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.

Ticks and Rickettsia on anteaters from Southeast and Central-West Brazil.

The giant anteater (Myrmecophaga tridactyla) and the collared anteater (Tamandua tetradactyla) are widespread in Brazil and found in all Brazilian biomes. These hosts frequently use domestic animal environments such as pastures, where tick and related microorganism interchange may occur between hosts. Reports of tick infestations of these animals are scattered and refer to small samples and/or are geographically restricted. We herein present data on a wide geographic distribution of ticks and their Rickettsia collected from 72 giant and 30 collared anteaters, mostly road killed, over a period of 18 years, from Southeast and Central-West Brazil encompassing four States and 46 Municipalities. Overall nine tick species (Amblyomma auricularium, A. calcaratum, A. nodosum, A. ovale, A. parvum, A. sculptum, A. triste, Rhipicephalus microplus and R. sanguineus sensu lato) were collected from anteaters. Amblyomma sculptum, A. nodosum, and A. calcaratum were the most prevalent corresponding to, respectively, 48.8%, 39.3% and 2.7% of all ticks (n = 1775). However, A. nodosum tick numbers on collared anteaters were significantly higher (P < 0.001) than those on giant anteaters. At the same time, an abundance of A. sculptum adults on giant anteaters was significantly higher (Z = 2.875; P = 0.004) than that of A. nodosum and only eight A. sculptum nymphs were found on collared anteaters. DNA samples from 20 ticks from nine different animals yielded a visible amplicon in PCR targeting gltA. The PCR products targeting spotted-fever Rickettsia gene (ompA) from five adults of A. nodosum were sequenced and were shown to be 100% identical to Rickettsia parkeri strain NOD (MF737635.1). The product of one nymph and one adult of A. sculptum yielded a sequence 99% identical to R. parkeri strain NOD. Further, Rickettsia bellii genes were found in three A. nodosum adults. Ecological, behavioral and anatomical traits of anteaters are discussed to explain reported tick infestations and Rickettsia DNA found.

Ectoparasitism and infections in the exoskeletons of large fossil cingulates.

Studies on paleopathological alterations in fossil vertebrates, including damages caused by infections and ectoparasites, are important because they are potential sources of paleoecological information. Analyzing exoskeleton material (isolated osteoderms, carapace and caudal tube fragments) from fossil cingulates of the Brazilian Quaternary Megafauna, we identified damages that were attributed to attacks by fleas and dermic infections. The former were compatible with alterations produced by one species of flea of the genus Tunga, which generates well-delimited circular perforations with a patterned distribution along the carapace; the latter were attributable to pathogenic microorganisms, likely bacteria or fungi that removed the ornamentation of osteoderms and, in certain cases, generated craters or pittings. Certain bone alterations observed in this study represent the first record of flea attack and pitting in two species of large glyptodonts (Panochthus and Glyptotherium) and in a non-glyptodontid large cingulate (Pachyarmatherium) from the Quaternary of the Brazilian Intertropical Region. These new occurrences widen the geographic distribution of those diseases during the Cenozoic and provide more evidence for the co-evolutionary interaction between cingulates and parasites registered to date only for a small number of other extinct and extant species.

CONJUNCTIVAL BACTERIAL FLORA, ANTIBIOGRAM, AND LACRIMAL PRODUCTION TESTS OF COLLARED ANTEATER (TAMANDUA TETRADACTYLA).

The collared anteater ( Tamandua tetradactyla ) is adapted to a variety of habitats. It is a solitary species for which no reference values for ophthalmic tests have been established. Eight animals ranging from 1 to 4 yr of age, two males and six females, were manually restrained for assessment. Ophthalmic tests included evaluation of tear production by Schirmer tear test 1 (STT1), endodontic absorbent paper point tear test (EAPPTT), palpebral fissure length (PFL), culture of the conjunctival bacterial flora, and antimicrobial susceptibility test. Median ± semi-interquartile range (S-IQR) STT1, EAPPTT, and PFL were 8.50 ± 4.13 mm/min, 14.13 ± 3.24 mm/min, and 15.91 ± 2.51 mm, respectively. Bacterial growth was present in 100% of the samples, with predominance of Gram-positive bacteria (70.27%). Staphylococcus spp. was the most frequently isolated genus. Antimicrobial susceptibility test showed sensitivity of Staphylococcus epidermidis and Staphylococcus spp. to neomycin, tobramycin, and gentamicin. The results in this study can benefit the determination of reference values for different diagnostic techniques, and may be used as a guide for diagnosis and treatment of ocular diseases in collared anteaters.

