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Picobirnavirus (PBV) is a family of non-enveloped double-stranded RNA viruses with bisegmented genomes. Segment 1 encodes the capsid protein and segment 2 encodes RNA-dependent RNA polymerase. They exhibit high genomic heterogeneity and infect a wide range of vertebrate hosts, including humans. The objective of this study was to expand our knowledge of the circulation of PBV in free-living animals from two regions (Brazil and Argentina) of the Atlantic Forest. Fecal samples were analyzed from free-living animals: tapir, brocket deer, peccary, and different species of rodents and marsupials. A total of 133 samples were collected and analyzed by RT-PCR, of which 44 (33.08%) were PBV-positive. Nine amplicons were sequenced, five species from Argentina and four from Brazil, and phylogenetic analysis was performed. The nucleotide and amino acid identities of the PBV strains detected in animals from Argentina and Brazil were between 66.3% and 82.5% and between 55.3% and 74.2%, respectively. The analysed strains presented conserved nucleotide blocks without distinction of the host species. The phylogenetic tree showed that PBV strains from Atlantic Forest animals belonging to genogroup I were grouped into different clusters, without defining groups according to host species (human or animal) or the geographical area of detection. This is the first study on PBV in free-living animals in the Atlantic Forest. Our analysis suggested that PBV strains can infect different animal species, leading to PBV transmission between animals and humans. This reinforces the hypothesis of previous crossover points in the ecology and evolution of heterologous PBV strains.

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Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45-90% and PG 35-70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45-90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35-70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45-90% was used for fertilization. PG 45-90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2-26.3%) and morula (8.1-10.5%) rates did not differ between the groups. Therefore, PG 45-90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

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Hepatitis E virus (HEV) genotype 3 is the most prevalent HEV genotype in Europe causing mostly asymptomatic infections in humans, but can also sporadically cause severe acute hepatitis, chronic liver disease, chronic hepatitis in immunocompromised patients and extra-hepatic manifestations. Although much is today known about the swine reservoir, no information is available on the occurrence of HEV from widely distributed deer species in Portugal. Here, we investigated the presence and characterized HEV in free-living deer in Portugal by screening stools from red deer (Cervus elaphus) (n = 95) and fallow deer (Dama dama) (n = 35) for HEV by a broad-spectrum nested RT-PCR, followed by sequencing and phylogenetic analysis. Two red deer females, sampled in central Portugal, showed to be shedding HEV (2.1%; 95% confidence interval: 0.58-7.35). Sequencing and genetic characterization showed that these two deer HEV sequences were 98.96% identical to each other, being both of HEV genotype 3 subgenotype 3e. The increasing numbers and distribution of deer in Portugal and the zoonotic features of the circulating HEV genotype 3 subgenotype 3e highlights the importance of continued surveillance directed to food-borne diseases, especially those involving wild animals and deer in particular.

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Sarcocystis spp. are intracellular protozoan parasites with heteroxenous life cycles. This study described Sarcocystis spp. infection in adult South American native deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda). Heart, diaphragm, tongue, and skeletal muscle samples were collected from 5 huemuls and 2 pudus, found dead in National Parks. Direct microscopic examination, transmission electron microscopy, PCR, and sequencing were performed. Sarcocystis spp. microscopic thin-walled cysts were identified in 3 huemuls and 1 pudu. Several cysts from 1 huemul and 1 pudu were observed by TEM; ultrastructure was similar to previously reported as cyst wall type 17 and types 2 and 8, respectively. Fragments of the 18S rRNA and cytochrome c oxidase subunit I (cox1) genes were amplified and sequenced from 3 individual cysts from 2 huemuls and 2 cysts from the pudu. The sequences from huemuls showed a high identity among them (> 99%) at both amplified targets. The highest identities were > 99.7% at 18S rRNA and 93% at cox1 with S. tarandivulpes sequences. The 18S rRNA gene sequences from pudus showed an identity > 99.7% with Sarcocystis sp., S. taeniata, and S. linearis sequences, while the cox1 sequences were different, one showing 99.42% identity with S. venatoria and the other 98.22% with S. linearis. A single species, similar to S. tarandivulpes, was identified in all huemul samples while 2 molecularly different Sarcocystis spp. were found in 1 pudu with high similarities to either S. venatoria or to S. linearis, S. taeniata-like, and S. morae. Based on the cox1 sequence identities, at least the Sarcocystis sp. in huemuls might represent a new species, primarily occurring in this host. Additional sarcocyst isolates from both hosts need to be examined molecularly in order to firmly establish whether these species are indeed native to huemuls and/or pudus or are derived from introduced deer species.

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Reported cases of neoplasia affecting hippopotamus are very scarce. This report describes a metastatic oral squamous cell carcinoma in a captive 44-year-old male common hippopotamus (Hippopotamus amphibius) that developed progressive loss of appetite, fibrinohemorrhagic oral discharge, and progressive weight loss. Gross, cytological, and histopathological findings were compatible with a conclusive diagnosis of an invasive oral squamous cell carcinoma with metastasis in the lung, adrenal, and spleen. To the best of our knowledge this is the first report of squamous cell carcinoma in a common hippopotamus.

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Reported cases of neoplasia affecting hippopotamus are very scarce. This report describes a metastatic oral squamous cell carcinoma in a captive 44-year-old male common hippopotamus (Hippopotamus amphibius) that developed progressive loss of appetite, fibrinohemorrhagic oral discharge, and progressive weight loss. Gross, cytological, and histopathological findings were compatible with a conclusive diagnosis of an invasive oral squamous cell carcinoma with metastasis in the lung, adrenal, and spleen. To the best of our knowledge this is the first report of squamous cell carcinoma in a common hippopotamus.(AU)

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