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.

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.

Comparative mitogenome phylogeography of two anteater genera (Tamandua and Myrmecophaga; Myrmecophagidae, Xenarthra): Evidence of discrepant evolutionary traits.

The species within Xenarthra (sloths, anteaters, and armadillos) are quintessential South American mammals. Of the three groups, Vermilingua (anteaters) contains the fewest extant and paleontological species. Here, we sampled and sequenced the entire mitochondrial genomes (mitogenomes) of two Tamandua species (Tamandua tetradactyla and Tamandua mexicana) (n=74) from Central and South America, as well as Myrmecophaga tridactyla (n=41) from South America. Within Tamandua, we detected three different haplogroups. The oldest (THI) contained many specimens with the T. tetradactyla morphotype (but also several with the T. mexicana morphotype) and originated in southeastern South America (currently Uruguay) before moving towards northern South America, where the THII haplogroup originated. THII primarily contained specimens with the T. mexicana morphotype (but also several with the T. tetradactyla morphotype) and was distributed in Central America, Colombia, and Ecuador. THI and THII yielded a genetic distance of 4%. THII originated in either northern South America or "in situ" in Central America with haplogroup THIII, which consisted of ~50% T. mexicana and 50% T. tetradactyla phenotypes. THIII was mostly located in the same areas as THII, i.e., Central America, Ecuador, and Colombia, though mainly in the latter. The three haplogroups overlapped in Colombia and Ecuador. Thus, T. tetradactyla and T. mexicana were not reciprocally monophyletic. For this reason, we considered that a unique species of Tamandua likely exists, i.e., T. tetradactyla. In contrast to Tamandua, M. tridactyla did not show different morphotypes throughout its geographical range in the Neotropics. However, two very divergent genetic haplogroups (MHI and MHII), with a genetic distance of ~10%, were detected. The basal haplogroup, MHI, originated in northwestern South America, whereas the more geographically derived haplogroup, MHII, overlapped with MHI, but also expanded into central and southern South America. Thus, Tamandua migrated from south to north whereas Myrmecophaga migrated from north to south. Our results also showed that temporal mitochondrial diversification for Tamandua began during the Late Pliocene and Upper Pleistocene, but for Myrmecophaga began during the Late Miocene. Furthermore, both taxa showed elevated levels of mitochondrial genetic diversity. Tamandua showed more evidence of female population expansion than Myrmecophaga. Tamandua experienced population expansion ~0.6-0.17 million years ago (Mya), whereas Myrmecophaga showed possible population expansion ~0.3-0.2 Mya. However, both taxa experienced a conspicuous female decline in the last 10 000-20 000 years. Our results also showed little spatial genetic structure for both taxa. However, several analyses revealed higher spatial structure in Tamandua than in Myrmecophaga. Therefore, Tamandua and Myrmecophaga were not subjected to the same biogeographical, geological, or climatological events in shaping their genetic structures.

Comparative masticatory myology in anteaters and its implications for interpreting morphological convergence in myrmecophagous placentals.

BACKGROUND: Ecological adaptations of mammals are reflected in the morphological diversity of their feeding apparatus, which includes differences in tooth crown morphologies, variation in snout size, or changes in muscles of the feeding apparatus. The adaptability of their feeding apparatus allowed them to optimize resource exploitation in a wide range of habitats. The combination of computer-assisted X-ray microtomography (µ-CT) with contrast-enhancing staining protocols has bolstered the reconstruction of three-dimensional (3D) models of muscles. This new approach allows for accurate descriptions of muscular anatomy, as well as the quick measurement of muscle volumes and fiber orientation. Ant- and termite-eating (myrmecophagy) represents a case of extreme feeding specialization, which is usually accompanied by tooth reduction or complete tooth loss, snout elongation, acquisition of a long vermiform tongue, and loss of the zygomatic arch. Many of these traits evolved independently in distantly-related mammalian lineages. Previous reports on South American anteaters (Vermilingua) have shown major changes in the masticatory, intermandibular, and lingual muscular apparatus. These changes have been related to a functional shift in the role of upper and lower jaws in the evolutionary context of their complete loss of teeth and masticatory ability. METHODS: We used an iodine staining solution (I2KI) to perform contrast-enhanced µ-CT scanning on heads of the pygmy (Cyclopes didactylus), collared (Tamandua tetradactyla) and giant (Myrmecophaga tridactyla) anteaters. We reconstructed the musculature of the feeding apparatus of the three extant anteater genera using 3D reconstructions complemented with classical dissections of the specimens. We performed a description of the musculature of the feeding apparatus in the two morphologically divergent vermilinguan families (Myrmecophagidae and Cyclopedidae) and compared it to the association of morphological features found in other myrmecophagous placentals. RESULTS: We found that pygmy anteaters (Cyclopes) present a relatively larger and architecturally complex temporal musculature than that of collared (Tamandua) and giant (Myrmecophaga) anteaters, but shows a reduced masseter musculature, including the loss of the deep masseter. The loss of this muscle concurs with the loss of the jugal bone in Cyclopedidae. We show that anteaters, pangolins, and aardvarks present distinct anatomies despite morphological and ecological convergences.

