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.

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.

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.

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.

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.

Evaluation of the origin of Mycobacterium leprae infections in the wild armadillo, Dasypus novemcinctus.

An enzyme-linked immunosorbent assay (ELISA) using the phenolic glycolipid-1 (PGL-1) antigen of Mycobacterium leprae and cross-reactive antisera specific for human IgM was developed to detect IgM antibodies to M. leprae in the nine-banded armadillo (Dasypus novemcinctus). Statistical definitions for positive and negative interpretations in the ELISA were developed by screening animals recently captured and experimentally inoculated with M. leprae. The ELISA was shown to have high sensitivity and specificity. Modern day armadillos of central Louisiana were observed to have a PGL-1 antibody prevalence rate as high as 20%, and a clinical disease rate as high as 5%. A retrospective serological survey of 182 armadillos taken in the years 1960-1964 and predating the use of armadillos in leprosy research was used to evaluate the 1968 environmental contamination hypothesis for the origin of M. leprae infections in the wild armadillo. Antibodies to the apparently species-specific PGL-1 antigen were detected in 17 of the samples taken in 1960-1964. Absorption with whole M. leprae, M. intracellulare, M. terrae, M. rhodesiae, M. scrofulaceum, M. diernhoferi, M. kansasii, M. phlei, M. avium, BCG, and 2 new armadillo-derived mycobacterial species showed these antibody reactions to be specific for PGL-1. Apparently, M. leprae was enzootic in armadillos as early as 1961, and original infection of these animals could not have occurred in 1968.

Panamanian forest mammals as carriers of Salmonella.

Enteric bacteria pathogenic to man were sought in a total of 974 forest mammals collected from a variety of sites in rural and jungle areas of Panamá. The highest incidence of infection among the mammals was observed during the Panamanian dry season, which normally extends from January through April. A minimum of 10 Salmonella serotypes including, three of the Arizona group and Ewardsiella tarda, was isolated. Opossums of the genera Philander, 11 of 54 (20.1%), and Didelphis, 12 of 102 (11.8%) demonstrated high infection rates. One sloth of the genus Choloepus and specimens of two genera of rodents also were infected to varying degrees: 1(11.1%) of 9 Choloepus, 8 (1.1%) of 704 Proechimys and 1 (16.7%) of 6 Diplomys.

Experimental St. Louis encephalitis virus infection of sloths and cormorants.

Experimental infection of 11 Bradypus variegatus and Choloepus hoffmanni sloths with St. Louis encephalitis (SLE) virus produced detectable viremias of seven to 27 (median 13) days duration and maximum titers of 2.7 to 6.5 (median 5.1) log10 median suckling mouse intracranial lethal doses (SMicLD50) per ml. Experimental SLE viremia onset was delayed and maximum titer depressed in two sloths concurrently infected with naturally acquired viruses. SLE viremias in four experimentally inoculated cormorants Phalacrocorax olivaceus were shorter, and of equal or lower titer, than in sloths. Colonized Culex pipiens quinquefasciatus mosquitoes were infected by feeding on sloths circulating at least 4.8 log10 SMicLD50 of SLE virus per ml, and subsequently transmitted the infection to mice and chicks. An uninoculated baby Bradypus became infected by contact transmission from its mother. The antibody response of sloths to SLE virus was slow, being undetectable until several weeks post-inoculation. However, both sloth species developed high and long-lasting neutralizing and hemagglutination-inhibition antibody titers. The complement-fixation antibody response in Bradypus was lower and slower to develop than in Choloepus. Sloths with naturally acquired SLE virus antibody did not become detectably viremic after experimental inoculation. Neither sloths nor cormorants become overly ill from SLE virus infection.

Viruses isolated from Panamanian sloths.

Seven virus strains were isolated in Vero cells from whole blood samples from 80 wild-caught sloths, Bradypus variegatus and Choloepus hoffmanni, from Central Panamá. Four strains of at least two different serotypes are related to Changuinola virus; two of these were associated with prolonged or recrudescent viremias. One strain is an antigenic subtype of Punta Toro virus, and another, described here as Bradypus-4 virus, is a new, antigenically ungrouped virus. A second new virus from sloths, Utive virus, forms an antigenic complex within the Simbu serogroup with Utinga and Pintupo viruses. Tests on sequential plasma samples from radio-marked free-ranging sloths and from recently captured animals maintained in captivity showed that both species develop neutralizing antibodies following naturally acquired virus infections. Antibodies against the Changuinola and Simbu serogroup viruses are widespread in both sloth species and are especially prevalent in Choloepus, but are virtually absent in all other wild vertebrate species tested.

Pneumocystis and Histoplasma infections in wild animals from the Amazon region of Brazil.

Routine examination of tissues from wild forest rodents from Amapá, north Brazil, revealed Pneumocystis carinii in lung smears from a newly captured Oryzomys capito (Cricetidae). Acute, fatal infections with this parasite are also recorded in a number of captive "coatimundis", Nasua narica (Carnivora: Procyonidae) and a sloth, Bradypus tridactylus (Edentata). Pneumocystis was also encountered in lung smears from a newly captured and apparently healthy sloth, Choloepus didactylus. The presence of infection in newly captured animals leads us to believe that the fatal, fulminating pneumocystosis seen in the captive Nasua and Bradypus was due to exacerbation of pre-existant infections acquired in their natural forest environment. Pneumocystis carinii is a well known cause of fatal, interstitial plasma-cell pneumonia in human infants and sometimes the weakened adult: the keeping of exotic pets such as the coatimundi is, therefore, not without some hazard in this respect. Histoplasma, another well known pathogen for man, was isolated from 4 rodents, Proechimys guyanensis (Echimyidae), all from virgin forest along the newly opened Trans Amazon Highway, Pará State, and from a single sloth, Choloepus didactylus, from near Belém, Pará. All these animals showed no symptoms of infection: isolation of the parasite was made by the inoculation of laboratory hamsters with saline suspensions of triturated liver and spleen.

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.

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).

Presence of "difficult to isolate" mycobacteria in armadillos.

Contrary to the findings with armadillos from Louisiana and Texas, armadillos from Florida are free of natural leprosy-like infection. Examination of ear clip, nasal, blood buffy coat, liver and spleen of inoculated armadillos from Florida did not reveal the presence of any acid fast bacteria. However, using massive inocula, 6 out of 77 armadillo tissues were found to contain very negligible proportions of cultivable mycobacteria. The significance of these isolates in relation to M. leprae and also to leprosy research is discussed.

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