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.

Reactivity against Sarcocystis neurona and Sarcocystis falcatula-like in horses from Southeastern and Midwestern Brazil.

Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona. Immunofluorescence antibody tests (IFATs) have been widely used to identify exposure of horses to S. neurona in Brazil. Here we used IFAT to search for IgG antibodies against Sarcocystis falcatula-like (Dal-CG23) and S. neurona (SN138) in sera from 342 horses sampled in Campo Grande, Mato Grosso do Sul state (Midwestern), and São Paulo, São Paulo state (Southeastern), Brazil. The 1:25 cutoff value was chosen to maximize sensitivity of the test. IgG antibodies against S. neurona were detected in 239 horses (69.88%), whereas IgG antibodies against S. falcatula-like were detected in 177 horses (51.75%). Sera from 132 horses (38.59%) reacted against both isolates. Absence of reactivity was evidenced in 58/342 horses (16.95%). The lower cutoff used, and the presence of opossums infected with S. falcatula-like and Sarcocystis spp. in the regions where the horses were sampled, might justify the high seroprevalence observed here. Owing to the similarity among antigens targeted in immunoassays, reports on S. neurona-seropositive horses in Brazil may also derive from the exposure of horses to other Sarcocystis species. The role of other Sarcocystis species in causing neurological diseases in horses in Brazil remains unclear.

Molecular diversity of Sarcocystis spp. in opossums (Didelphis spp.) from Southeastern and Midwestern Brazil.

South American opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, diverse studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS-1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Four alleles variants were found for SAG2, 13 for SAG3 and seven for SAG4, from which four, 13 and four, respectively, were novel. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Further studies are needed to clarify the role of Didelphis spp. as definitive hosts of Sarcocystis spp. other than that previous described.

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.

Molecular and serological detection of arthropod-borne pathogens in carnivorous birds from Brazil.

Rickettsiales, Haemosporida and Rhizobiales agents can cause diseases that affect various animal species, including humans. Due to predation behaviour, carnivorous birds may play an important role in spreading these etiological agentes across geographically distant areas, specially if they are migratory. The aim of this study was to investigate the occurrence and to access the phylogenetic relations among Anaplasmataceae (Ehrlichia, Anaplasma, Neorickettsia), Bartonellaceae (Bartonella spp.), and Haemosporida (Plasmodium, Haemoproteus and Leucocytozoon) agents in blood samples from 121 carnivorous birds sampled in the states of São Paulo and Rio de Janeiro, Brazil. Inclusions resembling hemoparasites were not observed in Giemsa-stained preparations. While three animals were seropositive for E. chaffeensis (3.41% [3/88]; 95% CI:1.17-9.55%), five showed antibodies to A. phagocytophilum (5.68% [5/88]; 95% CI: 2.45-12.62%). Despite the detection of rrs gene fragments closely related to E. chaffeensis (4.13% [5/121]; 95% CI: 1.78-9.31%), no positivity was observed in the qPCR based on the genes vlpt for the organism. Similarly, 12 (9.91% [12/121]; 95% CI: 5.76-16.74%) samples were positive in the qPCR for Anaplasma spp. based on groEL gene, but negative in the qPCR for A. phagocytophilum based on msp-2 gene. Three samples were positive in the nPCR for E. canis based on rrs gene. Three samples were positive for Haemoproteus spp. and one for Plasmodium spp. in the nPCR based on cytB gene. Four birds (3.3% [4/121]; 95% CI: 1.29-8.19%) presented co-positivity by Ehrlichia sp. and Anaplasma sp. in molecular assays. One (0.82% [1/121]; 95% CI:0.15-4.53%) bird showed to be seropositive for E. chaffeensis and and positive in PCR for Haemoproteus sp. All birds were negative in the qPCR assay for Bartonella spp. (nuoG). The present work showed the occurrence of Anaplasmataceae agents and hemosporidians in carnivorous birds from southeastern Brazil. The role of these animals in the dispersion of Anaplasmataceae agents should be further investigated.

Helminthfauna of road-killed cougars (Puma concolor) from the Northeastern Region of São Paulo State, Brazil.

As the second-largest Neotropical carnivore, cougars (Puma concolor) are especially important for maintenance of the biodiversity and ecosystem health. Five wild adult cougars (Puma concolor), found roadkilled in highways in the Northeastern region of São Paulo, were evaluated in search for parasites. Ten species representing nine families were identified. The most prevalent helminths were Uncinaria bidens, Lagochilascaris major, Spirometra sp., and Oncicola canis, followed by Cylicospirura subaequalis, Toxascaris leonina, Taenia omissa, Echinococcus sp., Filaroides sp. and Oncicola oncicola. It is important to note that some helminths found in this study, such as L. major, Spirometra sp., O. oncicola, O. canis, Echinococcus sp., T. leonina, C. subaequalis, and Filaroides sp. are known to affect domestic carnivores, which may indicate interaction between wild and domestic hosts. This study represents a new host record for four of the species found in cougars, U. bidens, L. major, O. canis, and Filaroides sp., and new locality records for U. bidens, T. leonina, C. subaequalis, and Filaroides sp.

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.

Necrotic Enteritis Caused by Clostridium perfringens in Blue and Gold Macaws (Ara ararauna).

