Comparative Analysis of How the Fecal Microbiota of Green-Winged Saltator (Saltator similis) Diverge among Animals Living in Captivity and in Wild Habitats.

The microbiota's alteration is an adaptive mechanism observed in wild animals facing high selection pressure, especially in captive environments. The objective of this study is to compare and predict the potential impact of habitat on the fecal bacterial community of Saltator similis, a songbird species that is a victim of illegal trafficking, living in two distinct habitats: wild and captivity. Nine wild and nine captive S. similis were sampled, and total bacterial DNA was obtained from the feces. Each DNA sample was employed to the amplification of the V4 region of the 16S rDNA following high-throughput sequencing. The most predominant phyla in all songbirds, irrespective of habitat, were Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteriota. Interestingly, a microbiota profile (phylogenetic and abundance relationship) related to habitat was identified. The genera "Candidatus Arthromitus", Acinetobacter, Kocuria, and Paracoccus were exclusively identified in animals living in captivity, which can be a potential biomarker associated with birds in captive environments. This study presents the first description of the fecal bacterial community composition of S. similis living two different lifestyles. Finally, our results suggest that the lifestyle of S. similis birds significantly impacts the composition of the fecal microbiota. The animals living in captivity showed dysbiosis in the microbiota, with some bacteria genera being indicated as biological markers of environmental behavior. Thus, the present research provides a new concept of life quality measure for songbirds.

New candidate genes potentially involved in Zika virus teratogenesis.

Despite efforts to elucidate Zika virus (ZIKV) teratogenesis, still several issues remain unresolved, particularly on the molecular mechanisms behind the pathogenesis of Congenital Zika Syndrome (CZS). To answer this question, we used bioinformatics tools, animal experiments and human gene expression analysis to investigate genes related to brain development potentially involved in CZS. Searches in databases for genes related to brain development and CZS were performed, and a protein interaction network was created. The expression of these genes was analyzed in a CZS animal model and secondary gene expression analysis (DGE) was performed in human cells exposed to ZIKV. A total of 2610 genes were identified in the databases, of which 1013 were connected. By applying centrality statistics of the global network, 36 candidate genes were identified, which, after selection resulted in nine genes. Gene expression analysis revealed distinctive expression patterns for PRKDC, PCNA, ATM, SMC3 as well as for FGF8 and SHH in the CZS model. Furthermore, DGE analysis altered expression of ATM, PRKDC, PCNA. In conclusion, systems biology are helpful tools to identify candidate genes to be validated in vitro and in vivo. PRKDC, PCNA, ATM, SMC3, FGF8 and SHH have altered expression in ZIKV-induced brain malformations.

Neuroinflammation, blood-brain barrier dysfunction, hippocampal atrophy and delayed neurodevelopment: Contributions for a rat model of congenital Zika syndrome.

The congenital Zika syndrome (CZS) has been characterized as a set of several brain changes, such as reduced brain volume and subcortical calcifications, in addition to cognitive deficits. Microcephaly is one of the possible complications found in newborns exposed to Zika virus (ZIKV) during pregnancy, although it is an impacting clinical sign. This study aimed to investigate the consequences of a model of congenital ZIKV infection by evaluating the histopathology, blood-brain barrier, and neuroinflammation in pup rats 24 h after birth, and neurodevelopment of the offspring. Pregnant rats were inoculated subcutaneously with ZIKV-BR at the dose 1 × 107 plaque-forming unit (PFU mL-1) of ZIKV isolated in Brazil (ZIKV-BR) on gestational day 18 (G18). A set of pups, 24 h after birth, was euthanized. The brain was collected and later evaluated for the histopathology of brain structures through histological analysis. Additionally, analyses of the blood-brain barrier were conducted using western blotting, and neuroinflammation was assessed using ELISA. Another set of animals was evaluated on postnatal days 3, 6, 9, and 12 for neurodevelopment by observing the developmental milestones. Our results revealed hippocampal atrophy in ZIKV animals, in addition to changes in the blood-brain barrier structure and pro-inflammatory cytokines expression increase. Regarding neurodevelopment, a delay in important reflexes during the neonatal period in ZIKV animals was observed. These findings advance the understanding of the pathophysiology of CZS and contribute to enhancing the rat model of CZS.