Molecular identification of Anaplasma marginale in two autochthonous South American wild species revealed an identical new genotype and its phylogenetic relationship with those of bovines.

BACKGROUND: Anaplasma marginale is a well-known cattle pathogen of tropical and subtropical world regions. Even though, this obligate intracellular bacterium has been reported in other host species different than bovine, it has never been documented in Myrmecophaga tridactyla (giant anteater) or Hippocamelus antisense (taruca), which are two native endangered species. METHODS: Samples from two sick wild animals: a Myrmecophaga tridactyla (blood) and a Hippocamelus antisense (blood and serum) were studied for the presence of A. marginale DNA through msp5 gene fragment amplification. Further characterization was done through MSP1a tandem repeats analysis and MLST scheme and the genetic relationship among previously characterized A. marginale sequences were studied by applying, eBURST algorithm and AMOVA analysis. RESULTS: Anaplasma marginale DNA was identified in the Myrmecophaga tridactyla and Hippocamelus antisense samples. Through molecular markers, we identified an identical genotype in both animals that was not previously reported in bovine host. The analysis through eBURST and AMOVA revealed no differentiation between the taruca/anteater isolate and the bovine group. CONCLUSIONS: In the present publication we report the identification of A. marginale DNA in a novel ruminant (Hippocamelus antisense) and non-ruminant (Myrmecophaga tridactyla) host species. Genotyping analysis of isolates demonstrated the close relatedness of the new isolate with the circulation population of A. marginale in livestock. Further analysis is needed to understand whether these two hosts contribute to the anaplasmosis epidemiology.

Presencia de anticuerpos contra serovares de Leptospira en Chaetophractus villosus (Mammalia, Dasypodidae), provincia de La Pampa, Argentina / Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina

Leptospirosis is a zoonosis of worldwide distribution. The aim of this study was to examine the presence of antibodies against 21 Leptospira reactive serovars in Chaetophractus villosus in La Pampa province, Argentina, using the microscopic agglutination test (MAT). Pathologic changes compatible with leptospirosis and in situ detection of the agent by immunohistochemistry were studied in 24 and 3 individuals respectively. Only 35/150 (23.3%) serum samples had antibodies against Leptospira sp. Six percent of the samples reacted with serovar Canicola, 4.7% with serovar Castellonis, 1.3% with serovar Icterohemorrhagieae and 0.7% with serovar Hardjo. Sixteen (10.6%) serum samples agglutinated with Castellonis­Icterohemorrhagiae and Canicola­Castellonis serovars, both with 4.7%, and Canicola­Hardjo and Castellonis­Canicola­Icterohemorrhagiae both with 0.6%. Fourteen animals had variable degrees of lesions, which were more severe in animals with higher serological titers (3200), and Leptospira sp. was detected in 3 animals by immunohistochemistry. These results represent the first record of the presence of Leptospira in C. villosus in La Pamp

La leptospirosis es una zoonosis de distribución mundial. Nuestro objetivo fue examinar la presencia de anticuerpos contra 21 serovares reactivos de Leptospira en Chaetopractus villosus en la provincia de La Pampa, Argentina, mediante la prueba de aglutinación microscópica (MAT). Se realizó el estudio histopatológico y la detección in situ del agente por inmunohistoquímica en 24 y 3 individuos, respectivamente. Solo 35/150 (23,3%) muestras de suero presentaron anticuerpos contra Leptospira sp. Seis por ciento reaccionaron al serovar Canicola; 4,7% a Castellonis; 1,3% a Icterohemorrhagieae y 0,7% a Hardjo. Dieciséis (10,6%) sueros aglutinaron con Canicola-Castellonis y Castellonis-Icterohemorrhagiae, ambos con 4,7%, y con Canicola-Hardjo y Castellonis-Canicola-Icterohemorrhagiae, ambos con 0,6%. En 14 animales se encontraron lesiones compatibles, las que resultaron más graves en animales con títulos serológicos elevados (3200). En 3 animales estudiados se detectó el agente causal por inmunohistoquímica. Estos resultados constituyen los primeros registros de la presencia de Leptospira en C. villosus en La Pampa

Convergence of gut microbiomes in myrmecophagous mammals.

