitatum collected from opossums and coatis in Brazil. On the other hand, the 18S rRNA sequence obtained from Dasypus novemcinctus was closely related to a Theileria sp. sequence previously detected in armadillos from Mato Grosso State, grouping in a subclade within the Theileria sensu stricto clade. In the phylogenetic analysis based on the ITS-1 region, the sequences obtained from Myrmecophaga tridactyla and Tamandua tetradactyla were placed into a single clade, apart from the other piroplasmid clades. The present study demonstrated the molecular occurrence of Piroplasmida in anteaters and Babesia sp. and Theileria sp. in armadillos from Brazil.

What a few hairs can tell us about the resource use of giant armadillos.

Understanding of resource requirements of vulnerable species is key for conservation planning. Here, we used stable carbon (δ13 C) and nitrogen isotopes (δ15 N) of hair from giant armadillos (Priodontes maximus) to understand individual resource use. We collected hair from 19 giant armadillos analyzing 34 samples from the Brazilian Pantanal and compared the resource use between sexes and age classes. We also assessed the relationship of isotopic values with individuals' body mass and the habitat type used. We observed a wide variation in resource use by giant armadillos, showing that individuals consume prey that feeds on distinct resources and occupies different habitat types, indicating that there is individual variation in foraging behavior. Most giant armadillos presented a mixed diet (C3 /C4 resources, forests/open areas), but a quarter of individuals had strictly C4 diets (open areas). Males are more prone to forage on C4 resources, presenting an isotopic niche 2.6-times larger than that of females. Subadults presented diets more associated with habitats with high arboreal cover (C3 resources, forests), while adults foraged more in open areas (C4 resources). This result is mirrored by the positive relationship between δ13 C values and body mass, suggesting that larger giant armadillos tend to feed more in open areas. We observed that δ13 C values decreased as individuals increased the use of habitats with high arboreal cover. We stress the importance of conserving the natural mosaic in the Pantanal landscape for the species persistence, given that giant armadillos require all habitat types to fulfill their dietary, spatial, and developmental needs.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

The role of environmental temperature on movement patterns of giant anteaters.

Mammals can show conspicuous behavioral responses to thermal variation, including changes in movement patterns. We used an integrative approach to understand how environmental temperature can drive the movement behavior of a mammal with low capacity for physiological thermoregulation, the giant anteater (Myrmecophaga tridactyla). We tracked 52 giant anteaters in 7 areas throughout the Brazilian savannah. We estimated the distance moved, area used, use of forest areas, and mean environmental temperature for each monitoring day of each individual. We modeled these data with Mixed Structural Equations - considering the possible interactions between our variables and controlling for sex and body mass. Giant anteaters reduced displacement and increased forest use with decreasing environmental temperature, probably because of their low body heat production. It is possible that they reduce distance moved and area used by reducing the duration of activity. With decreasing temperature, forest habitats become warmer than open ones, besides buffer rain and chilly winds. Reducing displacement and using forests are important strategies to reduce body heat loss and the energetic costs of thermoregulation. However, decreasing movement can limit food access and, consequently, fitness. Therefore, we highlight the importance of forests as thermal shelters. With increasing frequency and intensity of extreme weather events, we showed the need to preserve forest patches to offer suitable conditions for tropical mammals' behavioral thermoregulation. In this context, policies favoring deforestation on Brazilian territory are especially worrisome. Finally, we emphasize the need of integrative approaches to understand the complex interactions between organisms and the environment.

Novel gammaherpesvirus associated with primary gastric T-cell lymphoma in a free-ranging giant armadillo in Brazil.

The number of viral-associated neoplasms reported in wildlife has increased over the last decades, likely because of growing research efforts and a potentially greater burden of carcinogenic pathogens. Herein, we describe a primary gastric T-cell lymphoma in one free-ranging giant armadillo (Priodontes maximus) from Brazilian Pantanal infected by a novel gammaherpesvirus, proposed as Cingulatid gammaherpesvirus 1 (CiHV-1). By chromogenic in situ hybridisation against Epstein-Barr virus some neoplastic cells were labeled. Subsequently, a molecular screening was carried out to detect the occurrence of this pathogen in other giant armadillos in the same region. Overall, this novel virus was detected in 14.3% (3/21) of the tested giant armadillos. We suggest this herpesvirus, the first in Xenarthra, as a plausible aetiology of the neoplasm. The implications of CiHV-1 for this species are uncertain; while no outbreaks of disease have been recorded, the present study raises concerns. Further research is warranted to assess the real significance of CiHV-1 and its potential oncogenic role in this species.

Trypanosoma rangeli Genetic, Mammalian Hosts, and Geographical Diversity from Five Brazilian Biomes.