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.

Weight Gain, Lifestyle, and Cognition During the COVID-19 Pandemic in Southern Brazil.

BACKGROUND: During the COVID-19 pandemic, the world experienced social distancing that resulted in changes in habits and lifestyle. Such changes can compromise healthy eating habits and the practice of physical activities, known risk factors for developing weight gain and obesity. OBJECTIVE: The main objective of this study was to describe the change in eating habits, lifestyle, and cognition of the population of Rio Grande do Sul, a state in Southern Brazil, during social distancing due to COVID-19. METHODS: The study was conducted from July 21 to August 10, 2020, through a structured online questionnaire that asked for sociodemographic information (age, gender, and education), anthropometric (reported weight and height), change in eating habits, lifestyle (sleep quality and physical activity), and cognition. Chi-square, McNemar tests, and univariate and multivariate analysis were used to evaluate the variables. Confidence intervals were calculated with a significance level of 5%. RESULTS: Of a total of 1072 participants, 57.3% of respondents reported weight gain, and an increased percentage of people were classified as obese. Nearly half of the participants (46%) reported changes in their eating habits for the worse. Body mass index (BMI) was significantly associated with increased consumption of unhealthy foods. Our results identified high physical inactivity (46.9%) and obesity (19%) during social distancing. The changes in eating habits and lifestyle also increased the risk for decreased cognition. CONCLUSIONS: These findings highlighted that social distancing impacted eating habits and lifestyle, which increased obesity rates and might predispose to decreased cognition.

Zika Virus affects neurobehavioral development, and causes oxidative stress associated to blood-brain barrier disruption in a rat model of congenital infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with several neurodevelopmental outcomes after in utero infection. Here, we studied a congenital ZIKV infection model with immunocompetent Wistar rats, able to predict disabilities and that could pave the way for proposing new effective therapies. We identified neurodevelopmental milestones disabilities in congenital ZIKV animals. Also, on 22nd postnatal day (PND), blood-brain barrier (BBB) proteins disturbances were detected in the hippocampus with immunocontent reduction of ß_Catenin, Occludin and Conexin-43. Besides, oxidative stress imbalance on hippocampus and cortex were identified, without neuronal reduction in these structures. In conclusion, even without pups' microcephaly-like phenotype, congenital ZIKV infection resulted in neurobehavioral dysfunction associated with BBB and oxidative stress disturbances in young rats. Therefore, our findings highlighted the multiple impact of the congenital ZIKV infection on the neurodevelopment, which reinforces the continuity of studies to understand the spectrum of this impairment and to provide support to future treatment development for patients affected by congenital ZIKV.

Neutralizing antibodies to bovine and bubaline alphaherpesviruses in water buffaloes (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) have been introduced in many regions of the world as a source of animal protein. In many instances, bubaline cattle are reared close to or mixed with bovine or zebuine cattle. However, little is known about infectious diseases of bubaline and the interactions that may arise involving the microbiota of those species. Alphaherpesviruses of ruminants (bovine alphaherpesviruses types 1 and 5, BoHV-1, BoHV-5; bubaline alphaherpesvirus 1, BuHV-1) are highly cross-reactive in serological assays performed with bovine or zebuine sera. However, the profile of reactivity of bubaline cattle sera to alphaherpesviruses remains unknown. As such, it is not known which virus strain (or strains) would be most appropriate to be used as the challenge virus in the laboratory in search for alphaherpesvirus-neutralizing antibodies. In this study, the profile of neutralizing antibodies to alphaherpesviruses in bubaline sera was determined against different types/subtypes of bovine and bubaline alphaherpesviruses. Sera (n=339) were screened in a 24-h serum neutralization test (SN) against 100 TCID50 of each of the challenge viruses. From those, 159 (46.9 %) neutralized at least one of the viruses assayed; 131 (38.6%) sera neutralized the three viral strains used for screening. The viral strain that was neutralized by the largest number of sera was BoHV-5b A663 (149/159; 93.7%). A few sera neutralized only one of the challenge viruses: four sera neutralized BoHV-1 LA only; another neutralized BoHV-5 A663 only and four others neutralized BuHV-1 b6 only. SN testing with two additional strains gave rise to similar results, where maximum sensitivity (defined here as the largest number of sera that neutralized the challenge viruses) was obtained by adding positive results attained with three of the challenge strains. Differences in neutralizing antibody titers were not significant to allow inferences on which would be the most likely virus that induced the antibody responses detected here.

