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.

Ovarian Filariasis in a Wild Southern Tamandua (Tamanduatetradactyla; Mammalia: Myrmecophagidae).

Knowledge of reproductive health in wild southern tamanduas (Tamandua tetradactyla; Mammalia: Myrmecophagidae) is fragmentary. During necropsies of roadkill xenarthran species in Brazil, a case of ovarian filariasis in an adult female southern tamandua was observed. Macroscopically, both ovaries were irregularly enlarged and had numerous smooth protuberances. Histologically, the affected ovarian parenchyma presented adult nematodes (including females with microfilaria) surrounded by pleocellular inflammatory infiltrates. The morphological characteristics of the nematodes were consistent with the superfamily Filarioidea (order Spirurida). The adjacent ovarian parenchyma had developing and atretic follicles at different stages of maturation. Filarial nematodes were not observed in other tissues. The cause of death of this tamandua was fatal acute polytrauma as a consequence of the motor vehicle collision. This case adds to a prior report of ovarian filariasis in two southern tamanduas in Nicaragua and Guatemala, dating back almost 100 years, and suggests filarial infections could potentially have an impact on reproductive success in southern tamanduas and possibly other xenarthrans. Several xenarthran species are under different levels of threat and knowledge of their basic reproductive health is crucial for conservation programs.

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.

Morphology of the genital organs of male and female giant anteaters (Myrmecophaga tridactyla).

BACKGROUND: The giant anteater belongs to the supraorder Xenarthra which occupies a systematically isolated position among placental mammals. The species is categorized as Vulnerable by the International Union for Conservation of Nature, and understanding its reproductive characteristics is critical for future conservation efforts. METHODS: Gross and microscopic anatomy of the genital organs of 23 male and 21 female adult and young roadkill giant anteaters in Brazil were studied. RESULTS: Male giant anteaters presented a short conical penis, intraabdominal testes, and prostate, vesicular and bulbourethral glands. A tubular remnant of the partially fused Müllerian ducts extended from the seminal colliculus through the prostate gland, continued cranially in the genital fold, bifurcated, and attached with one elongation each to the left and right epididymal corpus. The structure presented a total length of up to 10 cm and contained a yellowish liquid in its lumen. Histologically, the caudal section of this structure resembled the female vagina, the middle portion corresponded to the uterus, and the extensions showed characteristics of uterine tubes. In adult female giant anteaters, ovoid ovaries with occasional seminiferous cord-like structures were observed. The animals possessed a simple uterus, which was directly continuous with the vaginal canal. The caudal portion of the vagina had two lumina, separated by a longitudinal septum and opening into two apertures into the vaginal vestibule, cranial to the urethral opening. In the urethral and the lateral vestibular wall, glandular structures with characteristics of male prostate and bulbourethral glands, respectively, were found. The vestibule opened through a vertical vulvar cleft to the exterior. A pair of well-differentiated Wolffian ducts with a central lumen originated ventrally at the vaginal opening into the vestibule and passed in a cranial direction through the ventral vaginal and uterine wall. Each duct extended highly coiled along the ipsilateral uterine tube until the lateral pole of the ovaries where it merged with the rete ovarii. DISCUSSION: The reproductive morphology of giant anteaters reveals characteristics shared with other Xenarthrans: intraabdominal testes, a simple uterus, and a double caudal vagina. The persistence of well-differentiated genital ducts of the opposite sex in both males and females, however, singles them out among other species. These structures are the results of an aberration during fetal sexual differentiation and possess secretory functions. The possibility of a pathological degeneration of these organs should be considered in reproductive medicine of the species. CONCLUSION: Knowledge of the unique reproductive characteristics of the giant anteater is essential for future reproductive management of the species. Additionally, further research on the peculiarities of the persisting genital duct structures might help to understand sexual differentiation in placental mammals in general.

Pulmonary adiaspiromycosis in armadillos killed by motor vehicle collisions in Brazil.

Knowledge of infectious diseases in wildlife provides important information for preventing potential outbreaks of zoonotic diseases. Adiaspiromycosis is a neglected human disease caused by dimorphic Onygenales fungi. The disease is produced by the inflammatory response against growing adiaspores, leading to granulomatous pneumonia. In humans, adiaspiromycosis is relevant in immunosuppressed patients. In animals, it is associated with pneumonia in fossorial species. Given the potential role of armadillos in the epidemiology of adiaspiromycosis, in this study, we sought to investigate the occurrence and pathological features of adiaspiromycosis in roadkilled armadillos. In total, 54 armadillo carcasses were suitable for postmortem pathologic examinations between February 2017 and 2020. Adiaspores, associated with granulomatous lesions, were observed in ten six-banded (Euphractus sexcinctus) and two southern naked-tailed armadillos (Cabassous unicinctus). A previously uncharacterized Onygenales species was molecularly identified in two E. sexcinctus. In summary, herein we report 12 cases of pulmonary adiaspiromycosis (PA) in two species of free-living armadillos in Brazil. Both, the morphology of the fungus, as well as the histopathological findings (granulomatous inflammatory response to adiaspores) are consistent with PA; however, as the molecular identification differs from the reported species, the potential impact of this fungus for human PA is unknown, and we cannot rule out its impact on public health.

Ehrlichia spp. and Anaplasma spp. in Xenarthra mammals from Brazil, with evidence of novel 'Candidatus Anaplasma spp.'

Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.

Multi-locus sequencing reveals a novel Bartonella in mammals from the Superorder Xenarthra.

The superorder Xenarthra consists of sloths, anteaters and armadillos, mammals originated from South America and currently distributed from the south of North America to the south of South America. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in blood and spleen samples from free-living Xenarthra mammals in the states of São Paulo (SP), Mato Grosso do Sul (MS), Rondônia (RO) and Pará (PA). Based on a quantitative real-time PCR (qPCR) assay, a Bartonella spp. nuoG gene fragment was detected in 1.51% (5/330) of the samples: 4 six-banded armadillos (Euphractus sexcinctus) sampled in the MS and 1 southern tamandua (Tamandua tetradactyla) sampled in the PA. Eight sequences (5 ftsZ, 2 gltA and 1 rpoB) were obtained in the conventional PCR assays. In both phylogenetic analyses based on Bayesian and distance (SplitsTree) methods, the obtained ftsZ, gltA and rpoB sequences were positioned in a distinct clade, but related to B. washoensis. The analysis of SplitsTree and genotype networks based on B. washoensis sequences from several hosts from various localities of the world showed that the sequences of the present study were allocated in a group separated from the other sequences, indicating that they probably originated from median vectors and large numbers of mutational events. Additionally, the analyses performed by BLAST showed low percentages of identities of the sequences obtained in the present study when compared to those previously deposited in GenBank. Therefore, we propose a new Candidatus to Bartonella occurring in Xenarthra in Brazil. The present study was the first to report the occurrence of Bartonella sp. in mammals of the superorder Xenarthra in the world, and it was the first to describe a new Candidatus related to B. washoensis in Brazil.