Characterization of coronary arteries in Giant Anteater (Myrmecophaga tridactyla: Myrmecophagidae) / Caracterização da artéria coronária em tamanduá-bandeira (Myrmecophaga tridactyla: Myrmecophagidae)]

Were used twelve (12) adult anteaters (Myrmecophaga tridactyla), adults, 6 (six) males and 6 (six) females, weighing from 20 to 27.32kg from free life. The thoracic cavity was opened until visualization of the whole heart and lungs and later injection of the coronary vessels. The right coronary artery emerged through a single coronary ostium of the aorta, 50%, emitting the intermediate branch and the subsinuous interventricular branch, had a path directed to the subsurface interventricular groove. In the other 50%, the right coronary artery was not present, showing only its branches, intermediate branch and subsurface interventricular branch with emergence of the aorta. Left coronary artery presented, in 83.33%, origin from the aorta in single ostium, issuing the circumflex and interventricular paraconal branches. In 16.66%, the left coronary artery was not evidenced originating from the aorta, but its branches, circumflex and interventricular paraconal.(AU)

Foram utilizados 12 tamanduás-bandeira (Myrmecophaga tridactyla), adultos, sendo seis machos e seis fêmeas, pesando de 20 a 27,32kg, provenientes de vida livre. Foi realizada abertura da cavidade torácica até a visualização do conjunto coração e pulmões e, posteriormente, injeção dos vasos coronários. A artéria coronária direita emergia, 50% dela, por um único óstio coronário da aorta, emitindo o ramo intermédio e o ramo interventricular subsinuoso; tinha trajeto direcionado ao sulco interventricular subsinuoso. Nos outros 50%, a artéria coronária direita não estava presente, exibindo somente seus ramos, ramo intermédio e ramo interventricular subsinuoso com emergência da aorta. A artéria coronária esquerda apresentou, em 83,33%, origem a partir da aorta em óstio único, emitindo os ramos circunflexo e interventricular paraconal. Em 16,66%, a artéria coronária esquerda não foi evidenciada originando-se da aorta, mas, sim, de seus ramos, circunflexo e interventricular paraconal.(AU)

Anatomia e segmentação pulmonar de tamanduá-bandeira (Myrmecophaga tridactyla - Linnaeus, 1758) de vida livre / Anatomy and pulmonary segmentation of Free-living Giant Anteater (Myrmecophaga tridactyla - Linnaeus, 1758)

O presente trabalho utilizou oito pulmões de tamanduá-bandeira (Myrmecophaga tridactyla), jovens e adultos, de ambos os sexos, provenientes do Setor de Atendimento Clínico Cirúrgico de Animais Selvagens (SACCAS), do Hospital Veterinário "Dr. Halim Atique"( Unirp, localizado na cidade de São José do Rio Preto, estado de São Paulo. Os animais foram encaminhados ao Laboratório de Anatomia Veterinária, onde se procedeu à separação dos órgãos, à lavagem em água corrente e à retirada do excesso de sangue, para serem fixados em formol a 10%. Após a fixação dos pulmões, estes foram dissecados para identificar os segmentos. Os animais apresentaram lobação pulmonar à direita em cranial, médio, caudal e acessório, sendo o lobo cranial dividido em partes cranial e caudal. A lobação pulmonar à esquerda apresentou cranial e caudal em 100% dos animais. Por sua vez, o lobo cranial, em 87,5%, dividiu-se em partes cranial e caudal, e em 12,5% não houve divisão. O pulmão direito apresentou maior tamanho do que o pulmão esquerdo. A árvore brônquica apresentou os brônquios principais direito e esquerdo, os quais se ramificam em brônquios lobares, que dão origem aos segmentos bronquiopulmonares. Em relação à segmentação bronquiopulmonar, foram observadas, à direita, de 14 (12,5%) a 18 segmentos (25%) e, à esquerda, de 10 (25%) a 30 segmentos (12,5%).(AU)

The study used eight lungs of giant anteaters (Myrmecophaga tridactyla), youth and adults of both sexes, from the Clinical Surgical Care Industry Wildlife (SACCAS), Veterinary Hospital "Dr. Halim Atique "- Unirp, located in the city of São José do Rio Preto, São Paulo. The animals were sent to the Veterinary Anatomy Laboratory, where organs were separated and washed under running waterto remove excess blood, then fixed in 10% formalin. After fixation, lungs were dissected to identify the segments. The animals have pulmonary lobations right cranial, middle, caudal and accessory; and the cranial lobe divided into cranial and caudal parts. Pulmonary lobation in the left cranial and caudal form were present in 100% of animals. The cranial lobe was evident in 87.5%, divided into cranial and caudal part, and 12.5% with no division. The right lung was shown to be larger than the left lung. Bronchial tree presented the main right and left bronchial tubes that branch into lobar bronchi that give rise to bronquiopulmonares segments. In relation to bronquiopulmonar targeting, 14 (12.5%) to 18 right segments (25%) and 10 (25%) to 30 (12.5%) left segments.(AU)