Clostridium perfringens types A and C, which are gram-positive, anaerobic, spore-forming bacteria, can cause necrotic enteritis in birds. Although Clostridium perfringens is considered a commensal organism in the avian intestinal tract, in association with severe stress, other infectious agents, or immunosuppressive conditions, it can cause disease outbreaks. This report describes a disease occurrence of necrotic enteritis caused by C perfringens in macaws (Ara ararauna). Two adult male blue and gold macaws maintained in a zoo exhibit were presented for postmortem examinations after histories of sudden death. Based on the gross examinations and microscopic evaluation of submitted tissue from both birds, the cause of death was determined to be necrotic enteritis. Microbiologic assays followed by polymerase chain reaction analyses identified the isolated strains as C perfringens type A, indicated by only being positive for the cpa gene that encodes the α-toxin. The birds were maintained in an exhibit in which patrons can interact with the animals within their environment. Thus, organisms, such as this pathogen, may present a danger for other birds because visitors could disperse the bacterium to other parts of the zoo.

Renal trematode infection in wild birds: histopathological, morphological, and molecular aspects.

Paratanaisia are eucotylidae digeneans that affect the upper urinary tract of birds. This genus contains three species (Paratanaisia bragai, P. robusta, and P. confusa) with similar morphological features. Macroscopic and microscopic damage caused by these parasites ranges from the irrelevant to significant lesions. This study aimed to describe the histological, morphological, and molecular features of the renal tissues and parasite specimens obtained from naturally infected free-ranging and captive wild birds in Brazil. Histopathological evaluations were performed on 103 slides containing kidney tissue sections from parasitized birds. Parasites were observed inside the collecting ducts, causing the dilation and destruction of the lining epithelial cells and alterations in other structures of the renal parenchyma. Such findings indicate that Paratanaisia have pathogenic potential in a wide range of hosts, suggesting low host specificity. The parasites recovered from the kidneys of 10 birds, including Columbiformes, Galliformes, Strigiformes, and Cuculiformes, were morphologically evaluated and identified as Paratanaisia sp. Formalin-fixed paraffin-embedded kidney fragments were subjected to conventional PCR assays targeting the 18S and 28S rDNA genes. A Bayesian inference analysis based on an 800-bp 18S rDNA gene fragment separated the trematode genus accurately, clustering all of the parasites tested with a previously described P. bragai specimen. Analyses on a small fragment of the 28S rDNA gene did not allow for accurately differentiating the Paratanaisia species. Therefore, further morphological studies with additional molecular markers are necessary to improve our understanding of the alpha-taxonomy of this group.

Occurrence of Paratanaisia spp. Freitas, 1951 in a domiciled cockatiel (Nymphicus hollandicus, Psittaciformes: Cacatuidae)

Abstract Digenetic eucotylids of the Paratanaisia genus are widely reported parasites of the avian renal system. The infection, commonly reported in free-living and zoo-housed birds, is described for the first time in a domiciled bird, a cockatiel kept as pet bird with free access to the yard outdoors. The bird was received at Veterinary Hospital, where clinical and radiographic evaluations suggested a case of heavy-metal poisoning. Although the bird received supportive care and a chelating agent, it died the next day. The necropsy showed friable kidneys and congestion of blood vessels in structures such as the skin, proventriculus, brain, and skullcap. The histopathological evaluation of the kidneys revealed multifocal hemorrhages, commonly found in cases of heavy-metal poisoning. Parasitic structures similar to those of the digenean trematodes Paratanaisia spp. were also observed inside dilated collecting ducts, which presented epithelial cell flattening and vacuolization. There was compression of adjacent tissue and discrete fibrotic areas. In the presence of intermediate hosts in the yard, the synanthropic nature of some wild free-living birds could make them a source of trematodes infection and dissemination for pet birds. Conversely, the infected cockatiel could also have served as a reservoir and carrier of trematodes to wild free-living birds.

Resumo Eucotilídeos digenéticos do gênero Paratanaisia são amplamente descritos como parasitas do trato urinário de aves. A infecção, comumente relatada em aves de vida livre e em zoológicos, é descrita pela primeira vez em uma ave domiciliada, uma calopsita mantida como ave de companhia com livre acesso ao jardim externo. A ave foi recebida no Hospital Veterinário onde avaliações clínicas e radiográficas sugeriram um quadro de intoxicação por metal pesado. Embora tenha recebido tratamento de suporte e agente quelante a ave veio a óbito no dia seguinte. A necropsia demonstrou rins de consistência friável e congestão dos vasos sanguíneos da pele, proventrículo, cérebro e calota craniana. A avaliação histopatológica dos rins revelou hemorragias multifocais comumente observadas em intoxicação por metais pesados. Estruturas parasitárias similares àquelas observadas em Paratanaisia spp. foram observadas no interior de ductos coletores dilatados, os quais apresentavam achatamento e vacuolização de células epiteliais. Havia compressão do tecido adjacente e discretas áreas de fibrose. Na presença de hospedeiros intermediários no jardim, a condição sinantrópica de algumas aves de vida livre poderia torná-las fonte de infecção e disseminação de trematódeos para aves domiciliadas. Em contrapartida, a calopsita infectada poderia também servir como reservatório e carreador de trematódeos para aves de vida livre.