An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera.

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Genomic evidence of SARS-CoV-2 reinfection cases in southern Brazil.

Cases of reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported worldwide. We investigated reinfection cases in a set of more than 30,000 samples, and the SARS-CoV-2 genomes from selected samples from four patients with at least two positive diagnoses with an interval ≥ 45 days between tests were sequenced and analyzed. Comparative genomic and phylogenetic analysis confirmed three reinfection cases and suggested that the fourth one was caused by a virus of the same lineage. Viral sequencing is crucial for understanding the natural course of reinfections and for planning public health strategies for management of COVID-19.

Characterization of novel of temperate phages of Staphylococcus aureus isolated from bovine milk.

Bovine mastitis is an important disease in dairy cows, and Staphylococcus aureus is the most prevalent microorganism. Bacteriophages are considered an alternative to treat bacterial infections due to antimicrobial resistance crisis. In this study, we isolated and characterized novel S. aureus temperate phages, namely B_UFSM4 and B_UFSM5, from bovine milk. The complete genomes of B_UFSM4 and B_UFSM5 have 41.396 bp and 41.829 bp, respectively. The viruses have double-stranded DNA and linear architecture. Phylogenic similarity was observed by proteome with Staphylococcus phage phiPV83, CN125 and JS01. Therefore, the phages were classified into the family Siphoviridae, genus Biseptimavirus and order Caudovirales. In the host range, the B_UFSM4 and B_UFSM5 had lytic activity of 45.8% and 54.16%, respectively, inclusive on isolates from Staphylococcus sciuri and Rothia terrae. Thus, in this study, species novel of S. aureus temperate phages was isolated and characterized, these phages reveal similarities to each other; however, they are distinct from other species of S. aureus phages of the family Siphoviridae.

Molecular survey of porcine respiratory disease complex pathogens in Brazilian wild boars.

Wild boar (Sus scrofa) is an exotic invasive species in Brazil and may be a reservoir for several pathogens, including those related to the porcine respiratory disease complex (PRDC), a critical infectious disease in pig production. The objective of this study was to investigate viral and bacterial pathogens related to PRDC in free-living wild boars from Brazil. Eighty animals were examined in search of genomes of porcine circovirus 2 (PCV2), Torque teno Sus virus 1a (TTSuV1a) and 1b (TTSuV1b), Influenza A virus (IAV), Actinobacillus pleuropneumoniae, Glaesserella parasuis, Pasteurella multocida, and Mycoplasma hyopneumoniae. The results demonstrated that 57.5% (46/80) of the animals had at least one detected pathogen, and 11.3% of them (9/80) were co-infected. TTSuV1a was the most prevalent genome, for which risk factors were associated with increased contact between wild boars and other animals. The other pathogens were detected at much lower frequencies or not detected (M. hyopneumoniae and IAV). An additional IAV serology search identified H1N1pdm09 antibodies in 35.5% (16/45) of the wild boars, bringing concern related to public health. In conclusion, wild boars are infected with pathogens that cause swine diseases, so their eventual contact with domestic pigs might risk animal production in Brazil.

The virome of the white-winged vampire bat Diaemus youngi is rich in circular DNA viruses.

In the Neotropical region, the white-winged vampire bat (Diaemus youngi) is the rarest of the three species of vampire bats. This bat species feeds preferentially on bird blood, and there is limited information on the viruses infecting D. youngi. Hence, this study aimed to expand the knowledge about the viral diversity associated with D. youngi by sampling and pooling the lungs, liver, kidneys, heart, and intestines of all animals using high-throughput sequencing (HTS) on the Illumina MiSeq platform. A total of three complete and 10 nearly complete circular virus genomes were closely related to gemykrogvirus (Genomoviridae family), smacovirus (Smacoviridae family), and torque teno viruses (TTVs) (Anelloviridae family). In addition, three sequences of bat paramyxovirus were detected and found to be closely related to viruses reported in Pomona roundleaf bats and rodents. The present study provides a snapshot of the viral diversity associated with white-winged vampire bats and provides a baseline for comparison to viruses detected in future outbreaks.