Serosurvey of Leptospira interrogans, Brucella abortus and Chlamydophila abortus infection in free-ranging giant anteaters (Myrmecophaga tridactyla) from Brazil / Inquérito sorológico de Leptospira interrogans, Brucella abortus e Chlamydophila abortus em tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre no Brasil

A serological survey for antibodies against Leptospira interrogans, Brucella abortus, and Chlamydophila abortus was conducted in 21 clinically healthy, free-ranging giant ant- eaters (Myrmecophaga tridactyla) from Parque Nacional das Emas (Goiás State, Brazil; n=6), Parque Nacional da Serra da Canastra (Minas Gerais State, Brazil; n=9), and RPPN SESC Pantanal (Mato Grosso State, Brazil; n=6) between July 2001 and September 2006. Sera were screened for antibodies against 22 serovars of Leptospira interrogans with a microscopic agglutination test. Twelve tested positive for L. interrogansserovars sentot (n=5 in PN Emas, n=2 in PN Serra da Canastra), butembo (n=2 in PN Serra da Canastra), autumnalis, bataviae, and shermani/icterohaemorrhagiae(n=1 each in SESC Pantanal)One adult female tested positive for B. abortus with the buffered plate antigen test. All sera were negative for C. abortususing the complement fixation text. This is the first report of pathogens that may interfere with the reproduction and population dynamics of free-ranging giant anteaters.

Inquéritos sorológicos para detecção de anticorpos contra Leptospira interrogans, Brucella abortus, e Chlamydophila abortus foram realizados em 21 tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre do Parque Nacional das Emas (Goiás, Brasil, n=6), o Parque Nacional da Serra da Canastra (Minas Gerais, Brasil, n=9) e RPPN SESC Pantanal (Mato Grosso, Brasil, n=6) entre julho de 2001 e setembro de 2006. Os sor os foram testados para anticorpos contra 22 sorotipos de Leptospira interrogans com um teste de aglutinação microscópica. Doze animais foram considerados positivos para L. interrogans sorovares sentot (n=5 em PN Emas, n=2 em PN Serra da Canastra), butembo (n=2 em PN Serra da Canastra), autumnalis, bataviae e shermani/icterohaemorrhagiae(n=1 para cada sorovar em SESC Pantanal). Uma fêmea adulta testou positivo para B. abortuscom o teste do antígeno tamponado. Todos os soros se mostraram negativos para C. abortusatravés do teste de fixação do complemento. Este é o primeiro relato de patógenos que podem interferir na dinâmica reprodutiva de populações de tamanduás em estado selvagem.

Ontogenetic criteria to distinguish vertebral types on the debated xenarthran synsacrum.

The presence of a synsacrum formed by the fusion of vertebrae that come into closed contact with the ilium and ischium is a feature that characterizes the clade Xenarthra. Nevertheless, the proper identity of each vertebral element that forms it is a matter of discussion. In this article, we provide ontogenetic information about skeletal ossification of the xenarthran synsacrum and define the position of the sacrocaudal limit within it. We analyzed the synsacrum of 25 specimens of nonadult and 101 adult armadillos and anteaters: Dasypus hybridus, D. novemcinctus, Chaetophractus vellerosus, C. villosus, Tamandua tetradactyla, and Myrmecophaga tridactyla. Two sets of vertebrae were identified: an anterior set, often attached to the iliac bones, in which transverse processes are originated mainly from an expansion of the base of the neural arches, and secondarily from a lateroventral ossification center. A posterior set is characterized by a series of vertebrae along which extra lateral ossifications (described here for the first time) are developed and form exclusively the transverse processes. Among armadillos, the sacrocaudal limit is set between the last vertebrae attached to the iliac bones and the first vertebrae that form the dorsal border of the sacroischial fenestra. In addition, anterior free caudals also showed extra lateral ossifications forming exclusively the transverse processes, supporting the notion that more posterior synsacrals are in fact caudal vertebrae that were incorporated to the synsacrum. In pilosans, the sacrocaudal limit is set between the first vertebrae that come into contact with the ischial bones and the immediately anterior one. However, the pattern of homologies is obscured by the low resolution in the ontogenetic sequence when compared to that of armadillos.

Novel 12S mtDNA findings in sloths (Pilosa, Folivora) and anteaters (Pilosa, Vermilingua) suggest a true case of long branch attraction

We sequenced 12S RNA mtDNA for the majority of the extant species of sloths and anteaters and compared our results with previous data obtained by our group using 16S RNA mtDNA in the same specimens and to GenBank sequences of the extinct giant sloth Mylodon. Our results suggest that pigmy-anteaters may be a case of the long-branch attraction phenomenon and also show the large genetic difference between the Amazonian and Atlantic forest three-toed sloths, contrasting with the small differences observed between the two non-Atlantic forest forms of sloths. These results have important implications for the taxonomy of sloths and anteaters and strongly suggest the placement of pigmy anteaters in their own family (Cyclopidae) and raising the taxonomic status of Bradypus torquatus to a genus.