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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., (© 2020 Wiley-VCH GmbH.)

Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Background: Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques., Methods/principal Findings: Aerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts., Conclusions/significance: Data may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.

Sporotrichosis is a polymorphic disease of man and animals caused by traumatic implantation of propagules into the skin and subcutaneous tissue. Pathogenic species includes S. brasiliensis, S. schenckii, S. globosa and S. luriei. The disease is remarkable for its occurrence as sapronoses and/or zoonosis outbreaks in tropical and subtropical areas; although, the ecology of the clinical clade is still puzzling. Here, we describe an anamorphic Sporothrix strain isolated from soil in an armadillo's burrow, which was located in a hyper endemic area of Paracoccidioidomycosis in Brazil. This isolate was identified as S. schenckii sensu stricto (Clade IIa) based on morphological and physiological characteristics and phylogenetic analyses of calmodulin sequences. We then discuss the role of the nine-banded armadillo Dasypus novemcinctus as a natural carrier of Sporothrix propagules to better understand Sporothrix sources in nature and reveal essential aspects about the pathogen's eco-epidemiology.

Leprosy is still a worldwide public health problem. Brazil and India show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in some regions of the United States. Identification of bacilli is difficult, particularly due to its inability to grow in vitro. The use of molecular tools represents a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, histopathology, microbiology, and PCR using specific primers for M. leprae repetitive sequences. PCR were performed using genomic DNA extracted from 138 samples of liver, spleen, lymph nodes, and skin of 44 D. novemcinctus, Euphractus sexcinctus, Cabassous unicinctus, and C. tatouay armadillos from the Middle Western region of the state of São Paulo and from the experimental station of Embrapa Pantanal, located in Pantanal da Nhecolândia of Mato Grosso do Sul state. Also, the molecular analysis of 19 samples from internal organs of other road killed species of wild animals, such as Nasua nasua (ring-tailed coati), Procyon cancrivoros (hand-skinned), Cerdocyon thous (dog-pity-bush), Cavia aperea (restless cavy), Didelphis albiventris (skunk), Sphigurrus spinosus (hedgehog), and Gallictis vittata (ferret) showed PCR negative data. None of the 157 analyzed samples had shown natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient presented PCR positive and its genomic sequencing revealed 100% identity with M. leprae. According to these preliminary studies, based on the used methodology, it is possible to conclude that wild mammals seem not to play an important role in the epidemiology of leprosy in the Middle Western region of the São Paulo state and in the Pantanal of Mato Grosso do Sul state.

Armadillos are primitive mammals used as food, mostly in rural areas. These animals may be sources of toxoplasmosis and leptospirosis infection for humans, but there is little information about their potential risk as reservoirs. In order to determine the prevalence of armadillos infected by Toxoplasma gondii and Leptospira spp., serum samples of 31 nine-banded armadillos (Dasypus novemcinctus), three six-banded armadillos (Euphractus sexcinctus), two naked-tailed armadillos (Cabassous tatouay) and two long-nosed armadillos (D. hybridus), captured in the mid-west region of the state of São Paulo, were analyzed for leptospirosis using the Microscopic Agglutination Test (MAT-l), and for toxoplasmosis using the Modified Agglutination Test (MAT-t). Only 4/31 (12.90%) nine-banded armadillos were positive for T. gondii, while 3/31 (9.68%) nine-banded armadillos and 1/3 (33.33%) six-banded armadillos presented antibodies to Leptospira spp., demonstrating the potential risk of T. gondii and Leptospira spp. transmission to humans, mainly due the habit of eating the meat of these animals in rural areas.

The knowledge on the biological aspects of Paracoccidioides brasiliensis has evolved greatly since the first description of the disease in 1908. From the pioneers, who were able to clearly demonstrate the fungal nature of the agent, to the recent genomic era, important advances have been achieved. P. brasiliensis is a true fungus, belonging to the Ascomycetous Division, although its sexual phase has not been demonstrated morphologically. A better understanding of the fundamental aspects of the agent, especially its phylogeny and evolutionary history, will provide us with valuable insights allowing a better comprehension of the disease and our capacity to deal with the problem. Concerning the fungus's ecology, although some progress had been observed, the ecological niche of the pathogen has not been determined yet. The aim of the present review is to focus on the biological aspects of P. brasiliensis from an evolutionary point of view, addressing the fungus's phylogenetic aspects, in those special points that might be relevant for the pathogen/host interactions, the biological forces that have been acting on its origin and maintenance of virulence, as well as in determining the fungus's ecology and epidemiology.