Genomic analysis of Enterococcus durans LAB18S, a potential probiotic strain isolated from cheese.

Gut microbiota exerts a fundamental role in human health and increased evidence supports the beneficial role of probiotic microorganisms in the maintenance of intestinal health. Enterococcus durans LAB18S was previously isolated from soft cheese and showed some desirable in vitro probiotic properties, for that reason its genome was sequenced and evaluated for genes that can be relevant for probiotic activity and are involved in selenium metabolism. Genome sequencing was performed using the Illumina MiSeq System. A variety of genes potentially associated with probiotic properties, including adhesion capability, viability at low pH, bile salt resistance, antimicrobial activity, and utilization of prebiotic fructooligosaccharides (FOS) were identified. The strain showed tolerance to acid pH and bile salts, exhibited antimicrobial activity and thrived on prebiotic oligosaccharides. Six genes involved in selenium metabolism were predicted. Analysis of the SECIS element showed twelve known selenoprotein candidates. E. durans LAB18S was the only food isolate showing absence of plasmids, virulence and antimicrobial resistance genes, when compared with other 30 E. durans genomes. The results of this study provide evidence supporting the potential of E. durans LAB18S as alternative for probiotic formulations.

Genomic characterization and production of antimicrobial lipopeptides by Bacillus velezensis P45 growing on feather by-products.

AIMS: To investigate the potential of novel Bacillus velezensis P45 as an eco-friendly alternative for bioprocessing poultry by-products into valuable antimicrobial products. METHODS AND RESULTS: The complete genome of B. velezensis P45 was sequenced using the Illumina MiSeq platform, showing 4455 protein and 98 RNA coding sequences according to the annotation on the RAST server. Moreover, the genome contains eight gene clusters for the production of antimicrobial secondary metabolites and 25 putative protease-related genes, which can be related to feather-degrading activity. Then, in vitro tests were performed to determine the production of antimicrobial compounds using feather, feather meal and brain-heart infusion (BHI) cultures. Antimicrobial activity was observed in feather meal and BHI media, reaching 800 and 3200 AU ml-1 against Listeria monocytogenes respectively. Mass spectrometry analysis indicates the production of antimicrobial lipopeptides surfactin, fengycin and iturin. CONCLUSIONS: The biotechnological potential of B. velezensis P45 was deciphered through genome analysis and in vitro studies. This strain produced antimicrobial lipopeptides growing on feather meal, a low-cost substrate. SIGNIFICANCE AND IMPACT OF STUDY: The production of antimicrobial peptides by this keratinolytic strain may represent a sustainable alternative for recycling by-products from poultry industry. Furthermore, whole B. velezensis P45 genome sequence was obtained and deposited.

Detection of coronavirus in vampire bats (Desmodus rotundus) in southern Brazil.

The vampire bat (Desmodus rotundus) is a haematophagous animal that feeds exclusively on the blood of domestic mammals. Vampire bat feeding habits enable their contact with mammalian hosts and may enhance zoonotic spillover. Moreover, they may carry several pathogenic organisms, including coronaviruses (CoVs), for which they are important hosts. The human pathogens that cause severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) and possibly coronavirus disease 2019 (SARS-CoV-2) all originated in bats but required bridge hosts to spread into human populations. To monitor the presence of potential zoonotic viruses in bats, the present work evaluated the presence of CoVs in vampire bats from southern Brazil. A total of 101 vampire bats were captured and euthanized between 2017 and 2019 in Rio Grande do Sul state, southern Brazil. The brain, heart, liver, lungs, kidneys and intestines were collected and macerated individually. The samples were pooled and submitted to high-throughput sequencing (HTS) using the Illumina MiSeq platform and subsequently individually screened using a pancoronavirus RT-PCR protocol. We detected CoV-related sequences in HTS, but only two (2/101; 1.98%) animals had CoV detected in the intestines by RT-PCR. Partial sequences of RdRp and spike genes were obtained in the same sample and the RdRp region in the other sample. The sequences were classified as belonging to Alphacoronavirus. The sequences were closely related to alphacoronaviruses detected in vampire bats from Peru. The continuous monitoring of bat CoVs may help to map and predict putative future zoonotic agents with great impacts on human health.

