Multi-Locus Sequencing Reveals Putative Novel Anaplasmataceae Agents, 'Candidatus Ehrlichia dumleri' and Anaplasma sp., in Ring-Tailed Coatis (Carnivora: Nasua nasua) from Urban Forested Fragments at Midwestern Brazil.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati's blood samples in Midwestern Brazil. Twenty-five samples (25/165-15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with 'Candidatus Anaplasma brasiliensis', respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely 'Candidatus Ehrlichia dumleri', and Anaplasma sp. closely related to the previously described 'Candidatus Anaplasma brasiliensis'.

Evaluation of HBV-Like Circulation in Wild and Farm Animals from Brazil and Uruguay.

The origin of the hepatitis B virus is a subject of wide deliberation among researchers. As a result, increasing academic interest has focused on the spread of the virus in different animal species. However, the sources of viral infection for many of these animals are unknown since transmission may occur from animal to animal, human to human, animal to human, and human to animal. The aim of this study was to evaluate hepadnavirus circulation in wild and farm animals (including animals raised under wild or free conditions) from different sites in Brazil and Uruguay using serological and molecular tools. A total of 487 domestic wild and farm animals were screened for hepatitis B virus (HBV) serological markers and tested via quantitative and qualitative polymerase chain reaction (PCR) to detect viral DNA. We report evidence of HBsAg (surface antigen of HBV) and total anti-HBc (HBV core antigen) markers as well as low-copy hepadnavirus DNA among domestic and wild animals. According to our results, which were confirmed by partial genome sequencing, as the proximity between humans and animals increases, the potential for pathogen dispersal also increases. A wider knowledge and understanding of reverse zoonoses should be sought for an effective One Health response.

Assessment of equine piroplasmids in the Nhecolândia sub-region of Brazilian Pantanal wetland using serological, parasitological, molecular, and hematological approaches.

Brazilian Pantanal is the world´s largest wetland ecosystem, where cattle's ranching is the most important economic activity. The objective of this study was to compile some epidemiological features on equine piroplasmids from the Nhecolândia sub-region of Pantanal wetland through the evaluation of the patterns of T. equi and B. caballi infections in different groups of horses; identification of the tick species that infest horses; and to study phylogenetic relationships among Theileria equi 18S rRNA gene sequences. During October 2015, blood and serum samples were collected from 170 horses in four different categories. Ticks, after identification, had their hemolymph and eggs examined for the presence of piroplasmid sporokinets. Also we searched parasites in the peripheral blood smears of the investigated horses. The number of red blood cells (RBCs) and the packed cell volume (PCV) ​​were determined to test for anemia in the infected animals, and exposure to B. caballi and T. equi was evaluated by enzyme-linked immunosorbent assay. "Catch all primers" based on 18S rRNA gene were used in polymerase chain reactions (PCR) to detect equine piroplasmids, followed by three nested PCRs for the phylogenetic analysis. The serological results showed that 61.8% and 52.9% of the horses sampled were exposed to T. equi and B. caballi, respectively. Piroplasmid DNA was detected in 43.5% of the horses analyzed. Our sequencing revealed 98-100% identity with some sequences previously published in GenBank for T. equi, and microheterogeneity among others. We found that 51.2% of the animals sampled were infested with Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus (Boophilus) microplus, singly or co-infested. Since positive and negative animals presented similar RBC and PCV values, and no sporokinets were found on blood smears, hemolymph and eggs of the ticks collected, we suggest that infected equines can act as asymptomatic carriers for piroplasmosis in the studied region. Our results together showed the enzootic characteristic of equine piroplasmids in Pantanal region highlighting the importance of using different methods for detection these parasites. Moreover, breeding mares and foals should be monitored since they displayed the greatest occurrences for molecular test (59.0% and 86.1% respectively) and tick infestations (87.2% and 63.9% respectively).

Health and epidemiological approaches of Trypanosoma evansi and equine infectious anemia virus in naturally infected horses at southern Pantanal.