Mammals have diversified into many dietary niches. Specialized myrmecophagous (ant- and termite-eating) placental mammals represent a textbook example of evolutionary convergence driven by extreme diet specialization. Armadillos, anteaters, aardvarks, pangolins and aardwolves thus provide a model system for understanding the potential role of gut microbiota in the convergent adaptation to myrmecophagy. Here, we expand upon previous mammalian gut microbiome studies by using high-throughput barcoded Illumina sequencing of the 16S rRNA gene to characterize the composition of gut microbiota in 15 species representing all placental myrmecophagous lineages and their close relatives from zoo- and field-collected samples. We confirm that both diet and phylogeny drive the evolution of mammalian gut microbiota, with cases of convergence in global composition, but also examples of phylogenetic inertia. Our results reveal specialized placental myrmecophages as a spectacular case of large-scale convergence in gut microbiome composition. Indeed, neighbour-net networks and beta-diversity plots based on UniFrac distances show significant clustering of myrmecophagous species (anteaters, aardvarks and aardwolves), even though they belong to phylogenetically distant lineages representing different orders. The aardwolf, which diverged from carnivorous hyenas only in the last 10 million years, experienced a convergent shift in the composition of its gut microbiome to become more similar to other myrmecophages. These results confirm diet adaptation to be a major driving factor of convergence in gut microbiome composition over evolutionary timescales. This study sets the scene for future metagenomic studies aiming at evaluating potential convergence in functional gene content in the microbiomes of specialized mammalian myrmecophages.

Importance of xenarthrans in the eco-epidemiology of Paracoccidioides brasiliensis

BACKGROUND: Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America. FINDINGS: This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild. CONCLUSION: Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment.

Clinical disorders observed in anteaters (Myrmecophagidae, Edentata) in captivity.

The major health problems found in 103 captive lesser anteaters (Tamandua tetradactyla) and giant anteaters (Myrmecophaga tridactyla), family Myrmecophagidae, are presented and correlated with management. The most common of 200 recorded clinical disorders involved the digestive system (26%), nutritional deficiency (20%), injury (15.5%), respiratory system (10%), skin (7%) and circulatory system (4.5%), but 13% of the cases were inconclusive. Parasites were identified in 48.5% of faecal samples, mainly the eggs of nematodes (40%), of which the commonest were Trichuris spp (28%) and Strongyloides spp (11%); protozoa (16%), of which the commonest were Eimeria spp (10%), Entamoeba spp (5%) and Giardia spp (1%); and cestodes (8%) and acanthocephalids (1%). Bacteria cultured from the various materials included Salmonella enteritidis, S. cholerasuis, Escherichia coli, Enterobacter aerogenes, Streptococcus spp and Staphylococcus spp. The ectoparasites found were Amblyomma spp and Otodectis spp (Arthropoda, Acaridae).

Light- and electron-microscopic study of M. leprae-infected armadillo nerves.

Lesions in peripheral nerves of armadillos experimentally infected with Mycobacterium leprae were studied by light- and electron-microscopy. Bacilli could be found clearly inside axons of unmyelinated nerve fibers. Heavily bacillated Schwann cells were seen embracing unmyelinated axons with interrupted cytoplasmic membranes. This indicated the initiation of rupture of those cells which were responsible for the liberation of bacilli into the axons. The nerve lesions were divided into three grades according to their severity: grade I showed lesions focalized in the perineurium; grade II lesions were scattered inside nerve tissue; and in grade III lesions the nerve tissues were diffusely affected. No regressive changes, such as fibrosis or scar formation, were seen in the nerve lesions. Bacillated macrophages were not as foamy as those of human lesions, indicating that these bacillated cells were younger or more easily disrupted with a higher turnover than the cells in human lesions. This would promote the spread of lesions in armadillos, and would explain the less foamy appearance of the cells. We found bacilli inside lymphatics surrounding the nerves, substantiating the opinion that lesions spread to peripheral nerves not only by a hematogenous route but also by the lymphatics.

Interrelaçäo entre acari Ixodidae e hospedeiros Edentata da Serra da Canastra, Minas Gerais, Brasil / Interelationships between acari Ixodidae and Edentata hosts form Serra da Canastra, Minas Gerais State, Brazil

Recebemos para exame uma pequena coleçäo de carrapatos no Parque Nacional da Serra da Canastra (MG), entre 1979 e 1980. Os autores demosntraram a existência de uma larga co-acomodaçäo de Amblyomma pseudoconcolor em edentados da familia Dasypodidae, sendo Dasypodini a tribo mais ajustada a sta infestaçäo. De acordo com as Figs. 1 e 2, Dasypodini säo, provavelmente, os hospedeiros reais de A. pseudoconcolor e também os hospedeiros mais antigos. Pela primeira vez, A. pseudoconcolor é também registrado e, Cabassous tatouay, C. unicinctus, priododntes maximus e Euphractus sexcinctus. Também, pela primeira vez, A. pseudoconcolor e A. calcaratum foram registrados no Estado de Minas Gerais. os ectoparasitos estäo depositados no Departamento de Parasitologia da Universidade Federal de Minas Gerais, Brasil

[Interrelatio of acari Ixodidae and hosts of Edentata of the Serra da Canastra, Minas Gerais, Brazil]. / Interrelações entre acari ixodidae e hospedeiros Edentata da serra da canastra, minas gerais, brasil.