SARS-CoV-2 introduction and lineage dynamics across three epidemic peaks in Southern Brazil: massive spread of P.1.

Genomic surveillance of SARS-CoV-2 is paramount for understanding viral dynamics, contributing to disease control. This study analyzed SARS-CoV-2 genomic diversity in Rio Grande do Sul (RS), Brazil, including the first reported case in each Regional Health Coordination and cases from three epidemic peaks. Ninety SARS-CoV-2 genomes from RS were sequenced and analyzed through comparison with SARS-CoV-2 datasets available in GISAID for phylogenetic inference and mutation analysis. Among the first reported cases, we found the following lineages: B.1 (33.3%), B.1.1.28 (26.7%), B.1.1 (13.3%), B.1.1.33 (10.0%), and A (6.7%), evidencing SARS-CoV-2 introduction by both international origin and community-driven transmission. We found predominance of B.1.1.33 (50.0%) and B.1.1.28 (35.0%) during the first epidemic peak (July-August 2020), emergence of P.2 (55.6%) in the second peak (November-December 2020), and massive spread of P.1 and related sequences (78.4%), such as P.1-like-II, P.1.1 and P.1.2 in the third peak (February-April, 2021). Eighteen novel mutation combinations were found among P.1 genomes, and 22 different spike mutations and/or deletions among P.1 and related sequences. This study shows the dispersion of SARS-CoV-2 lineages in Southern Brazil and describes SARS-CoV-2 diversity during three epidemic peaks, highlighting the spread of P.1 and the high genetic diversity of currently circulating lineages. Genomic monitoring of SARS-CoV-2 is essential to guide health authorities' decisions to control COVID-19 in Brazil.

Zika Virus Envelope Domain III Recombinant Protein Delivered With Saponin-Based Nanoadjuvant From Quillaja brasiliensis Enhances Anti-Zika Immune Responses, Including Neutralizing Antibodies and Splenocyte Proliferation.

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis, was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses.

Detection of multiple viruses in oropharyngeal samples from Brazilian free-tailed bats (Tadarida brasiliensis) using viral metagenomics.

In this study, we analyzed the viral population in oropharyngeal samples from T. brasiliensis using a viral metagenomic approach. Genomes corresponding to members of the families Circoviridae, Genomoviridae, Herpesviridae, Paramyxoviridae, Coronaviridae, and Astroviridae were detected. This study provides the first preliminary understanding of the oropharyngeal virome of T. brasiliensis, which may guide the discovery and isolation of novel viruses in the future and highlights the need for continuing investigations in this regard.

Zika virus-induced brain malformations in chicken embryos.

BACKGROUND: Zika virus (ZIKV) was confirmed to be related to microcephaly in 2016. However, there is still a need for understanding the embryonic morphological changes induced by ZIKV and when they occur. Here, chicken embryos were chosen as experimental model of ZIKV to evaluate virus-associated morphological alterations that might take place during embryonic development. METHODS: A screening with different viral doses was conducted in embryos at HH Stage 10-12 (E1.5) as well as a follow up of the first 5 days postinfection (dpi) was performed to observe the main morphologic changes post ZIKV infection. RESULTS: ZIKV exposed embryos presented a higher prevalence of mortality and defects such as brain malformation when compared to controls. Moreover, we observed that the phenotypes become more evident at 4dpi, when the viral load quantification reaches a peak. CONCLUSIONS: We found that ZIKV exposed embryos presented a high prevalence of mortality and central nervous system (CNS) abnormalities in a dose-dependent manner. The phenotype was more evident 4 days postinfection, when the viral load quantification reached a peak.