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Toxoplasmosis is a worldwide distributed zoonosis that affects man and most warm-blooded animals, with a great economic impact in animal and public health. Serum samples from nine 9-banded armadillos, three 6-banded armadillos, three coatimundis, two opossums and one nutria were submitted for anti-Toxoplasma gondii antibody detection by means of a modified direct agglutination method. Encephalic tissue of three 6-banded armadillos, one 9-banded armadillo, one coatimundi and one nutria were digested in acid pepsin solution and inoculated into Swiss mice for parasite isolation. Only one serum sample from a nine-banded armadillo and two from six-banded armadillos reacted producing titers equal to 256, 512 and 512, respectively. T. gondii was isolated in two 6-banded armadillos, one of which was not positive in the serological test.

Coccidioidomycosis, listed as a priority mycosis by the WHO, is endemic in the United States but often overlooked in Central and South America. Employing a multi-institutional approach, we investigate how disease characteristics, pathogen genetic variation, and environmental factors impact coccidioidomycosis epidemiology and outcomes in South America. We identified 292 cases (1978-2021) and 42 outbreaks in Piauí and Maranhão states, Brazil, the largest series outside the US/Mexico epidemic zone. The male-to-female ratio was 57.4:1 and the most common activity was armadillo hunting (91.1%) 4 to 30 days before symptom onset. Most patients (92.8%) exhibited typical acute pulmonary disease, with cough (93%), fever (90%), and chest pain (77%) as predominant symptoms. The case fatality rate was 8%. Our negative binomial regression model indicates that reduced precipitation levels in the current (p = 0.015) and preceding year (p = 0.001) predict heightened incidence. Unlike other hotspots, acidic soil characterizes this region. Brazilian strains differ genomically from other C. posadasii lineages. Northeastern Brazil presents a distinctive coccidioidomycosis profile, with armadillo hunters facing elevated risks. Low annual rainfall emerges as a key factor in increasing cases. A unique C. posadasii lineage in Brazil suggests potential differences in environmental, virulence, and/or pathogenesis traits compared to other Coccidioides genotypes.

Paracoccidioidomycosis is an endemic fungal disease to Latin America caused by at least five species-level genotypes of Paracoccidioides, named P. lutzii, P. brasiliensis (S1a and S1b populations), P. americana, P. restrepiensis, and P. venezuelensis. In this manuscript, we report on Paracoccidioides sp. sampling efforts in armadillos from two different areas in Brazil. We sequenced the genomes of seven Paracoccidioides isolates and used phylogenomics and populations genetics for genotyping. We found that P. brasiliensis and P. lutzii are both present in the Amazon region. Additionally, we identified two Paracoccidioides isolates that seem to be the result of admixture between divergent populations within P. brasiliensis sensu stricto. Both of these isolates were recovered from armadillos in a P. lutzii endemic area in Midwestern Brazil. Additionally, two isolates from human patients also show evidence of resulting from admixture. Our results suggest that the populations of P. brasiliensis sensu stricto exchange genes in nature. More generally, they suggest that population structure and admixture within species is an important source of variation for pathogenic fungi. [ABSTRACT FROM AUTHOR]

Abstract: The habitat of the mycelial saprobic form of Paracoccidioides brasiliensis, which produces the infectious propagula, has not been determined and has proven difficult for mycologists to describe. The fungus has been rarely isolated from the environment, the disease has a prolonged latency period and no outbreaks have been reported. These facts have precluded the adoption of preventive measures to avoid infection. The confirmation of natural infections in nine-banded armadillos (Dasypus novemcinctus) with P. brasiliensis, in high frequency and wide geographic distribution, has opened new avenues for the study and understanding of its ecology. Armadillos belong to the order Xenarthra, which has existed in South America ever since the Paleocene Era (65 million years ago), when the South American subcontinent was still a detached land, before the consolidation of what is now known as the American continent. On the other hand, strong molecular evidence suggests that P. brasiliensis and other dimorphic pathogenic fungi – such as Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum – belong to the family Onygenaceae sensu lato (order Onygenales, Ascomycota), which appeared around 150 million years ago. P. brasiliensis ecology and relation to its human host are probably linked to the fungal evolutionary past, especially its long coexistence with and adaptation to animal hosts other than Homo sapiens, of earlier origin. Instead of being a blind alley, the meaning of parasitism for dimorphic pathogenic fungi should be considered as an open two-way avenue, in which the fungus may return to the environment, therefore contributing to preserve its teleomorphic (sexual) and anamorphic (asexual) forms in a defined and protected natural habitat. [Copyright &y& Elsevier]