We received for examination a small colection of ticks captured in the National Park of the Serra da Canastra (MG), between 1979 and 1980. The authors demonstrated the existence of a broad co-accomodation of Amblyomma pseudoconcolor on Edentata of the family Dasypodidae, being Dasypodini the tribe more adjusted to this infestation. In conformity to the Figs 1 and 2, Dasypodini are probably the real hosts of A. pseudoconcolor and also the oldest hosts. For the first time, A. pseudoconcolor is also recorded on Cabassous tatouay, C. unicinctus, Priodontes maximus and Euphractus sexcincuts. Also for the first time A. pseudoconcolor and Amblyomma calcaratum were recorded in the State of Minas Gerais. The ectoparasites are deposited in the "Departamento de Parasitologia da Universidade Federal de Minas Gerais, Brasil".

[Mycobacteria isolated from armadillos Dasypus novemcinctus inoculated with mycobacterium leprae]. / Micobactérias isoladas de tatus Dasypus novemcinctus inoculados com Mycobacterium leprae.

The authors report the isolation of two mycobacteria from armadillos livers and spleens, inoculated with Mycobacterium leprae in the Kato medium. They discuss this results.

Partial nucleotide sequence of 16S ribosomal RNA isolated from armadillo-grown Mycobacterium leprae.

Ribosomal RNA (rRNA) was isolated from Mycobacterium leprae recovered from infected tissue of the Nine-banded Armadillo, and nucleotide sequences near the 3' end of the 16S species were determined by primer extension in the presence of dideoxynucleotides. Previously published data for bacterial 16S rRNAs show a pattern of conserved and non-conserved sequences that fit a common secondary structure. Our data for M. leprae fits this general pattern.

Separation of Mycobacterium leprae from contamination with armadillo-liver-derived "pigment" particles.

Mycobacterium leprae isolated from armadillo liver by the widely used IMMLEP protocol is sometimes contaminated with a particulate "pigment." This paper describes a simple, efficient, and rapid method for purifying large quantities of contaminated bacteria, which may readily be used as an additional step added at the end of the protocol when necessary. The process involves a discontinuous Percoll gradient and generates an essentially pure fraction containing greater than 90% of the original bacteria, and a fraction of "pigment" slightly contaminated with bacteria. Use of the system should release large additional numbers of pure M. leprae suitable for use in human vaccine trials.

Thorns in armadillo ears and noses and their role in the transmission of leprosy.

Both ears from 494 wild nine-banded armadillos (Dasypus novemcinctus) and nose specimens from 224 animals were collected and histopathologically studied. Lepromatous granulomas were present in the ear specimens of ten of 494 animals. There were thorns in the ears of 22.5% of animals, and in 36.6% of the nose specimens. In one armadillo, there was evidence to suggest that Mycobacterium leprae entered the tissue through the thorn pricks. In the normal habitat of the armadillo in Louisiana there are thorny bushes consisting mostly of the green briar and the southern dewberry. Thorn pricks as a means of transmission of leprosy in the wild armadillos is suggested.

Naturally acquired leprosy in the nine-banded armadillo: a decade of experience 1975-1985.

A decade has passed since our first report of naturally acquired leprosy in the nine-banded armadillo. Our studies and those of others during this period confirm the identification of the etiologic agent as Mycobacterium leprae. Confirmation is based on the results of histopathologic examination and microbiologic evaluations that included attempts to culture the organism, flourescent antibody studies, mycolic acid analysis, and DNA determinations demonstrating complete relatedness between the natural agent and M. leprae. Surveys involving large numbers of animals demonstrate a significant prevalence of the disease in armadillos captured in Louisiana and Texas. The discovery of naturally acquired leprosy in a chimpanzee in 1977 and a sooty mangabey monkey in 1979 reinforce the concept of leprosy as a zoonosis. Extensive contact with armadillos has been implicated by other observers in seven patients with leprosy in Texas. We believe the prevalence of leprosy in wild armadillos requires that they be considered a source of infection in patients from geographic areas where leprosy and armadillos co-exist.

A cooperative taxonomic study of mycobacteria isolated from armadillos infected with Mycobacterium leprae.

Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.