Equine infectious anemia virus (EIAV) and Trypanossoma evansi are endemic in Brazilian Pantanal Biome, an important area for livestock production. In this sense, we evaluated the epidemiological single and co-infection effects of T. evansi and EIAV in naturally infected horses in the southern Pantanal wetland by serological tests and hematological assays. Both higher seroprevalence and heath poor condition of the sampled animals were associated with differences in horse management between farms. We found that the negative animals for both infectious agents (NN) represented the major group in F1 (37%), and the smallest group in F2 (19%). Furthermore, we recorded higher EIAV seroprevalence (56%) in F2, compared to F1 (38%). We observed that T. evansi infection was mostly related to young horses, as seen by their higher seroprevalence, ranging from 70.7% in the beginning of the rainy season to 81% in the end of flood period, in comparison with the values of 42% and 68%, respectively, in working animals. on the other hand, working animals showed a higher seroprevalence for EIAV (48%) in both seasons than young horses. We observed that the management of working horses could be a risk factor of EIAV infection. On the other hand, as T. evansi is maintained in the study region by many species of wild mammals, the mechanical transmission through blood-sucking vectors ensures the infection to horses since early. Our results showed that single or co-infection by EIAV and T. evansi caused different degree of anemia in the infected animals. Moreover, the health of horses in Brazilian Pantanal is also influenced by differences in horse management and environmental circumstances.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil.

OBJECTIVES: The objective of this study was to develop a quantitative 5' nuclease real-time polymerase chain reaction (PCR) assay to diagnose infections caused by Bartonella species. METHODS: Between January and April 2013 whole blood samples were collected by convenience from 151 cats (86 domiciled and 65 stray cats). The feline blood samples were subjected to a novel quantitative 5' nuclease real-time PCR (qPCR) for Bartonella species targeting the nictonamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) and conventional PCR assays targeting intergenic transcribed spacer, ribC, gltA, pap31 and rpoB, followed by sequencing and basic local alignment search tool analysis. RESULTS: The qPCR assay detected as few as 10 copies of plasmid per reaction. Forty-six (54.4% domiciled and 45.6% stray cats) of 151 sampled cats showed positive results in nuoG qPCR for Bartonella species. The absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 1.1 × 10(4) to 1.3 × 10(4). Eighteen (39.1%) of 46 positive samples in the qPCR were also positive in conventional PCR assays. The sequencing confirmed that Bartonella henselae and Bartonella clarridgeiae circulate in cats in midwestern Brazil. CONCLUSIONS AND RELEVANCE: The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species.

Distinct Leishmania species infecting wild caviomorph rodents (Rodentia: Hystricognathi) from Brazil.

BACKGROUND: Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. METHODOLOGY: We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. PRINCIPAL FINDINGS: In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. CONCLUSIONS/SIGNIFICANCE: The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the "tip of the iceberg."

Domestic and wild mammals infection by Trypanosoma evansi in a pristine area of the Brazilian Pantanal region.

The objective of this study was to estimate the Trypanosoma evansi infection rate and epizootical status of wild and domestic animals from the Brazilian Pantanal region using a standardized polymerase chain reaction (PCR). We used a simple DNA extraction method based on Chelex resin (BioRad, USA) on blood eluted from filter paper confetti. Primers directed to repetitive nuclear DNA sequences were used in the PCR, and could detect 30 fg of T. evansi DNA. The analytical specificity of the assay was evaluated using T. evansi, T. rangeli, T. cruzi, Leishmania braziliensis, Crithidia fasciculata and Herpetomonas muscarum DNAs as templates and the technique showed the expected 164 bp specific band solely for Trypanozoon trypanosomes. The application of the standardized PCR protocol in 274 field samples from domestic and wild mammals from the Rio Negro (Brazilian Pantanal region), showed a general infection rate of 10.2% while the traditional parasitological technique (direct search of the protozoan by the microematocrit centrifugue technique) was able to determine infection in only 1.1% of the animals. The peccaries and feral pigs were found to be the animals most frequently infected with T. evansi (24.4% and 30.7%, respectively). Both sampling and extraction methods used herein, showed to be simple and efficient to be applied in epidemiological surveys using PCR.