Trypanosoma rangeli is a generalist hemoflagellate that infects mammals and is transmitted by triatomines around Latin America. Due to its high genetic diversity, it can be classified into two to five lineages. In Brazil, its distribution outside the Amazon region is virtually unknown, and knowledge on the ecology of its lineages and on host species diversity requires further investigation. Here, we analyzed 57 T. rangeli samples obtained from hemocultures and blood clots of 1392 mammals captured in different Brazilian biomes. The samples were subjected to small subunit (SSU) rDNA amplification and sequencing to confirm T. rangeli infection. Phylogenetic inferences and haplotype networks were reconstructed to classify T. rangeli lineages and to infer the genetic diversity of the samples. The results obtained in our study highlighted both the mammalian host range and distribution of T. rangeli in Brazil: infection was observed in five new species (Procyon cancrivorous, Priodontes maximum, Alouatta belzebul, Sapajus libidinosus, and Trinomys dimidiatus), and transmission was observed in the Caatinga biome. The coati (Nasua nasua) and capuchin monkey (S. libidinosus) are the key hosts of T. rangeli. We identified all four T. rangeli lineages previously reported in Brazil (A, B, D, and E) and possibly two new genotypes.

Zoonotic parasites infecting free-living armadillos from Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild-caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen's disease). Forty-three free-living armadillos from Pantanal and seven road-killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti-T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

ANESTHESIA AND SURGERY PROTOCOLS FOR INTRA-ABDOMINAL TRANSMITTER PLACEMENT IN FOUR SPECIES OF WILD ARMADILLO.

The anatomy and physiology of armadillos make anesthetic procedures and the placement of telemetry devices challenging. From June 2011 to November 2019, a total of 73 free-living armadillos were captured and anesthetized in the Pantanal, Brazil. Giant (Priodontes maximus), six-banded (Euphractus sexcinctus), southern naked-tailed (Cabassous unicinctus), and nine-banded (Dasypus novemcinctus) armadillos were divided into two groups. In group 1, 30 armadillos were anesthetized for collection of biological samples, body measurements, and placement of a microchip tag. Anesthetic combination BDM was applied: butorphanol tartrate, detomidine hydrochloride, and midazolam hydrochloride, each at 0.1 mg/kg. In group 2, 43 armadillos received ketamine hydrochloride at 10 mg/kg 20 min after BDM injection, and intra-abdominal radio transmitters were surgically implanted. The transmitter was inserted freely into the abdominal cavity. Vital signs were monitored during anesthesia every 10 min and varied within species and between groups. Rectal temperature varied from 33.1 ± 1.36 to 35.34 ± 1.21°C, heart rate (beats/min) from 19 ± 2.14 to 84.71 ± 9.25, respiratory rate (breaths/min) from 11 ± 4.16 to 31 ± 2.82, and oxygen saturation values (SPO2%) from 84.17% ± 2.39 to 98% ± 1.20. Both groups received the antagonist combination NYF: naloxone hydrochloride (0.02 mg/kg), yohimbine hydrochloride (0.125 mg/kg), and flumazenil (0.01 mg/kg). Recovery varied according to intravenous or intramuscular injection from 2 ± 4 to 8.08 ± 2.93 min respectively. BDM protocol was considered satisfactory and provided enough time to complete the procedures (60 ± 85 to 133.20 ± 9.12 min) according to the species and group. Ketamine added to the BDM provided enough time and a surgical plane of anesthesia (97 ± 22 to 137 ± 39.5 min). The surgical procedure technique chosen did not appear to have a negative impact on armadillos studied. Implantable transmitters provide a cost-effective method for long-term monitoring of wild individuals.

Survey of Leptospira spp. and Brucella abortus in Free-Ranging Armadillos from Pantanal, Brazil.

Out of the 20 recognized species of armadillos in the world, 11 are found in Brazil, and five of them are found in Pantanal, one of the world's largest wetlands. Beef cattle (Bos taurus) farming is the main economic activity in this region, which promotes intense wildlife-livestock contact and increases the likelihood of pathogen exposure, including to agents with zoonotic and economic relevance. Previous studies demonstrated that several wildlife species in Pantanal have been exposed to Brucella abortus and Leptospira spp.; however, little is known regarding the exposure and/or prevalence of zoonotic pathogens in armadillos. We used conventional PCR, the rose Bengal test (RBT), and the microscopic agglutination test (MAT) to investigate the exposure to and infection by Brucella spp. and Leptospira spp. using blood samples from four species of armadillos: nine-banded armadillo (Dasypus novemcinctus, n=2), southern naked-tailed armadillo (Cabassous unicinctus, n=8), yellow armadillo (Euphractus sexcinctus, n=16), and giant armadillo (Priodontes maximus, n=22), captured in Nhecolândia, Pantanal, Brazil. Samples were PCR- and RBT-negative for Brucella spp. infection and exposure. However, MAT revealed a Leptospira spp. seroprevalence of 31% (5/16; 95% confidence interval [CI]=0.11-0.58) in yellow armadillo and 18% (4/22; 95% CI=0.05-0.40) in giant armadillo specimens to serogroups Autumnalis, Cynopteri, and Pomona, with titers ranging from 200 to 1,600. Our results contribute to the understanding of zoonotic pathogens in armadillos in Pantanal and reinforce the importance of wildlife health surveillance in this area.